Journal: Frontiers in Immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: The effect of acute ethanol-exposure on mouse bone marrow derived macrophages (BMDM). Phagocytosis in BMDM exposed to vehicle or ethanol ± LPS to study phagocytosis using pHrodo bioparticles and SIRT2 expression. (A) Representative images of intracellular pHrodo bioparticles in vehicle or Ethanol-exposed WT-BMDM ± LPS. (B) Fluorescence quantification of pHrodo bioparticles in WT-BMDM (n=4 repetitions/group; * p<0.05). (C) SIRT2 protein expression detected by western blot in Vehicle- or Ethanol-exposed BMDM cells ± LPS. (D) Western blot image quantification of SIRT2 protein blot in vehicle or ethanol-exposed BMDM cells ± LPS (n = 4 blots/group; * p < 0.05). (E) Representative images of pHrodo bioparticles in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. (F) Fluorescence quantification of pHrodo bioparticles in Ethanol-exposed WT-BMDM, and SIRT2KO-BMDM ± LPS. * p < 0.05. (G) Representative images of pHrodo bioparticles in Ethanol-exposed WT-BMDM, co-treated with SIRT2 inhibitor AK-7 or DMSO ± LPS. (H) Fluorescence quantification of pHrodo bioparticles in Ethanol-exposed WT-BMDM with AK-7/DMSO ± LPS stimulation. * p < 0.05.

Article Snippet: PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Derivative Assay, Expressing, Fluorescence, Western Blot

Journal: Frontiers in Immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: The effect of acute ethanol-exposure-induced SIRT2 on PFKP expression in macrophages. (A) PFKP expression in WT-BMDM exposed to vehicle or ethanol ± LPS by western blot analysis. (B) PFKP western blot image quantification of PFKP protein in vehicle vs. ethanol exposed WT-BMDM, Y axis represents fold of vehicle-LPS (fold of vehicle control) (n = 4 blots; * p < 0.05). (C) PFKP expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. (D) Western blot image quantification of PFKP protein in ethanol exposed WT-BMDM and SIRT2KO-BMDM. Y axis represents fold of Ethanol-exposed WT-LPS (fold of WT- ethanol control) (n = 4 blots; * p < 0.05). (E) PFKP expression in Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. (F) Western blot image quantification of PFKP in AK-7 vs. DMSO treated-Ethanol-exposed WT-BMDM ± LPS, Y axis represents fold of Ethanol-exposed WT-LPS (fold of WT- ethanol control) (n = 4 blots; * p < 0.05).

Article Snippet: PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: SIRT2-PFKP in vivo and in vitro interaction. (A) RAW264.7 cell macrophages (RAW) ± LPS. IP of whole-cell lysates using an anti-SIRT2 antibody followed by IB analysis of PFKP and SIRT2. IP with isotype IgG control antibody was used as a negative control. (B) Western blot analysis of PFKP and SIRT2 in the whole cell lysate used as input for the SIRT2 IP. (C) In-vitro interaction between SIRT2 and PFKP using SPR. SIRT2 protein immobilized onto sensor chip and the PFKP was flowed at various concentration (15.62, 31.25, 62.5, 125, 250, 500 and 1000nM). The response units on Y axis (RU) represent quantitative assessment of protein-protein interaction. (D) wtPFKP and control plasmid transfection and IP, using turbo-GFP-trap in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of acetyl lysine, turbo-GFP-wtPFKP and control for PFKP (turbo-GFP). HEK293T cell lysate without transfection used as a negative control. (E) Western blot analysis of control-PFKP (Turbo-GFP), wtPFKP (Turbo-GFP-wtPFKP), DDK-SIRT2 and CPA in whole cell lysate used as input for the turbo-GFP IP. (F) wtPFKP transfection and IP using magnetic-TUBEs in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of ubiquitination. (G) Western blot analysis of turbo-GFP wtPFKP, DDK-SIRT2, turbo-GFP and CPA in whole cell lysate used as an input for the TUBE IP.

