Journal: Vaccines

Article Title: N-Terminal Fatty Acids of NEF MUT Are Required for the CD8 + T-Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles

doi: 10.3390/vaccines8020243

Figure Lengend Snippet: Structure of the DNA molecular constructs expressing Nef mut -based products. On the top, shown are both nucleotide and amino acid N-terminal sequences of both Nef mut /E7 and GC-AG Nef mut /E7. Mutated sequences of the latter are highlighted in red. At the bottom, the structure of both DNA vectors is reported, including the restriction sites where the HPV16-E7 open reading frame was inserted. IE-CMV: immediate-early CMV promoter; SD: major splice donor site; SA major splice acceptor site; polyA: polyadenylation site.

Article Snippet: A total of eight mice for each experiment were inoculated i.m. two times in fourteen-day intervals with 50 μg for each quadriceps of DNA vectors purified through endotoxin-free Qiagen kit (Hilden, Germany).

Techniques: Construct, Expressing

Journal: Vaccines

Article Title: N-Terminal Fatty Acids of NEF MUT Are Required for the CD8 + T-Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles

doi: 10.3390/vaccines8020243

Figure Lengend Snippet: Analysis of expression of GC-AG Nef mut /E7. Western blot analysis of total cell lysates from HEK-293T cells transfected with DNA vectors expressing the indicated Nef mut -based fusion products. As control, conditions from mock-transfected cells, as well as cells transfected with Nef mut , were included. Polyclonal anti-Nef Abs served to detect Nef mut -based products, while β-actin levels were detected as a reference standard. Nef-specific signals are highlighted. Molecular markers are given in kilodaltons (kDa). Signal intensity ratios compared to the Nef mut control condition, considering the respective β-actin signals, were 1.48 and 1.43 for GC-AG Nef mut /E7 and Nef mut /E7, respectively. The results are representative of five independent experiments. Uncropped blots showing all the bands with all molecular weight markers are shown in .

Article Snippet: A total of eight mice for each experiment were inoculated i.m. two times in fourteen-day intervals with 50 μg for each quadriceps of DNA vectors purified through endotoxin-free Qiagen kit (Hilden, Germany).

Techniques: Expressing, Western Blot, Transfection, Molecular Weight

Journal: Vaccines

Article Title: N-Terminal Fatty Acids of NEF MUT Are Required for the CD8 + T-Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles

doi: 10.3390/vaccines8020243

Figure Lengend Snippet: Analysis of stability of GC-AG Nef mut /E7. Comparative stability analysis between Nef mut /E7 and GC-AG Nef mut /E7 fusion proteins in total lysates from HEK-293T cells transfected with the respective DNA vectors. Forty-eight hours after transfection, cells were washed and reseeded in complete medium containing 1 μg/mL of cycloheximide. Transfected cells were harvested at the indicated times, and total lysates from the same number of cells were analyzed by Western blot assay. Filters were revealed by both anti-Nef and anti- β-actin polyclonal antibodies. Time zero refers to cells harvested at the time of cycloheximide treatment. Signal intensity ratios compared to respectve time zero are indicated. Molecular markers are given in kDa. The results are representative of two independent experiments. Uncropped blots showing all the bands with all molecular weight markers are shown in .

Article Snippet: A total of eight mice for each experiment were inoculated i.m. two times in fourteen-day intervals with 50 μg for each quadriceps of DNA vectors purified through endotoxin-free Qiagen kit (Hilden, Germany).

Techniques: Transfection, Western Blot, Molecular Weight

Journal: Vaccines

Article Title: N-Terminal Fatty Acids of NEF MUT Are Required for the CD8 + T-Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles

doi: 10.3390/vaccines8020243

Figure Lengend Snippet: Detection of Nef mut /E7 based fusion products in transfected cells and exosomes. Shown is the Western blot analysis of total lysates from the same number of HEK293T cells transfected with DNA vectors expressing either Nef mut /E7 or GC-AG Nef mut /E7 (left panels). Equal volumes of buffer, where purified exosomes were resuspended after differential centrifugations of the respective supernatants, were also analyzed (right panel). As control, conditions from mock-transfected cells, as well as cells transfected with Nef mut alone, were included. Polyclonal anti-Nef Abs served to detect Nef mut -based products, while β-actin and Alix were revealed as markers for cell lysates and exosomes, respectively. Concerning signals from cell lysates, intensity ratios compared to the Nef mut control condition, considering the respective β-actin signals, were 1.05 and 0.91 for GC-AG Nef mut /E7 and Nef mut /E7, respectively. Regarding exosomes, the intensity ratio between Nef mut and Nef mut /E7 conditions, considering the respective Alix signals, was 0.81. Molecular markers are given in kDa. The results are representative of five independent experiments. Uncropped blots showing all the bands with all molecular weight markers are shown in .

