EMBO/soda-nel huggingface dataset:
USP22 controls necroptosis by regulating receptor-interacting protein kinase 3 ubiquitination (2020)
Figure 5

Figure 5: Identification of novel, USP22-regulated RIPK3 ubiquitination sites

A Schematic representation of the experimental strategy for quantitative analysis of ubiquitination sites in USP22 KO HT-29 cells versus HT-29 CRISPR/Cas9 control (ctr) cells under untreated and necroptosis-induced conditions. Control cells were grown in SILAC medium containing Arg0/Lys0 ("Light") labeled, whereas USP22 KO cells were labeled in SILAC medium containing Arg10/Lys8 ("heavy") labeled. In the treated conditions, cells were incubated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 6 h. Cells were lysed, and equal amounts of extracted proteins extracted from "Light" or "Heavy" were pooled and digested in-solution with trypsin. Ubiquitin remnant peptides were enriched using di‐glycine‐lysine‐specific antibodies, fractionated by Micro‐SCX and analyzed by LC‐MS/MS.

B Bar graphs demonstrating the number of up‐, non‐, and downregulated ubiquitination sites in USP22 KO HT-29 cells versus control under TBZ-treatment.

C Schematic representation of RIPK3. Indicated are the two major protein domains, the kinase domain (KD), the RHIM and the three different potential ubiquitination sites identified by ubiquitin remnant profiling.

D HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K42R, K351, K518R, 2xKR or 3xKR were pre‑incubated with 1 µg/ml Dox, 20 µM Dab, or 10 µM NSA overnight, followed by pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 4 h. Cell death was measured by quantification of PI-positive nuclei.

E HeLa cells expressing Dox-inducible RIPK3 mutants were treated with 1 µg/ml Dox overnight. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. GAPDH served as loading control.

Data information: Data represent mean ± SD; **P < 0.01; ***P < 0.001, by unpaired 2‐tailed Student's t‐test. Three independent experiments performed in triplicate are shown.
USP22 controls necroptosis by regulating receptor-interacting protein kinase 3 ubiquitination (2020)
Figure 6

Figure 6: USP22-regulated modification of RIPK3 lysine residue 518 affects necroptosis in RIPK3-reconstituted HeLa cells

A HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox and/or 10 µM NSA and 20 µM Dab overnight. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were examined by Western blotting. β-Actin was used as a loading control.

B HeLa cells expressing Dox-inducible RIPK3 WT or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3 and 4 h. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were analyzed by Western blotting. β-Actin served as loading control.

C HeLa cells expressing Dox-inducible RIPK3 WT, 2xKR or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control.

D HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Whole cell lysates were generated using RIPA lysis buffer containing 2% SDS. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control.

E HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K518R or 3xKR were incubated overnight with 1 µg/ml Dox and pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1 and 2 h. Strep-RIPK3 was immunoprecipitated by using anti-Strep-beads. Co-immunoprecipitated phosphorylated MLKL and RIPK1, as well as protein expression of indicated proteins were analyzed by Western blotting. β-Actin served as a loading control.

F HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.
USP22 controls necroptosis by regulating receptor-interacting protein kinase 3 ubiquitination (2020)
Figure 7

Figure 7: RIPK3 K518R hypersensitize HT‑29 cells for TBZ-induced necroptosis

A HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 4 h. Cell death was measured by analysis of PI-positive nuclei.

B HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight, as indicated. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 2 h and analyzed by Western blotting for RIPK3 and phosphorylated MLKL expression levels. β-Actin served as a loading control.

C Schematic overview of the putative mechanistic roles of USP22 in necroptosis. Activation of TNFR1 by TNFα, upon caspase-8 inhibition by zVAD.fmk and cIAP1/2 inactivation by BV6, induces RIPK1/3 activation, necrosome formation and execution of necroptosis. In the absence of USP22 KO, TBZ-induced necroptosis is delayed, accompanied by increased RIPK3 phosphorylation and ubiquitination of RIPK3 at lysine 518. This USP22-mediated necroptotic signaling could be caused either by direct USP22-mediated (de)ubiquitination of RIPK3 and/or indirectly by, for example, regulating RIPK3 autophosphorylation or the activity of RIPK3-associated E3 ligases or kinases. Additionally, USP22-mediated retrograde effects on RIPK1 phosphorylation and RIPK3 oligomerization might be involved as well.