Article Snippet: PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: In Vivo, In Vitro, Control, Negative Control, Western Blot, Concentration Assay, Plasmid Preparation, Transfection, Ubiquitin Proteomics

Journal: Frontiers in Immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: Effect of K394R mutation on PFKP. (A, B) HEK293T cells transfected with wtPFKP/mtPFKP in the presence or absence of SIRT2. Western blot analysis of Turbo-GFP-wtPFKP, DDK-SIRT2, turbo-GFP (control for wtPFKP plasmid transfected) and CPA. (C) mtPFKP transfection and IP using turbo-GFP-trap in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of acetyl lysine, turbo-GFP-mtPFKP and turbo-GFP (control for wtPFKP plasmid transfected). Pulldown with HEK293T cell lysate without transfection was used as a negative control. (D) Western blot analysis of turbo-GFP mtPFKP, DDK-SIRT2, turbo-GFP and CPA in whole cell lysate, used as an input for the turbo-GFP IP. (E) mtPFKP transfection and IP using magnetic-TUBEs in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of ubiquitination. (F) Western blot analysis of turbo-GFP mtPFKP, DDK-SIRT2, turbo-GFP and CPA in whole cell lysate used as input for the TUBE IP.

Article Snippet: PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Mutagenesis, Transfection, Western Blot, Control, Plasmid Preparation, Negative Control, Ubiquitin Proteomics

Journal: Frontiers in Immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: The effect of acute ethanol-exposure on SIRT2 expression and the effect of AK-7 on LC3-associated phagocytosis in Ethanol-exposed Human macrophages. Human macrophages were exposed to vehicle or ethanol ± LPS to study SIRT2 expression and LAP. SIRT2 expression was analyzed by immunostaining (A) Representative images of SIRT2 immunostaining in vehicle or Ethanol-exposed human macrophages ± LPS. (B) Fluorescence quantification of SIRT2 immunostaining in human macrophages (n=4; *p<0.05). (C) LAP in human macrophages with vehicle or ethanol-exposure ± LPS. Representative images of intracellular pHrodo bioparticles during phagocytosis and co-stained for LC3. (D) For each image, the co-localization of intracellular pHrodo (red) and LC3 (green) were determined as LAP and divided by the total numbers of nuclei. Graph represents fluorescence quantification of LAP in human macrophages (n=4; * p<0.05). (E) Ethanol-exposed human macrophages were co-treated with SIRT2 inhibitor AK-7 or DMSO ± LPS. Representative images of intracellular pHrodo bioparticles during phagocytosis and stained for LC3 to study LAP. (F) Graph represents fluorescence quantification of LAP in Ethanol-exposed human macrophages ± AK-7 ± LPS stimulation (n=4; * p<0.05).

Article Snippet: PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Expressing, Immunostaining, Fluorescence, Staining

Journal: British Journal of Pharmacology

Article Title: Tubulin acetylation promoting potency and absorption efficacy of deacetylase inhibitors

doi: 10.1111/bph.12946

Figure Lengend Snippet: Multiple interactions of tubulin deacetylases with tubulin and TPPP/p25 followed by elisa. (A) The plate was coated with HDAC6 or SIRT2, then it was incubated with TPPP/p25 at different concentrations. Normalized absorbance was calculated as the absorbance at a given TPPP/p25 concentration divided by the absorbance at saturation. (B, C) The plate was coated with HDAC6 (B) or SIRT2 (C), then it was incubated with tubulin at different concentrations in the absence or in the presence of 200 nM TPPP/p25, the interaction was detected by tubulin antibody. The apparent dissociation constants (Kd) characteristic for the interactions were evaluated by non-linear fitting of the hyperbolic saturation curves using the Microcal Origin 8.0 software (OriginLab Corporation, Northampton, MA, USA). The different symbols indicate independent sets of experiments. (D) Scheme of multiple interactions of tubulin, TPPP/p25 and deacetylases with the affinity constants, Kd.

Article Snippet: Human recombinant HDAC6 (50006) and SIRT2 (50013) were purchased from BPS Bioscience (Camarillo, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Software

Journal: British Journal of Pharmacology

Article Title: Tubulin acetylation promoting potency and absorption efficacy of deacetylase inhibitors

doi: 10.1111/bph.12946

Figure Lengend Snippet:

Article Snippet: Human recombinant HDAC6 (50006) and SIRT2 (50013) were purchased from BPS Bioscience (Camarillo, CA, USA).

