Journal: Metabolites

Article Title: A Novel Ketone-Supplemented Diet Improves Recognition Memory and Hippocampal Mitochondrial Efficiency in Healthy Adult Mice

doi: 10.3390/metabo12111019

Figure Lengend Snippet: Primary antibody information and dilutions.

Article Snippet: OPA1 , 1:1000 , Rabbit , Novus Biologicals , NB110-55290.

Techniques:

Journal: bioRxiv

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1101/2023.03.23.533672

Figure Lengend Snippet: ( A ) Interrogation of ChIP-seq data indicated binding of TAp73α, TAp73β and p53 to the putative OPA1 promoter region. Sequencing read files were obtained from the GEO data set GSE15780, and tracks shown for the indicated transcription factors at selected genes. ( B-C ) Targeted ChIP of TAp73 bound chromatin. RT-qPCR primers were designed in the promoter region of the OPA1 gene (OPA1 ‘A’ and OPA1 ‘B’). Red squares indicate regions enriched for the Trp73 motif (p<0.001). qPCR was performed to quantify the fold enrichment of the OPA1 promoter region in IP sample relative to IgG control. Enrichment of MDM2 and SAT2 promoter regions was assayed as positive and negative controls, respectively. qPCR was carried out on 3 independent ChIP experiments and data shown as individual data points ± SD (n=3). ( D ) Representative Western blot of mitochondrial fusion proteins in TAp73 KO and WT control. Cells were transfected with either EV or TAp73α expression construct for 24h. ( E ) RT-qPCR was performed against OPA1 , MFN2 and CDKN1A genes and expression values calculated using the ΔΔCt method, relative to WT empty vector control. Data shown as mean ± SD (n=3). (*) P ≤ 0.05 (Student’s t-test, comparison of indicated condition with WT EV control).

Article Snippet: Expression plasmid for OPA1 was purchased from Origene (#SC128155, Rockville, MD, USA), and HA- TAp73 cloned into the pcDNA3 backbone.

Techniques: ChIP-sequencing, Binding Assay, Sequencing, Quantitative RT-PCR, Control, Western Blot, Transfection, Expressing, Construct, Plasmid Preparation, Comparison

Journal: bioRxiv

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1101/2023.03.23.533672

Figure Lengend Snippet: (A) WT or TAp73 KO H1299 cells were transfected with the indicated plasmids for 24h and IF carried out against ATP5B (green) with DAPI nuclear counterstain (blue). Cells transfected with HA-TAp73α expression plasmid were stained for HA as a transfection control (red). Scale bar = 10 μm. (B) Representative western blot of OPA1 expression following transfection of WT and TAp73 KO cells with pCMW-OPA1 construct. (C) Quantification of mitochondrial morphology from (a) using Zeiss Intellesis module, trained to segment individual mitochondria. Statistical significance compared to WT EV control was calculated using Student’s T-test; (*) p < 0.1, (ns) not significant (n=3). (D) Transmission electron micrographs of mitochondrial morphology from WT and TAp73 KO cells. Scale bar = 100 nm. (E) Mitochondrial length measurements obtained from (D). (****) p<0.0001 in Student’s t-test. A minimum of 100 mitochondria were measured from n=3 independent biological replicates. ( F-G ) Mitochondrial stress test performed on Seahorse XFe96 analyser. Canonical mitochondrial inhibitors injected sequentially as labelled (Oligomycin = 2μM, FCCP = 500nM, Antimycin A/ Rotenone = 2μM). The indicated mitochondrial stress test parameters were calculated from OCR data. Data were corrected for non- mitochondrial OCR, normalised to cell number, and are shown as mean ± SD (n=3). (*) P ≤ 0.05 and (**) P ≤ 0.01 in Student’s T-test relative to WT control. (H) Western blot of the indicated ETC subunits in Wild-type and TAp73 KO cells, obtained using OXPHOS antibody cocktail. (I) qPCR against mt-CO2 , expressed relative to expression of nuclear encoded β 2- microglobulin . Relative expression was calculated using the ΔΔCt method and expressed as a percentage of wild-type control (n=2). n.s = not significant in Student’s t-test.

Article Snippet: Expression plasmid for OPA1 was purchased from Origene (#SC128155, Rockville, MD, USA), and HA- TAp73 cloned into the pcDNA3 backbone.

