C. elegans strains
C. elegans strains were cultured at 20 °C on standard Nematode Growth Medium seeded with OP50 Escherichia coli70. On day 1 of adulthood, worms were transferred to plates containing OP50 E. coli (or HT115 E. coli for RNAi experiments) supplemented with 100 μg ml−1 5-fluoro-2′-deoxyuridine to prevent progeny development, except in lifespan assays. All experiments were conducted using hermaphrodite worms, and the age of the worms is indicated in the corresponding figures and figure legends.
WT (N2) and AM141 (rmIs133[unc-54p::Q40::YFP]) strains were obtained from the Caenorhabditis Genetics Center (CGC), supported by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). RB751 (eps-8(ok539)) was generated by the C. elegans Gene Knockout Consortium and acquired from the CGC. AM23 (rmIs298[F25B3.3p::Q19::CFP]) and AM716 (rmIs284[F25B3.3p::Q67::YFP]) strains were gifted by Richard I. Morimoto24. MAH602 (sqIs61[vha-6p::Q44::YFP + rol-6(su1006)]) was provided by Malene Hansen71. ZM5838 (hpIs223[rgef-1p::FUSWT::GFP]), ZM5844 (hpIs233[rgef-1p::FUSP525L::GFP]) and ZM5842 (hpIs228[rgef-1p::FUSR522G::GFP]) were provided by Peter St. George-Hyslop45. CK405(Psnb-1::TDP-43WT,myo-2p::dsRED) and CK423 (Psnb-1::TDP-43M337V,myo-2p::dsRED) were provided by Brian C. Kraemer33.
From these strains, we generated NFB2862 (Psnb-1::TDP-43WT,myo-2p::dsRED;juIs76[unc-25p::GFP + lin-15(+)]II) and NFB2863 (Psnb-1::TDP-43M337V,myo-2p::dsRED;juIs76[unc-25p::GFP + lin-15(+)]II). NFB2858 (rmIs298[F25B3.3p::Q19::CFP];otIs549[unc-25p::unc-25(partial)::mChopti::unc-54 3′ untranslated region (UTR) + pha-1(+)];him-5(e1490)V), NFB2859 (rmIs284[F25B3.3p::Q67::YFP];otIs549[unc-25p::unc-25(partial)::mChopti::unc-54 3′ UTR + pha-1(+)];him-5(e1490)V), NFB2860 (hpIs223[rgef-1p::FUSWT::GFP];otIs549[unc-25p::unc-25(partial)::mChopti::unc-54 3′ UTR + pha-1(+)];him-5(e1490)V) and NFB2861 (hpIs233[rgef-1p::FUSP525L::GFP];otIs549[unc-25p::unc-25(partial)::mChopti::unc-54 3′ UTR + pha-1(+)];him-5(e1490)V) were generated by crossing the respective polyQ and FUS-expressing strains with the OH13526 strain72. For RNAi in the neurons of polyQ67 worms, we used the DVG196 strain (rmIs284[F25B3.3p::Q67::YFP];sid-1(pk3321)V;uIs69[pCFJ90(myo-2p::mCherry) + unc-119p::sid-1]).
Worms expressing endogenous WT EPS-8::3xHA (VDL05, eps-8(syb2901)IV) or mutant EPS-8(K524R/K583R/K621R::3×HA) (VDL06, eps-8(syb2901, syb3149)IV) were previously generated via CRISPR–Cas9 (ref. 14). The strains DVG344 (rmIs284[pF25B3.3::Q67::YFP]);eps-8(syb2901) and DVG363 (rmIs133[unc-54p::Q40::YFP]);eps-8(syb2901) were generated by crossing VDL05 with AM716 and AM141, respectively. DVG345 (rmIs284[pF25B3.3::Q67::YFP]);eps-8(syb2901, syb3149) and DVG364 (rmIs133[unc-54p::Q40::YFP]);eps-8(syb2901, syb3149) were generated by crossing VDL06 to AM716 and AM141, respectively. These strains were validated by sequencing using the following primers: eps-8(syb2901): 5′-TTTGTTCGAAGCATGAACGA-3′ and 5′-AGCAGCCCCTGAAATAGTGA-3′; eps-8(syb2901, syb3149): 5′-AACGAGCTAGCAATCCGAAA-3′ and 5′-AGTGCTCTGCCGTCATTAAT-3′. DVG365 (rmIs284[pF25B3.3::Q67::YFP];eps-8(ok539)) was generated by crossing RB751 to AM716. The strain was outcrossed two times to AM716 and validated by polymerase chain reaction with 5′-TCTCCACCACCACAACGTAA-3′ and 5′-GCGGAGCAACTCTTCCATAG-3′ primers.