Article Snippet: A total of eight mice for each experiment were inoculated i.m. two times in fourteen-day intervals with 50 μg for each quadriceps of DNA vectors purified through endotoxin-free Qiagen kit (Hilden, Germany).

Techniques: Transfection, Western Blot, Expressing, Purification, Molecular Weight

Journal: Vaccines

Article Title: N-Terminal Fatty Acids of NEF MUT Are Required for the CD8 + T-Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles

doi: 10.3390/vaccines8020243

Figure Lengend Snippet: HPV16-E7-specific CD8 + T-cell immunity induced in mice after i.m. DNA injection. CD8 + T-cell immune response in C57 Bl/6 mice inoculated i.m. with the DNA vectors expressing either Nef mut /E7 or GC-AG Nef mut /E7 DNA vectors. As controls, mice were inoculated with void vector. At the time of sacrifice, 2.5 × 10 5 splenocytes were incubated o.n. with or without 5 μg/mL of either unrelated or E7-specific peptides in triplicate IFN-γ ELISpot microwells. Shown are the numbers of IFN-γ spot-forming units (SFU)/well as mean values of triplicates after subtraction of mean values measured in wells of splenocytes treated with the unspecific peptide. Reported are cumulate data from two independent experiments. Intragroup mean values + standard deviations are also reported.

Article Snippet: A total of eight mice for each experiment were inoculated i.m. two times in fourteen-day intervals with 50 μg for each quadriceps of DNA vectors purified through endotoxin-free Qiagen kit (Hilden, Germany).

Techniques: Injection, Expressing, Plasmid Preparation, Incubation, Enzyme-linked Immunospot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Mitofilin Mitigates Myocardial Damage in Acute Myocardial Infarction by Regulating Pyroptosis of Cardiomyocytes

doi: 10.3389/fcvm.2022.823591

Figure Lengend Snippet: Reduced mitofilin expression is associated with the upregulation of pyroptosis-related factors in an acute myocardial infarction (AMI) model. (A) The percentage of myocardial infarction (MI) area increased in the AMI group. (B,C) The malondialdehyde (MDA) level and the lactate dehydrogenase (LDH) level were increased upon AMI induction. (D,E) The levels of interleukin (IL)-1β and IL-18 were increased in the AMI group. (F) Mitofilin protein levels were significantly decreased in the AMI group, while the protein levels of cleaved-caspase-1, nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing (ASC), Gasdermin D (GSDND) were increased. ** p < 0.01 and *** p < 0.001. ns: not significant.

Article Snippet: A recombinant adenoviral vector encoding mitofilin cDNA and its negative control were obtained from GeneChem (Montreal, Quebec Canada).

Techniques: Expressing, Binding Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Mitofilin Mitigates Myocardial Damage in Acute Myocardial Infarction by Regulating Pyroptosis of Cardiomyocytes

doi: 10.3389/fcvm.2022.823591

Figure Lengend Snippet: Overexpression of mitofilin alleviates AMI. (A) The administration of adenovirus carrying mitofilin (ad-mitofilin) significantly increased the expression level of mitofilin in the myocardium. (B) The area of MI was partially reduced after the administration of ad-mitofilin. (C,D) MDA levels and LDH levels were partially reduced after the administration of ad-mitofilin. (E,F) IL-1β and IL-18 levels were partially reduced after the administration of ad-mitofilin. (G) After the administration of ad-mitofilin, the mitofilin protein level was significantly increased upon AMI induction, while the protein levels of cleaved- caspase-1, NLRP3, ASC, and GSDND protein levels were partially decreased. Sham vs AMI: ** P < 0.01 and *** P < 0.001. AMI vs AMI+ad-mitofilin: ### P < 0.001.