Techniques: Transfection, Expressing, Plasmid Preparation, Staining, Control, Western Blot, Construct, Transmission Assay, Injection

Journal: bioRxiv

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1101/2023.03.23.533672

Figure Lengend Snippet: (A) Immunofluorescence staining performed against the cilia marker Ac-α-tubulin (green) with DAPI nuclear stain in tracheal cross-sections from WT and Trp73 -/- mice (blue). Scale bar = 20 μm. (B) RT-qPCR for OPA1 mRNA expression in dissociated tracheal epithelial cells. Mouse trachea (n=3 of each WT and Trp73 -/- ) were pooled together to obtain a sufficient number of cells. (C) Multiplexed IHC on mouse trachea against Ac-a-tubulin (yellow), OPA1 (red) and DAPI (blue). Cyan arrows indicate MCCs with low OPA1 expression. (D) Quantification of OPA1 expression in ciliated and non-ciliated cell populations from WT and Trp73 -/- mice. (E) Images of individual sections of MCCs from WT and Trp73 -/- ciliated epithelium obtained by SBF-SEM. Scale bar = 200 nm. (F) Mitochondrial length measurements were obtained from (E), including ciliated and non-ciliated cell populations. n≥100 mitochondria from two samples of each genotype. (***) P ≤ 0.001, n.s = not significant, in t-test.

Article Snippet: Expression plasmid for OPA1 was purchased from Origene (#SC128155, Rockville, MD, USA), and HA- TAp73 cloned into the pcDNA3 backbone.

Techniques: Immunofluorescence, Staining, Marker, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1101/2023.03.23.533672

Figure Lengend Snippet: (A) Trp73 expression data from healthy and COPD individuals from previously described Lung Genomics Research Consortium (LGRC) cohort (GSE47460; Affymetrix array data from n=157 healthy and n=220 COPD patients). (B) Heatmap of Trp73, CDKN1A, OPA1 and FOXJ1 expression in healthy and COPD individuals from the LGRC cohort. The heatmap was generated with the PulmonDB tool using Scipy library in Python, utilising cosine distance and average linkage. Red/blue cells represent positive/negative values. (C) Row similarity matrix indicating the association between each gene across patient data shown in (B). Red shading indicates a positive similarity (measured as 1 - cosine- distance, with similarity values indicated). ( D-E ) Expression data showing the correlation between Trp73 and OPA1 (D), or Trp73 and CDKN1A (E) in COPD patients (GSE47460). Expression values are shown as Log2 fold change for the indicated genes relative to healthy control. The strength of the correlation was calculated using Pearson’s coefficient (r).

Article Snippet: Expression plasmid for OPA1 was purchased from Origene (#SC128155, Rockville, MD, USA), and HA- TAp73 cloned into the pcDNA3 backbone.

Techniques: Expressing, Generated, Control

Journal: bioRxiv

Article Title: TAp73 regulates mitochondrial dynamics and multiciliated cell homeostasis through an OPA1 axis

doi: 10.1101/2023.03.23.533672

Figure Lengend Snippet: Schematic illustration showing the identified role of TAp73 in regulating mitochondrial dynamics. TAp73 expression is required for mitochondrial homeostasis in vitro and in the ciliated epithelium (green nuclei) (left). Conversely, TAp73 ablation leads to decreased OPA1 expression, mitochondrial fission, and impaired mitochondrial function (right). The Trp73 -/- tracheal epithelium maintains a limited expression of FOXJ1 positive cells (purple), indicating additional mechanisms, such as the observed mitochondrial dysfunction drives MCC loss and COPD pathogenesis.

Article Snippet: Expression plasmid for OPA1 was purchased from Origene (#SC128155, Rockville, MD, USA), and HA- TAp73 cloned into the pcDNA3 backbone.

Techniques: Expressing, In Vitro

Journal: Brain

Article Title: Sustained OMA1-mediated integrated stress response is beneficial for spastic ataxia type 5

