recombinant human α-syn monomer  (Millipore)

Journal: Biomedicines

Article Title: Astemizole, a Second-Generation Histamine H1-Receptor Antagonist, Did Not Attenuate the Aggregation Process of α-Synuclein In Vitro

doi: 10.3390/biomedicines12030611

Figure Lengend Snippet: The absorbance spectra of Congo red expressed as arbitrary units (au) in the presence of increasing molar concentrations of astemizole (AST) and human recombinant amyloid-β(1-42) (Aβ42) or α-synuclein (α-syn). The absorbance spectra of Aβ42 or α-syn aggregates formed in the absence (black line) and presence of increasing molar concentrations of AST (colored lines). The absorbance spectra of Congo red alone (dotted yellow line) are also presented. ( A , B ) Congo red binding absorbance spectra at 400–600 nm and the binding absorbance of 40 μM human Aβ42 monomer at 500 nm in the presence of various concentrations of astemizole. ( C , D ) The absorbance spectra of Congo red in the presence of 10 μM α-syn monomer with or without various concentrations of AST. Data represent the mean ± standard error from three replicates. * = p < 0.05, significant compared with Aβ42 (40 μM) or α-syn (10 μM) only; n.s. = not significant.

Article Snippet: Recombinant human α-syn monomer was purchased from Sigma-Aldrich (St. Louis, MO, USA) and Abcam Corporation (Cambridge, MA, USA).

Techniques: Recombinant, Binding Assay

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: SARS-CoV-2 differently affects SNCA mRNA expression and α-syn protein levels at 24 and 48 h post-infection in A549-hACE2 and CaLu-3 epithelial lung cells. A qPCR-based relative quantification of SNCA mRNA in Mock- and SARS-CoV-2-infected cells (MOI 0.05), either in the presence or absence of exogenous IFN-β, 24 and 48 h post-infection. Results are shown as mean ± SEM from n = 3 independent experiments. Statistical analysis was performed by applying Two-way ANOVA, followed by multiple testing correction. To avoid graphs overcrowding, p values are shown for statistically significant groups of interest only. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. B and C Representative immunofluorescence images for SARS-CoV-2 Spike (S) and α-syn proteins in Mock- and SARS-CoV-2-infected (MOI 0.05) in A549-hACE2 ( B ) and CaLu-3 ( C ) epithelial lung cells 24 and 48 h post-infection, in the absence (CTR) or presence of IFN-β. Cells were fixed in 4% Formaldehyde solution for 15 min and permeabilized with 0.1% of Triton X-100 for 15 min. Images are representative of n = 3 independent experiments. Bars correspond to 20 μm

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Expressing, Infection, Immunofluorescence

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: A1 and A2 Exogenous administration of α-syn monomers increases SARS-CoV-2 replication in A549-hACE2 and CaLu-3 epithelial cells, which is prevented by IFN-β. Quantification of viral replication from cell supernatants through RT-qPCR for the SARS-CoV-2 N gene in infected A549-hACE2 ( A1 ) and CaLu-3 cells ( A2 ), 24 and 48 h post-infection (MOI 0.05). A1 In A549-hACE2 cells, exogenous α-syn monomers significantly increased viral replication compared with infected CTR both at 24 and 48 h post-infection. A2 In CaLu-3 cells the pro-viral effect of exogenous α-syn monomers was maximally detected at 24 h, while it declined at 48 h post-infection. In both cases, IFN-β, either alone or in combination with exogenous α-syn, completely prevented SARS-CoV-2 replication. Results show SARS-CoV-2 N gene copy numbers quantified from the RNA isolated from 100 μl of cell supernatants. Results are shown as mean ± SEM from n ≥ 5 independent experiments. Values for 24 and 48 h post-infection were analyzed by applying One-Way ANOVA followed by multiple testing correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. B1 and B2 Representative immunofluorescence images of SARS-CoV-2 Nucleocapsid (N) and α-syn proteins in Mock- and SARS-CoV-2-Infected A549-hACE2 epithelial lung cells 48 h post-infection (MOI 0.05), in the absence and presence of IFN-β, exogenous α-syn monomers, or both. SARS-CoV-2 replication is enhanced in the presence of exogenous α-syn monomers, which impair accumulation of permeabilization-resistant α-syn species that are instead increased by IFN-β. B1 For visualization of large, permeabilization-resistant α-syn species, that potentially correspond to multimers or intracellular membrane-bound species, cells were fixed for 15 min in 4% Formaldehyde solution and permeabilized with 0.3% of Triton X-100 for 15 min. Bars correspond to 20 μm. B2 For better preservation, and visualization of small, highly-soluble α-syn species potentially corresponding to monomers, cells were fixed for 15 min in 4% Paraformaldehyde (PFA) and permeabilized with 0.1% of Triton X-100 for 10 min. Bars correspond to 20 μm. Images are representative of n = 3 independent experiments. C Quantification of SARS-CoV-2-N-positive cells in SARS-CoV-2-infected CTR vs α-syn-treated SARS-CoV-2-infected cells. Values are expressed as the percentage of N-positive cells ± SD calculated as the number of N-positive cells/total cells per microscopic field from n = 12 microscopic fields per experimental group, from n = 3 independent experiments. Data were analyzed by applying the Student’s t-test for comparison between VIRUS CTR and VIRUS α-syn groups. **p < 0.01

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Quantitative RT-PCR, Infection, Isolation, Immunofluorescence, Membrane, Preserving, Comparison, Virus