Techniques: Histone Deacetylase Assay

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Plasma and gene expression levels of SIRT2 are associated with viral parameters in chronic untreated HIV infection. Proteomic array applied to plasma samples from untreated chronically infected individuals with different degrees of HIV control ( n = 40). (A) Heatmap showing the relative plasma levels of the most differentially detected soluble factors between untreated HIV-infected individuals with high viral loads (HIV-high, with more than 50,000 viral copies/mL, n = 20) and those with low viral loads (HIV-low, viral loads < 10,000 viral copies/mL, n = 20). Red indicates high protein abundance in plasma, and green indicates reduced protein levels. (B) Volcano plot representing the relative expression of the 185 molecules measured, identifying SIRT2 as the most significant and differentially detected soluble factor between HIV-high and HIV-low (Log 2 FC = 1.039; log 10 P value = 2.478). The log 2 fold change is shown on the x axis and the −log 10 of the P value on the y axis. (C) Scatterplot showing the relative plasma protein levels of SIRT2 in both study groups (HIV-high [ n = 20, orange dots] and HIV-low [ n = 20, green dots]). (D) SIRT2 gene expression in PBMCs from chronic untreated HIV-infected individuals grouped as HIV-high ( n = 16, orange dots), HIV-low ( n = 30, green dots), and elite controllers (i.e., untreated HIV-infected individuals with undetectable HIV viral load in plasma, n = 12, blue dots). Study groups are shown on the x axis, and relative SIRT2 gene expression corrected for CD4 counts is shown on the y axis. (E and F) Correlation between relative SIRT2 gene expression and plasma viral load (E) and HIV proviral DNA levels (F) in PBMC for all three groups. HIV-high patients ( n = 16, orange dots), HIV-low patients ( n = 30, green dots), and elite controllers ( n = 12, blue dots) are indicated in the plot. Relative SIRT2 gene expression corrected for CD4 counts is shown on the x axis, and viral load (HIV RNA copies/mL) and proviral levels (HIV DNA copies/10 6 PBMCs) are shown on the y axis. Plasma proteome data were analyzed using the t test. SIRT2 plasma levels between HIV-high and HIV-low were analyzed using the Mann-Whitney test. SIRT2 gene expression levels between HIV-high, HIV-low, and EC were analyzed using ANOVA test for multiple comparisons corrected by original FDR method of Benjamini and Hochberg. The Spearman's rank test was applied for the correlation analysis. Statistical significance was set at P < 0.05.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Clinical Proteomics, Gene Expression, Infection, Control, Quantitative Proteomics, Expressing, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Top candidates identified by plasma proteomic analysis

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Clinical Proteomics

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Plasma levels of SIRT2 are associated with neurological factors in chronic untreated HIV infection. (A) Correlation matrix showing the significant relationship between SIRT2 and proteins measured in the antibody array considering all HIV-infected groups (HIV-high and HIV-low). The color scale shows positive correlations in blue and negative correlations in red. (B) The functional analysis performed using the STRING webpage represents the interaction between SIRT2 and the remaining correlated factors. Several functions derived from Gene Ontology, Uniprot, Reactome, and PubMed were identified as follows: Regulation of Cell Death category (GO, 0010941; FDR = 0.0030) in blue, Innate Immunity (KW0399; FDR = 3.57 × 10E7) in red, Complement Cascade (HSA-166658; FDR = 3.44 × 10E6) in yellow, and epigenetic mechanisms involved in neurological disorders (PMID25774124; FDR = 0.00025) in green. (C to E). Correlation plots showing the associations between sirtuin-2 and plasma levels of SNCA (C), MAPT (D), and BDNF (E) measured in the antibody array across all chronic untreated HIV-infected individuals. HIV-high ( n = 20, orange dots) and HIV-low ( n = 20, green dots) groups are indicated in the plot. The x axis shows relative plasma SIRT2 levels, and the y axis shows the relative plasma levels for SNCA, BDNF, and MAPT, respectively. The Spearman's rank test was applied for the correlation analysis. Statistical significance was set at P < 0.05.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Clinical Proteomics, Infection, Ab Array, Functional Assay, Derivative Assay