doi: 10.1093/brain/awad340

Figure Lengend Snippet: OMA1 induces the activation of ISR in vivo in Afg3l2 −/− mice cerebellum. ( A ) Western blot analysis performed on Afg3l2 +/+ and Afg3l2 −/− cerebellar lysates collected from mice at postnatal Day 14, showing levels of OMA1 and OPA1, with relative quantification. Calnexin was used to verify equal loading. Bars represent means ± standard error of the mean (SEM); n ≥ 4 mice. Unpaired Student's t- test: * P < 0.05, *** P < 0.001, **** P < 0.0001. CRB = cerebellum. ( B ) Schematic representation of the stress cascade downstream OMA1 activation. Created with BioRender.com . ( C ) Western blot analysis performed on Afg3l2 +/+ and Afg3l2 −/− cerebellar lysates collected from mice at postnatal Day 14, showing levels of P-eIF2α and TOT-eIF2α, with relative quantification. CKAP4 was used to verify equal loading. Bars represent means ± SEM, n ≥ 6 mice. Unpaired Student's t -test with Welch correction: ** P < 0.01, ns = not significant. ( D ) qRT-PCR showing the expression of Atf4 , Chop , Chac1 , Ppp1r15a , Herpud1 , Eif2s2 and Fgf21 in cerebellum of Afg3l2 +/+ and Afg3l2 −/− mice at postnatal Day 14. Expression levels were normalized to the housekeeping gene Hprt1 . Bars represent means ± SEM, n ≥ 4 mice. Unpaired Student's t -test with Welch correction: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( E ) Western blot analysis performed on Afg3l2 +/+ and Afg3l2 −/− cerebellar lysates collected from mice at postnatal Day 1, showing levels of P-eIF2α and TOT-eIF2α, with relative quantification. CKAP4 was used to verify equal loading. Bars represent means ± SEM, n = 3 mice. Unpaired Student's t -test, ns = not significant. ( F ) qRT-PCR showing the expression of Atf4 , Chop and Fgf21 in cerebellum of Afg3l2 +/+ and Afg3l2 −/− mice at postnatal Day 1. Expression levels were normalized to the housekeeping gene Hprt1 . Bars represent means ± SEM, n ≥ 5 mice. Unpaired Student's t -test, ns = not significant. ISR = integrated stress response.

Article Snippet: Primary antibodies were diluted in 5% milk TBS-T and blots were incubated for 3 h at room temperature or overnight at 4°C with OPA1 (BD Transduction Laboratories), OMA1 (Novus Biologicals), AFG3L2 C-terminal (previously generated in the lab), CKAP4 (Novus Biologicals), P-eIF2α (Cell Signaling), TOT-eIF2α (Cell Signaling), Calnexin (Sigma), ATF4 (Santa Cruz Biotechnology), HA (Covance) and GAPDH (Santa Cruz Biotechnology).

Techniques: Activation Assay, In Vivo, Western Blot, Quantitative Proteomics, Quantitative RT-PCR, Expressing

Journal: Brain

Article Title: Sustained OMA1-mediated integrated stress response is beneficial for spastic ataxia type 5

doi: 10.1093/brain/awad340

Figure Lengend Snippet: Activation of OMA1-mediated ISR in SPAX5 patient fibroblasts . ( A ) AFG3L2 protein scheme with functional domains, reporting the mutations described here. ( B ) Brain MRI scan of Patient 2: MRI T 2 -weighted, axial view, performed at age 3 years, showing symmetric hypersignal intensity in both globi pallidi (white arrow) and in the pyramidal tract at the midbrain level (double white arrow) as well as volume loss in both globi pallidi. ( C ) Analysis of ΔΨ m by live-imaging measurement of TMRM fluorescence intensity in SPAX5-Patient 2 primary fibroblasts. Bars represent mean ± standard error of the mean (SEM), n = 300 cells from three independent experiments. Two-way ANOVA with Tukey correction: **** P < 0.0001. ( D ) Western blot analysis performed on cell lysates obtained from SPAX5-Patient 2 primary fibroblasts and controls, showing levels of OMA1 and OPA1, with relative quantification. Calnexin was used to verify equal loading. Bars represent means ± SEM of three independent experiments. Unpaired Student's t -test: * P < 0.05; *** P < 0.001. ( E ) Representative images of SPAX5-Patient 2 and relative control human primary fibroblasts, infected with mtDsRed2, and relative quantification of major axis, average size and circularity index. Bars represent means ± SEM of 300 cells from three independent experiments. Unpaired Student's t -test: * P < 0.05; ** P < 0.01. ( F ) Representative western blot analysis performed on cell lysates obtained from SPAX5-Patient 1 and Patient 2 primary fibroblasts and controls, showing levels of AFG3L2, P-eIF2α and TOT-eIF2α, with relative quantification. Calnexin was used to verify equal loading. Bars represent mean ± SEM of at least three independent experiments (we employed four different age-matched control human primary fibroblasts). One-way ANOVA with Tukey correction: * P < 0.05; *** P < 0.001; **** P < 0.0001. ( G ) Western blot analysis performed on three different AFG3L2 knockdown HEK 293 T clones and wild-type controls upon overexpression of DELE1-HA and relative quantification of S-DELE1 on L-DELE1 and OMA1. GAPDH was used to verify equal loading. Asterisks highlight aspecific bands. Bars represent means ± SEM of three independent experiments. One-way ANOVA with Tukey correction: ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Primary antibodies were diluted in 5% milk TBS-T and blots were incubated for 3 h at room temperature or overnight at 4°C with OPA1 (BD Transduction Laboratories), OMA1 (Novus Biologicals), AFG3L2 C-terminal (previously generated in the lab), CKAP4 (Novus Biologicals), P-eIF2α (Cell Signaling), TOT-eIF2α (Cell Signaling), Calnexin (Sigma), ATF4 (Santa Cruz Biotechnology), HA (Covance) and GAPDH (Santa Cruz Biotechnology).