Data information: Data represent mean ± SD; **P < 0.01, by unpaired 2‐tailed Student's t‐test. Three independent experiments performed in triplicate are shown.

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Endothelial IGF-1 receptor mediates crosstalk with the gut wall to regulate microbiota in obesity (2021)
Figure 1

Figure 1. Endothelial IGF-1R overexpression prevents high fat diet (HFD) induced weight gain.

A, Schematic representation of feeding time course.

B, C, In primary endothelial cells isolated from human IGF-1 receptor endothelial overexpressing mice (hIGFREO) and wildtype littermates (WT), quantitative polymerase chain reaction (qPCR) shows that hIGFREO have increased expression of human IGF-1R but similar levels of murine insulin receptor (IR) gene expression as WT (n=3-5 mice per group).

D, E, In primary endothelial cells isolated from WT and hIGFREO, immunoblotting shows that hIGFREO have increased expression of IGF-1R but similar levels of IR protein expression (n=3-4 mice per group).

F, Chow fed hIGFREO had similar body mass to WT, however, hIGFREO did not gain as much weight as WT after 8 weeks of HFD (n=6-10 mice per group).

G, Representative images of difference in fat and water distribution shown by magnetic resonance (MR) imaging in hIGFREO and WT. Scale bar = 1cm.

H, subcutaneous white adipose tissue (sWAT) and visceral white adipose tissue (vWAT) volumes were reduced in hIGFREO (n=4 per genotype)..

I, hIGFREO had reduced white epididymal adipose depot weight compared to WT, there was no difference in heart, spleen or liver weight (n=7-11 mice per group).

J, hIGFREO had similar whole-body and femur length as WT (n=7-9 mice per group).

Data information: Data shown as mean ±SEM, individual mice are shown as data points, P<0.05 taken as being statistically significant using student t-test and denoted as * (** denotes P≤0.01, ns denotes not significant).
Endothelial IGF-1 receptor mediates crosstalk with the gut wall to regulate microbiota in obesity (2021)
Figure 2

Figure 2. Endothelial IGF-1R overexpression prevents high fat diet (HFD) induced glucose intolerance.

A, human IGF-1R endothelial overexpressing mice (hIGFREO) had significantly lower fasting blood glucose compared to wildtype littermates (WT) after HFD (n=5-7 mice per group).

B, C, hIGFREO had reduced glucose intolerance compared to WT (as measured by glucose tolerance test and area under the curve (AUC)) (n=5-7 mice per group).

D, hIGFREO had improved insulin sensitivity compared to WT as shown by lower HOMA-IR score (n=9-10 mice per group).

E, F, hIGFREO and WT had similar fasting plasma IGF-1 and insulin concentrations (n=6-12 mice per group).

G, H, Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve (n=10-12 mice per group).

Data information: Data shown as mean ±SEM and individual mice are shown as data points, P<0.05 taken as being statistically significant using student t-test and denoted as *(** denotes P≤0.01,ns denotes not significant).
Endothelial IGF-1 receptor mediates crosstalk with the gut wall to regulate microbiota in obesity (2021)
Figure 3

Figure 3. Protection from high fat diet (HFD) induced weight gain in human IGF-1R endothelial overexpressing mice (hIGFREO) is not due to changes in activity, food intake, energy expenditure, adipose browning or gut transit time.

A-C, hIGFREO exhibit no difference in activity levels, food consumption or energy expenditure using indirect calorimeter assessment after HFD compared to wildtype littermates (WT) after HFD. (n=4 per genotype).

D, Adipose expression of browning markers are also no different in white epididymal adipose tissue and brown adipose tissue compared to WT (n= 6 per genotype).

E, Core body temperature is no different in hIGFREO compared to WT (n=6-8 mice per group).

F, G, Gut transit time is also unaltered in hIGFREO compared to WT as shown by no change in small intestine length F (n=7-9 mice per group), or total gut transit time after a carmine red gavage G (n=12-13 mice per group).