Article Snippet: A recombinant adenoviral vector encoding mitofilin cDNA and its negative control were obtained from GeneChem (Montreal, Quebec Canada).

Techniques: Over Expression, Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: Mitofilin Mitigates Myocardial Damage in Acute Myocardial Infarction by Regulating Pyroptosis of Cardiomyocytes

doi: 10.3389/fcvm.2022.823591

Figure Lengend Snippet: Mitofilin knockdown exacerbates hypoxia/reoxygenation (H/R)-induced cell damage of H9C2 by regulating pyroptosis. (A) The expression level of mitofilin was strongly reduced after the administration of ad-si-mitofilin. (B,C) H/R induced the increase of MDA level and LDH level in H9C2 cells, while levels were further increased after the administration of ad-si-mitofilin. The presence of AC-YVAD-CMK (an inhibitor for caspase-1) suppressed the effect of mitofilin knockdown. (D,E) H/R induced the increase of IL-1β and IL-18 levels in H9C2 cells, which were further increased after the mitofilin knockdown. The presence of AC-YVAD-CMK suppressed the effect of mitofilin knockdown. (F) Mitofilin knockdown increased the protein levels of cleaved-caspase-1, NLRP3, ASC, and GSDND in H9C2 cells upon H/R induction, which was rescued by AC-YVAD-CMK. (G) H/R induction promoted cell death by increasing the percentage of cells with impaired membrane integrity [revealed by propidium iodide (PI) staining]. Mitofilin knockdown exacerbated cell death (with typical morphology of cell swelling and membrane rupture). AC-YVAD-CMK suppressed the effect of mitofilin knockdown. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: A recombinant adenoviral vector encoding mitofilin cDNA and its negative control were obtained from GeneChem (Montreal, Quebec Canada).

Techniques: Expressing, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Mitofilin Mitigates Myocardial Damage in Acute Myocardial Infarction by Regulating Pyroptosis of Cardiomyocytes

doi: 10.3389/fcvm.2022.823591

Figure Lengend Snippet: Mitofilin regulates the PI3K/AKT signaling pathway. (A) The levels of p-PI3K and p-AKT in H9C2 cells were decreased after H/R induction, which were further decreased after the mitofilin knockdown. (B) The levels of p-PI3K and p-AKT in H9C2 cells were rescued after mitofilin overexpression. Con vs H/R: ** P < 0.01 and *** P < 0.001. H/R vs H/R+ad-si-mitofilin or ad-mitofilin: ### P < 0.001.

Article Snippet: A recombinant adenoviral vector encoding mitofilin cDNA and its negative control were obtained from GeneChem (Montreal, Quebec Canada).

Techniques: Over Expression

Journal: Frontiers in Cardiovascular Medicine

Article Title: Mitofilin Mitigates Myocardial Damage in Acute Myocardial Infarction by Regulating Pyroptosis of Cardiomyocytes

doi: 10.3389/fcvm.2022.823591

Figure Lengend Snippet: The protective effect of mitofilin in the H/R-induced H9C2 damage is dependent on PI3K activity. (A) Hypoxia/reoxygenation induced a decrease in the mitofilin expression level in H9C2 cells. The level of mitofilin protein was increased after mitofilin overexpression. (B,C) Hypoxia/reoxygenation induced increase of the MDA level and LDH level was partially reduced after mitofilin overexpression. The presence of GDC-0941 (a PI3K inhibitor) abrogated the rescue effect. (D,E) Hypoxia/reoxygenation induced the increase of IL-1β and IL-18 levels in H9C2 cells, and mitofilin overexpression reduced their levels. The presence of GDC-0941 abrogated the rescue effect. (F) Mitofilin overexpression suppressed the protein levels of cleaved-caspase-1, NLRP3, ASC, and GSDND in H9C2 cells upon H/R induction, and the effects were abrogated by GDC-0941. * P < 0.05, ** P < 0.01, and *** P < 0.001. ##: H/R vs H/R+ad-mitofilin: P < 0.01.

Article Snippet: A recombinant adenoviral vector encoding mitofilin cDNA and its negative control were obtained from GeneChem (Montreal, Quebec Canada).

Techniques: Activity Assay, Expressing, Over Expression