Techniques: Activation Assay, Functional Assay, Imaging, Fluorescence, Western Blot, Quantitative Proteomics, Control, Infection, Knockdown, Clone Assay, Over Expression

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Summary of q RT-PCR oligonucleotide primers used in measuring mRNA expression in mitochondrial structural, biogenesis, synaptic genes and autophagy and mitophagy genes.

Article Snippet: OPA1 Rabbit polyclonal (#NBP2-59770) 1:100 , Novus Biological, Littleton, CO , Donkey anti-rabbit HRP 1:200 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Expressing, Sequencing

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Summary of antibody dilutions and conditions used in the immunofluorescence analysis of mitochondrial dynamics, mitochondrial biogenesis, synaptic mitophagy and autophagy proteins in mitophagy enhancer-treated and -untreated hAbKI mice.

Article Snippet: OPA1 Rabbit polyclonal (#NBP2-59770) 1:100 , Novus Biological, Littleton, CO , Donkey anti-rabbit HRP 1:200 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Immunofluorescence

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Summary of antibody dilutions and conditions used in the immunoblotting analysis of mitochondrial dynamics, mitochondrial biogenesis, synaptic mitophagy and autophagy proteins in mitophagy enhancer-treated and -untreated hAbKI mice.

Article Snippet: OPA1 Rabbit polyclonal (#NBP2-59770) 1:100 , Novus Biological, Littleton, CO , Donkey anti-rabbit HRP 1:200 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Western Blot

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Summary of mRNA fold changes comparison in Urolithin A-treated hAbKI mice and Urolithin A+EGCG treated hAbKI mice.

Article Snippet: OPA1 Rabbit polyclonal (#NBP2-59770) 1:100 , Novus Biological, Littleton, CO , Donkey anti-rabbit HRP 1:200 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Comparison

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Immunoblotting analysis of mitochondrial dynamic proteins . Immunoblotting analysis was assessed using lysates prepared from post-mortem brains of seven-month-old hAbKI mice and treated hAbKI mice with Uralithin A and EGCG. ( A ) Representative immunoblots for hAbKI mice and treated hAbKI mice with Urolithin A and EGCG. ( B ) Quantitative-densitometry analysis for mitochondrial fission genes Drp1 and Fis1 and fusion proteins, which were significantly decreased in the treated hAbKI mice Urolithin A and EGCG as compared to the hAbKI mice. Mitochondrial fusion proteins Mfn1, Mfn2 and Opa1 were significantly increased in urolithin A-treated hAbKI mice and combined treatment of urolithin A+EGCG in 7-month-old hAbKI mice.

Article Snippet: OPA1 Rabbit polyclonal (#NBP2-59770) 1:100 , Novus Biological, Littleton, CO , Donkey anti-rabbit HRP 1:200 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Astrocyte–neuron crosstalk through extracellular vesicle-shuttled miRNA-382-5p promotes traumatic brain injury

doi: 10.1038/s12276-024-01355-3

Figure Lengend Snippet: a Overlap of potential targets of miRNA-382-5p predicted by the TargetScan, miRWalk, miRDB, RNAhybrid, and miRanda databases. b Venn diagram of putative target genes of miRNA-382-5p overlapping with mitochondria-related genes. c Wild-type and mutant OPA1 3′-UTR reporter constructs. d Luciferase reporter assay results for HEK293T cells cotransfected with the indicated wild-type or mutant 3’-UTR constructs and the miRNA-382-5p mimic ( n = 6 per group; one-way ANOVA). e , f WB analysis and densitometric quantification of OPA1 levels in primary neurons transfected with or without the miRNA-382-5p mimic ( n = 6 per group; Student’s t test). g MitoTracker was used to label mitochondria in primary neurons, mitochondrial morphology was examined via fluorescence microscopy, and mitochondrial morphological features were quantified (aspect ratios) using ImageJ software. h The boxed area next to each micrograph shows an magnified version of the white square ( n = 6 per group, one-way ANOVA). i , j Representative images of MitoSOX fluorescence and quantitative comparison of mitochondria-derived ROS levels ( n = 6 per group; one-way ANOVA). k , l Representative images of fluorescence staining and quantitative comparison of JC-1 aggregates (red fluorescence) and JC-1 monomers (green fluorescence) in cultured primary neurons from different experimental groups ( n = 6 per group; one-way ANOVA). The data are presented as the means ± SDs; ** p < 0.01, *** p < 0.001, and NS not significant.