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: SIRT2 levels in the CNS of HIV-infected individuals. (A to C) Scatterplot showing the relative plasma protein levels of SNCA (A), MAPT (B), and BDNF (C) in both study groups (HIV-high [ n = 20, orange dots] and HIV-low [ n = 20, green dots]). (D) Plot representing normalized protein expression (NPX) levels of SIRT2 in the plasma (left) and CSF (right) in HIV-high ( n = 10, red bar), HIV-low ( n = 10, green bar), and seronegative individuals ( n = 5, black bar). The mean and standard deviation are shown. (E and F) Correlation between relative SIRT2 levels and relative CSF levels of MAPT (E) and NFL (F) in HIV-high ( n = 10, red dots), HIV-low ( n = 10, green dots), and seronegative individuals ( n = 5, black dots). Relative MAPT and NFL levels are shown on the y axis, and relative SIRT2 levels are shown on the x axis. (G) Plot representing SIRT2 gene expression in postmortem brain tissues measured by microarray in the GSE28160 study performed in seronegative ( n = 6), cART-treated HAD (HIV-associated disorders, n = 6), and cART-untreated HAD individuals ( n = 8). Values are expressed as mean and standard deviation. (H) Correlation between gene expression levels of SIRT2 ( x axis) and MAPT ( y axis) in postmortem brain tissue samples in HIV-infected individuals (cART-treated HAD [ n = 6] and cART-untreated HAD individuals [ n = 8]). For comparisons between groups, ANOVA test for multiple comparisons corrected by original FDR method of Benjamini and Hochberg was applied. The Spearman's rank test was applied for the correlation analysis. Statistical significance was set at P < 0.05.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Infection, Clinical Proteomics, Expressing, Standard Deviation, Gene Expression, Microarray

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Plasma SIRT2 levels are associated with neurological dysfunction in treated HIV infection. (A) Schematic representation of the following two study arms included in the ARBRE study: early-cART ( n = 9) and later-cART ( n = 10) HIV-infected individuals who started cART at different time points after estimated HIV acquisition and underwent longitudinal neurological evaluation (neuropsychological tests and neuroimaging) and quantification of SIRT2 in plasma. Early-cART patients initiated treatment within a maximum of 3 months since the estimated date of infection, and later-treated patients initiated cART at least 6 months after the estimated date of infection. Study visits were at baseline (BSL), 1 month (4 weeks), and 1 year after initiation of cART. (B to D) Plots representing results of the NPZ12 and global gray matter and medial orbitofrontal cortex volume (peak coordinate at MNI, x = 6, y = 44, z = −29) in early- and later-cART individuals at baseline and after 1 year on treatment (early-cART, n = 9, green group; later-cART, n = 9, red group). The values on the y axis represent the NPZ12 score, global gray matter (cm 3 ) and medial orbitofrontal cortex volume (a.u.). The box plot shows the median and minimum to maximum values for each group. (E to H) Absolute plasma NFL, GFAP, UCHL1, and MAPT levels at baseline (BSL) and after 1 year on treatment in early-cART ( n = 9, green group) and later-cART ( n = 10, red group) individuals expressed in picograms per milliliter. The upper limit of the bar is the median value of protein plasma levels. (I) Absolute MAPT (tau protein) plasma levels represented longitudinally and expressed in picograms per milliliter. (J) Correlation between longitudinal plasma MAPT (tau protein) levels ( x axis, pg/mL) and the results for medial orbitofrontal cortex volumetry ( y axis) expressed as the difference between 1 year and baseline in the ARBRE study including early- ( n = 9, green dots) and later-cART individuals ( n = 5, red dots). (K and L) Plots representing the absolute plasma SIRT2 in early-cART ( n = 9, green line) and later-cART ( n = 10, red line) individuals cross-sectionally (K) and longitudinally (L) at BSL (baseline) and 1 year time points. Weeks of treatment are shown on the x axis, and absolute plasma levels of SIRT2 (ng/mL) are shown on the y axis. (M) Correlation between longitudinal plasma SIRT2 ( x axis, ng/mL) and the results for medial orbitofrontal cortex volumetry expressed as the difference between 1 year on cART and baseline in the ARBRE study including early- ( n = 9, green dots) and later-cART individuals ( n = 9, red dots). Differences between the groups were analyzed using the Mann-Whitney U test, changes over time were assessed using the paired t test, and the correlation analysis was based on the Spearman's rank test. Statistical significance was set at P < 0.05.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Clinical Proteomics, Infection, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Clinical information of neurologically evaluated HIV cohort (ARBRE study)