Data information: The light/dark cycle for graphs A-C are shown as follows; light in yellow and dark in brown. Data shown as mean ±SEM and individual mice are shown as data points.For indirect calorimiety, ANOVA testing was performed using mass as a co-variant (ANCOVA testing) using calrapp.org. ns denotes not significant.
Endothelial IGF-1 receptor mediates crosstalk with the gut wall to regulate microbiota in obesity (2021)
Figure 4

Figure 4. Endothelial IGF-1R overexpression alters the gut microbiota and augments the abundance of the beneficial genus Akkermansia.

A, B, Faith's phylogenetic diversity (PD) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF-1R endothelial overexpressing mice (hIGFREO) mice and wildtype littermates (WT) mice after high fat diet feeding (n=4-5 mice per group).

C, D, Chao-1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT (n=4-5 mice per group).

E, F, Partial least squares discriminant analysis (PLS-DA) model and used the variable importance in projection (VIP) score was used to assess the contribution of each genus, shown as a scores plot in (E), and a loadings plot of PLS-DA of genus abundances in F. VIP score cut off of 1(n=4-5 mice per group).

Data information: Data shown as mean ±SEM and individual mice are shown as data points. Diversity analyses were run on the resulting OTU/feature .biom tables to provide both phylogenetic and non-phylogenetic metrics of alpha and beta diversity. Additional data analysis (PLS-DA) and statistics were performed with R . P<0.05 taken as being statistically significant using student t-test and denoted as *.
Endothelial IGF-1 receptor mediates crosstalk with the gut wall to regulate microbiota in obesity (2021)
Figure 5

Figure 5. Antibiotic administration in the setting of high fat diet (HFD) eliminates the anti-obesity and anti-diabetic actions of endothelial IGF-1R overexpression.

A, Schematic representation of antibiotic dosing and feeding time course.

B, Bi, human IGF-1R endothelial overexpressing mice (hIGFREO) had comparable weight gain as wildtype littermates (WT) after 8 weeks of HFD + antibiotics (ABs) when compared to WT. Bii, both hIGFREO and WT gained significant weight compared to chow fed mice (n=7=9 mice per group).

C, There was no difference in fasting blood glucose in hIGFREO compared to WT (n=7-9 mice per group).

D, E, There was no difference in hIGFREO and WT glucose tolerance (as measured by glucose tolerance test and area under the curve (AUC)) (n=7-9 mice per group).

F, Wet organ weights were similar in hIGFREO and WT(n=7-9 mice per group).

G, Chao-1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates no difference between hIGFREO and WT after HFD + antibiotic treatment (n=3-5 mice per group).

H, Alpha diversity P values using Kruskal-Wallis pairwise comparisons shows there is no difference in microbial diversity.

Data information: Data shown as mean ±SEM and individual mice are shown as data points, P<0.05 taken as being statistically significant using student t-test and denoted as * or ** for P P<0.01 and NS denotes not significant. Diversity analyses were run on the resulting OTU/feature .biom tables to provide both phylogenetic and non-phylogenetic metrics of alpha and beta diversity. Additional data analysis (PLS-DA) and statistics were performed with R .

Expanded View Figure Legends
Characterising a homozygous two-exon deletion in UQCRH: comparing human and mouse phenotypes (2021)
Figure 2

Figure 2: Uqcrh-/- mice displayed several essential characteristics of human CIII disorders

A Representative picture of a WT and age- and sex-matched Uqcrh-/- mouse at 4 weeks of age. Scale expressed in centimetres.

B Body weight of Uqcrh -/- mice and wild-type littermates at 1-2 weeks (n = 5 to 10), during the post-weaning period from 5-6 weeks of age (n = 3 to 7) and weekly from 6 to 10 weeks (n = 13 to 66), data are shown as mean ± SD. **** p < 0.0001, 2-Way ANOVA, from 7.5wk of age for Uqcrh-/- males and females versus sex-matched controls.

C, D Clinical chemistry plasma concentrations of parameters related to acid-base balance (Lactate and Ammonia) and metabolic state (Glucose) n = 15 Uqcrh-/- and 16 WT pups 1-2-week-old (C); n = 15 f, 15 m Uqcrh-/- and 15 f,15 m WT mice aged 8-9 weeks (D); sex not differentiated. Data shown as scatter dot plots with line at median. * p < 0.05, ** p < 0.01, **** p < 0.0001, Wilcoxon rank sum test.