Article Snippet: Neuron-specific OPA1 knockout mice were generated by crossing Opa1 f/f mice (Cyagen, USA, Inc., CA, USA) with Map2-CreERT2 mice (Shanghai Model Center) and then treating the offspring with 75 mg/kg tamoxifen (#S1238, Selleck) for 5 days.

Techniques: Mutagenesis, Construct, Luciferase, Reporter Assay, Transfection, Fluorescence, Microscopy, Software, Comparison, Derivative Assay, Staining, Cell Culture

Journal: Experimental & Molecular Medicine

Article Title: Astrocyte–neuron crosstalk through extracellular vesicle-shuttled miRNA-382-5p promotes traumatic brain injury

doi: 10.1038/s12276-024-01355-3

Figure Lengend Snippet: a Cartoon illustrating the process used to construct OPA1 conditional knockout mice. b Results showing the identification of the neuron-specific OPA1 conditional knockout mice. c TEM images showing cortical mitochondrial crista remodeling in the Opa1 f/f group or Opa1 CKO group subjected to sham surgery or TBI. d Fifty randomly selected mitochondria per sample were scored in three categories ( n = 6 per group, one-way ANOVA): more than four cristae (Class I), between two and three cristae (Class II), and one or no cristae (Class III) per mitochondrion. e Fifty mitochondria per sample were scored according to matrix density and swelling ( n = 6 per group, one-way ANOVA): dense matrix (Class A) and swollen mitochondria with a hypodense matrix (Class B). f The length of the mitochondria in each group, which is represented by the mean length of 50 randomly selected mitochondria ( n = 6 per group; one-way ANOVA). g , h Representative MR images and statistical analysis of histological impairments at 24 h after TBI ( n = 6 per group; Student’s t test). i Statistical analysis of brain edema. n = 6 per group; Student’ s t test) j – l The neurological function deficit scores of the Opa1 f/f group or Opa1 CKO group ( n = 12 per group, two-way ANOVA). The data are presented as the means ± SDs; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Neuron-specific OPA1 knockout mice were generated by crossing Opa1 f/f mice (Cyagen, USA, Inc., CA, USA) with Map2-CreERT2 mice (Shanghai Model Center) and then treating the offspring with 75 mg/kg tamoxifen (#S1238, Selleck) for 5 days.

Techniques: Construct, Knock-Out

Journal: Experimental & Molecular Medicine

Article Title: Astrocyte–neuron crosstalk through extracellular vesicle-shuttled miRNA-382-5p promotes traumatic brain injury

doi: 10.1038/s12276-024-01355-3

Figure Lengend Snippet: a , b WB analysis and densitometric quantification of the levels of the OPA1 protein in the perilesional cortex of mice injected with or without RVG-miR-382-5pi-EVs ( n = 6 per group; one-way ANOVA). c TEM images showing that the loss of mitochondrial cristae in the perilesional cortex was inhibited in the RVG-NC-EVs group and the RVG-miR-382-5pi-EVs group. d Fifty mitochondria per sample were assigned to three categories: more than four cristae (Class I), between two and three cristae (Class II), and one or no cristae (Class III) per mitochondrion ( n = 6 per group, one-way ANOVA). e Fifty mitochondria per sample were scored according to matrix density and swelling: dense matrix (Class A) and swollen mitochondria with a hypodense matrix (Class B) ( n = 6 per group, one-way ANOVA). f Representative quantitative results of the length of 50 mitochondria per experiment ( n = 6 per group; one-way ANOVA). g , h Representative MR images and statistical analysis of histological impairments at 24 h after TBI ( n = 6 per group; one-way ANOVA). i Statistical analysis of brain edema ( n = 6 per group, one-way ANOVA). j‒l Neurological function deficit scores of the RVG-NC-EVs group and RVG-miR-382-5pi-EVs group ( n = 12 per group, two-way ANOVA). The data are presented as the means ± SDs; * p < 0.05, ** p < 0.01, *** p < 0.001, and NS not significant.

Article Snippet: Neuron-specific OPA1 knockout mice were generated by crossing Opa1 f/f mice (Cyagen, USA, Inc., CA, USA) with Map2-CreERT2 mice (Shanghai Model Center) and then treating the offspring with 75 mg/kg tamoxifen (#S1238, Selleck) for 5 days.

Techniques: Injection