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Transmission Assay, Infection, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Longitudinal neurological evaluation and SIRT2 plasma levels in ARBRE study

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Effect of in vitro SIRT2 inhibition on HIV replication. (A to C) Inhibition of HIV replication in the presence of SIRT2 inhibitor (AK-1), tested in HIV-infected PHA blasts (seven independent experiments) (A and B) with HIV NL4-3 strain, at day 3 and 1 week postinfection, respectively, and HIV-infected monocyte-derived macrophages (MDMs) (six independent experiments) (C) infected with HIV BaL strain at day 4 postinfection. (D to F) Cell viability in the presence of SIRT2 inhibitor (AK-1), tested in HIV-infected PHA blasts (seven independent experiments) (D and E) with HIV NL4-3 strain, at day 3 and 1 week, respectively, and monocyte-derived macrophages (MDMs) (six independent experiments) (F) infected with HIV BaL strain at day 4 postinfection. Experimental conditions are shown on the x axis; quantification of viability (% live cells) is shown on the y axis. (G) Glial cells infected with the HIV NLAD8 virus strain (MOI, 0.01) in the presence of different doses of AK-1 inhibitor. Experimental conditions are shown on the x axis; quantification of absolute p24 supernatant (pg/mL) is shown on the y axis. The data presented correspond to the mean of two duplicates performed in a single experiment. (H) HIV-infected microglial cells (six independent experiments) with the NLAD8 virus strain in the presence of different doses of AK-1 inhibitor. Experimental conditions are shown on the x axis; quantification of p24 levels (pg/mL) is shown on the y axis. For results from HIV-infected PHA blasts, MDMs, and primary glial cells, ANOVA test for multiple comparisons corrected by original FDR method of Benjamini and Hochberg was used to analyzed differences between conditions. For all comparisons, P < 0.05 was considered significant. The plots show the median of all experiments for each condition.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: In Vitro, Inhibition, Infection, Derivative Assay, Virus

Journal: Journal of Virology

Article Title: Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction

doi: 10.1128/jvi.01655-22

Figure Lengend Snippet: Effect of in vitro SIRT2 inhibition on HIV reactivation. (A) Histogram plot showing green fluorescent protein (GFP) cellular counts of three different conditions of the experiment, including nonstimulated cells (black), PMA-stimulated cells (orange), and cells stimulated with PMA in the presence of AK-1 (blue) from one representative experiment. (B and C) HIV reactivation measured by the percentage of GFP expression measured by flow cytometry in J-LAT A2 cells is shown on the y axis; different conditions of the experiment, including nonstimulated cells, PMA-stimulated cells, and cells stimulated with PMA in the presence of AK-1 are shown on the x axis. (D) Gating strategy of HIV virus reactivation in the J-LAT A2 cell line experiment. For multiple comparisons, ANOVA test corrected by original FDR method of Benjamini and Hochberg was used to analyze differences between conditions. For all comparisons, P < 0.05 was considered significant. The plots show the median of all experiments for each condition.

Article Snippet: Human SIRT2 enzyme-linked immunosorbent assay (ELISA) kit (Aviva Systems Biology) was used to measure the SIRT2 levels in plasma, and the absolute levels were quantified by applying a 4-parameter logistic curve analysis.

Techniques: In Vitro, Inhibition, Expressing, Flow Cytometry, Virus