E, F Measures related to behaviour and neurological function, data generated by the Open Field test (8 weeks old, E) and SHIRPA (9 weeks old, F), n = 7f, 8m Uqcrh-/- and 8f, 9m WT animals. Prepulse inhibition of the acoustic startle as a parameter associated with sensorimotor gating (10 weeks, E right panel), n = 7 f/5 m Uqcrh-/- and 8 f/ 9 m WT mice. Sex is not differentiated. Data are shown as scatter dot plots with a line representing the median. *** p < 0.001, **** p < 0.0001, Wilcoxon rank-sum test.

G Parameters related to heart function generated by Echocardiography (ECHO) and Electrocardiography (ECG), n = 15 f, 15 m Uqcrh-/- and 15 f, 15 m WT mice, 6 weeks old.

Data are shown as single data points and median, or categorical data, males and females pooled, since genotype-related effects were comparable for both sexes from different cohorts. * p < 0.05, *** p <0.001, Wilcoxon rank-sum test.
Characterising a homozygous two-exon deletion in UQCRH: comparing human and mouse phenotypes (2021)
Figure 3

Figure 3: Biochemical assessment of patient fibroblasts and Uqcrh-/- mice show impaired complex III activity and stability

A Respiratory chain enzyme activities from human control (green) and patient (red) fibroblasts. Mean activities of controls (n = 8) are set to 100% and error bars represent 1 standard deviation. Data are normalised to citrate synthase (CS) activity.

B Respiratory chain enzyme activities in heart, brain and liver, of wild-type and Uqcrh-/- mice. Data are normalised to citrate synthase (CS) activity. Values are given as mean ± SD, n = 4 WT, 5 Uqcrh-/- (Heart), 3 WT, 3 Uqcrh-/- (Brain), 1 WT and 2 Uqcrh-/- (Liver). * p < 0.05, **** p < 0.0001, 2-way ANOVA (Multiple comparison). Data to show CS activity/total protein concentration can be found in Appendix Fig S1A.

C Western blot analysis of OXPHOS components in the patient (P) and control (n = 2, C1-2) fibroblasts.

D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from patient and control fibroblasts (n = 2, C1-2) solubilised with DDM. Immunoblotting was performed using antibodies to a subunit of each complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A])

E Western blot analysis of liver lysates derived from 6 wild-type (WT) and 6 Uqcrh-/- animals at 4 weeks of age. The upper band (97 kDa) refers to loading control VCP protein, the lower band (11 kDa) indicates UQCRH.

F Immunohistochemical staining of UQCRC2 was performed on liver, heart and kidney derived from wild-type and Uqcrh-/- mice at 9 weeks of age. Staining of VDAC1/porin was also carried out (Appendix Fig S1B).

G Graphs indicate score values of immunohistochemical staining intensity of both VDAC1/porin and UQCRC2. Data are given as mean ± SEM. * p < 0.05; n = 3, Student's t-test (unpaired samples).
Characterising a homozygous two-exon deletion in UQCRH: comparing human and mouse phenotypes (2021)
Figure 4

Figure 4: Lentiviral transduction of patient fibroblasts with wild-type UQCRH, but not UQCRHL ameliorates the CIII defect

A Western blot analysis of control (n = 2, C1-2) and patient fibroblasts transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Expression of wild-type UQCRH was induced using various concentrations of doxycycline (dox) up to 20 ng/ml for 72 hours.

B BN-PAGE analysis of control (n = 2, C1-2) and patient fibroblasts transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Transduced fibroblasts were either uninduced or induced with 20 ng/ml doxycycline (dox) for 72 hours.

C Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts.

D Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts.

E Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts transduced with wild-type UQCRH.

F Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts transduced with wild-type (WT) UQCRH.

G Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts transduced with the pseudogene UQCRHL.

H Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts transduced with the pseudogene UQCRHL.