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: In HUVECS, absence of productive SARS-CoV-2 infection is associated with α-syn accumulation. A Quantification of SARS-CoV-2 replication in HUVECs in the absence and presence of α-syn and IFN-β (MOI 1). Results are shown as mean ± SEM from n = 4 independent experiments. Data were analyzed by applying Two-Way ANOVA. **p < 0.01, ***p < 0.001. B In the absence of productive viral replication, overall α-syn immunostaining in HUVECs is not decreased by either SARS-CoV-2 infection or the addition of exogenous α-syn monomers. Representative immunofluorescence images for SARS-CoV-2-Nucleocapsid (N), and α-syn in Mock- and SARS-CoV-2-Infected HUVECs 72 h post-infection (MOI 1), in the absence and presence of IFN-β, or exogenous α-syn monomers. Cells were fixed for 15 min in Formaldehyde solution, followed by 15 min permeabilization with 0.1% Triton X-100. Bars correspond to 20 μm. C In HUVECs, absence of productive SARS-CoV-2 infection is associated with the occurrence of juxtanuclear α-syn foci. 3D reconstruction of immunofluorescence images for SARS-CoV-2-Nucleocapsid (N), and α-syn proteins in Mock- and SARS-CoV-2-Infected HUVECs 72 h post-infection (MOI 1) shows the presence of juxtanuclear α-syn foci (arrows) which are induced by SARS-CoV-2 and potentiated by IFN-β and exogenous α-syn monomers

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Infection, Immunostaining, Immunofluorescence

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: SARS-CoV-2 synergizes with an excess of exogenously added α-syn monomers to reduce α-syn multimer:monomer ratio, which is prevented by IFN-β. A Representative western blot, and B quantification of total α-syn, α-syn monomers, α-syn multimers, and C α-syn multimer:monomer ratio in A549-hACE2 cells 48 h post- Mock and SARS-CoV-2 infection in the presence of exogenously added α-syn. Quantification was performed by normalizing to total protein (Loading control). Results show raw normalized values presented as mean ± SEM from n = 3 independent experiments. Multimer:monomer ratio is expressed as absolute value calculated as “multimers/monomers”. Data were analyzed by applying Two-Way ANOVA (for total, multimer, and monomer α-syn quantification) or One Way ANOVA (for multimer:monomer ratio). *p < 0.05, ***p < 0.001, ****p < 0.0001

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Western Blot, Infection

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: A Gene expression changes associated with the pro-viral and anti-viral effects of exogenous α-syn monomers and IFN-β, respectively . qPCR-based transcriptional analyses were performed in A549-hACE2 cells 48 h post-infection. Results are expressed as mean ± SEM from n = 3 independent experiments. Data for each gene were analyzed by One Way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. SNCA synclein alpha; IFNB Interferon beta; STAT1 Signal transducer and activator of transcription 1; MX1 Myxovirus Resistance Protein 1; MX2 Myxovirus Resistance Protein 2; OAS1 2'-5'-oligoadenylate synthetase 1, RIG-I retinoic acid-inducible gene I; TNFA tumor necrosis factor alpha, TNFRSF1A tumor necrosis factor receptor superfamily 1A, TLR8 Toll-like receptor 8; Toll-like receptor 9 (TLR9). B SNCA mRNA expression is negatively correlated with viral titers at 3 and 5d post-infection of epithelial lung cells . The analyses were performed in CaLu-3 and A549-hACE2 cells at 3 and 5d post-infection with SARS-CoV-2 at 3 different MOI (0.01, 0.005, 0.001). Correlation between relative SNCA mRNA expression and SARS-CoV-2 N gene mRNA expression in A549-hACE2 and CaLu-3 cells was calculated through the Pearson’s r coefficient. Statistical significance was calculated through the two-tailed, paired Student’s t test . n = 12 for A549-hACE2 cells; n = 9 for CaLu3 cells. C1 and C2 SNCA mRNA expression is increased in human PBMCs stimulated with SARS-CoV-2 and is negatively correlated with SARS-CoV-2 replication in co-cultured CaLu-3 cells. C1 qPCR-based quantification of SNCA mRNA expression in in vitro SARS-CoV-2-exposed human PBMCs compared with Mock CTR. Statistical significance was calculated through the two-tailed, paired Student’s t test . The graph shows individual values (n = 8) with mean ± SD. ** p < 0.01. C2 Correlation analysis between relative SNCA mRNA expression within SARS-CoV-2-exposed PBMCs, and SARS-CoV-2 replication (N gene copy number) in co-cultured CaLu-3 epithelial cells. Correlation was calculated through the Pearson’s r coefficient. Statistical significance was calculated through the two-tailed, paired Student’s t test. n = 8

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Expressing, Infection, Two Tailed Test, Cell Culture, In Vitro

Journal: Biological Research

Article Title: Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

doi: 10.1186/s40659-023-00482-x

Figure Lengend Snippet: Summary cartoon depicting the opposite effects of extracellular α-syn monomers and IFN-β upon cellular internalization, and related α-syn multimer:monomer dynamics during SARS-CoV-2 infection of susceptible epithelial lung cells

Article Snippet: Type-I IFN (human recombinant IFN-β, 500 IU/ml, BEI resources) and human recombinant α-syn monomers (1 μM, Merck-Sigma), were exogenously pre-administered to cells, both alone and in combination, at 24 h pre-infection.

Techniques: Infection