I Graph indicates the staining intensity of UQCRC2 in control (NHDF) fibroblasts (green) and patient fibroblasts (red), those respective cell lines transduced (T) with wild-type UQCRH (diagonal lines) and those cell lines transduced with the pseudogene UQCRHL (vertical lines) measured using Image J, Data are given as mean ± SEM. One-way ANOVA (Kruskal-Wallis test), * p < 0.05; n = 3 macroscopic field (10-14 cells for macroscopic field were measured).
Characterising a homozygous two-exon deletion in UQCRH: comparing human and mouse phenotypes (2021)
Figure 5

Figure 5: Two-exon deletion in UQCRH alters supercomplex stoichiometries and stability

Mitochondrial membranes from fibroblasts of control (WT) and patient fibroblasts (i, immortalised) (upper panels) and heart tissue from wild-type (WT) and Uqcrh-/- mice (lower panels) were solubilised with digitonin and separated on native gradient gels.

A BN-PAGE gel stained with Coomassie showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I)=immortalised fibroblasts)

B BN-PAGE gel stained with NADH:NTB reductase activity stain showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I)=immortalised fibroblasts)

C Further complexome analysis of the BN-PAGE gels shown in A and B. Each native lane was cut into even fractions from high to low molecular mass. Proteins in each slice were digested with trypsin and analysed by quantitative mass spectrometry. Shown here are average IBAQ values of all identified subunits from complexes III, I and IV (full data including all individual subunits shown in Fig EV4).

D Profile of complex III (averaged IBAQ values) in the patient (dashed line) and control (solid line) fibroblasts.

E Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in control (solid lines) and patient (dashed lines) fibroblasts.

F BN-PAGE gel stained with Coomassie (left) and NADH:NTB reductase activity stain (right) showing molecular weight marker (BHM) and heart tissue samples from wild-type (WT) and Uqcrh-/- mice.

G BN-PAGE and immunoblot using the antibody to CIII subunit UQCRC2 in heart tissue samples from wild-type (WT) and Uqcrh-/- mice.

H Further complexome analysis of the BN-PAGE gels shown in F, shown here are average IBAQ values of subunits from complexes III, I and IV (full data including all individual subunits shown in Fig EV4).

I Profile of complex III (averaged IBAQ values) in wild-type (+/+, solid line) and Uqcrh-/- (-/-, dashed line) heart tissue.

J Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in wild-type (+/+, solid line) and Uqcrh-/- (-/-, dashed line) heart tissue.
Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat (2010)
Figure 1

Transcriptional profiling of polyQ Atrophins. (A) Illustration of the crossing and ageing scheme used to obtain total RNA extracts from fly heads for the transcriptional profiling and all successive qPCR assays. Expression of different Atrophin forms with the GMR driver was induced, owing to a temperature‐sensitive mutant Gal80 repressor. F1 flies were allowed to develop at 18°C; at this temperature the Gal80 repressor keeps transgenes silent. Newly eclosed flies (0-48 h) were collected and killed immediately (0d) or aged for 2 or 14 days at 29°C. This inactivates Gal80 and transgenes are switched on by GMR‐Gal4. This protocol allows comparing siblings that differ exclusively in their age and transgenes expression. Control flies crossed to GMR‐Gal4; UbiGal80ts in all experiments are from the w1118 stock in which all UAS transgenes have been generated. (B) Tangential eye sections of flies representative of all the different populations used in the microarray analysis at all different time points. Weak degeneration is only visible after 14 days with polyQ Atro; in particular with Atro75QN there is an initial loss of photoreceptors (PR, arrow), 30.7% of the ommatidia has lost at least 1 PR, that is only 5.1% of all neuronal PR have been lost at this stage (N=333).
Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat (2010)
Figure 2

Global analysis of transcript profiling. (A) Heat maps for three subsets of the pattern clustering analysis generated using the Euclidean Distance metric. Upregulated genes are in red and downregulated genes are in green. The downregulating transcriptional activity increases with time. The main branch in clustering of the different Atro mutants is along time points. See also Supplementary Figure 4. (B) Venn diagrams for the subset of genes affected at the two time points for the different Atro versions with respect to control flies. The overlap between all Atro forms and the two polyQ versions increases with time. The genes in common for all Atro versions or between the polyQ Atro mutants at both times are listed.