RNAi constructs
Adult worms were fed HT115 E. coli carrying either an empty control vector (L4440) or vectors expressing double-stranded RNAi. The RNAi constructs targeting eps-8, ifb-2, jnk-1, kgb-1, mig-2 and otub-3 were obtained from the Vidal library. The csn-6, F07A11.4, math-33, rac-2, usp-4, usp-5 and usp-48 RNAi constructs were obtained from the Ahringer library. All RNAi constructs were sequence verified. The RNAi sequences are listed in Supplementary Table 2.
Lifespan assay
Larvae were synchronized using the egg-laying protocol and grown on OP50 E. coli at 20 °C until day 1 of adulthood. Adult hermaphrodites were then transferred to plates with HT115 E. coli carrying either an empty vector or RNAi constructs for lifespan assays. All lifespan assays were performed at 20 °C. Each condition included 96 worms, scored daily or every other day73. Worms that were lost, burrowed into the medium, had a protruding vulva or underwent bagging were censored73.
Nose touch assay
Age-synchronized worms were assessed for nose touch response as previously described74,75,76. In brief, worms were placed on a thin bacterial lawn, and an eyelash pick was positioned in front of a forward-moving animal. A lack of response was recorded when the worm continued moving forward to crawl under or over the pick. For each condition, 30–40 animals were tested by monitoring the number of responses to a total of 10 gentle eyelash touches.
Chemotaxis assay
Freshly prepared agar plates (2% agar, 5 mM KPO4 (pH 6.0), 1 mM CaCl2, 1 mM MgSO4) were divided into four equal quadrants, along with an inner circle measuring approximately 1 cm across diagonally. A test solution (0.5% benzaldehyde (Sigma-Aldrich, B1334) in ethanol + 0.25 M sodium azide) and a control solution (ethanol + 0.25 M sodium azide) were added to two opposing diagonal quadrants. On the indicated days of adulthood (as shown in the corresponding figures), worms were collected in S-Basal medium, washed three times to remove residual bacteria and placed at the center of the chemotaxis plate. The plates were sealed with parafilm and incubated at 20 °C for 90 minutes. The number of worms in each quadrant was counted, excluding those that did not cross the inner circle. The chemotaxis index was calculated using the following formula: chemotaxis index = ((number of animals in test quadrants) − (number of animals in control quadrants)) / total number of animals77.
Motility assays
C. elegans were synchronized on OP50 E. coli using the egg-laying method and grown until day 1 of adulthood and then randomly transferred to plates with HT115 E. coli containing either empty vector or RNAi for the remainder of the experiment. For experiments with Ub-less EPS-8 mutants or DUB inhibitor treatment, worms were instead transferred to fresh plates containing OP50 E. coli. On the indicated day of adulthood (as shown in the corresponding figures), worms were randomly picked and transferred to a drop of M9 buffer, allowing 30 seconds for recovery24. Body bends were then recorded for 30 seconds and analyzed using ImageJ software (version 1.53k) with the wrMTrck plugin (https://www.phage.dk/plugins/)78,79. The locomotion velocity data were used to calculate body bends per second.
Microscopy
For imaging GABAergic neurons, fluorescent reporter worms were anesthetized with a drop of 0.5 M sodium azide (Sigma-Aldrich, 26628-22-8) on 4% agarose pads (diluted in distilled water) placed over a standard microscope glass slide (Rogo-Sampaic, 11854782). These preparations were sealed with 24 × 60-mm coverslips (RS France, BPD025). To score the number of GABAergic neurons and ventral nerve cord projections, we used a Zeiss Axio Imager.M2 microscope with a ×40 objective. Whole-body worm images were acquired using a Leica THUNDER Imager microscope with Tile Scan function and a ×40 objective.
Human cell lines
HEK293T/17 cells (American Type Culture Collection (ATCC), CRL-11268) were plated on 0.1% gelatin-coated plates and grown in DMEM (Thermo Fisher Scientific, 11966025), supplemented with 1% MEM non-essential amino acids (Thermo Fisher Scientific, 11140035), 1% GlutaMAX (Life Technologies, 35050038) and 10% FBS (Thermo Fisher Scientific, 10500064) at 37 °C with 5% CO2. ALS-iPSCs (FUSP525L/P525L) were kindly provided by Irene Bozzoni and Alessandro Rosa37. iPSCs were cultured on Geltrex (Thermo Fisher Scientific, A1413302) using mTeSR1 medium (STEMCELL Technologies, 85850) at 37 °C with 5% CO2. All cell lines were routinely tested for mycoplasma contamination, and no contamination was detected.
Motor neuron differentiation
Motor neurons were derived from ALS-iPSCs using a monolayer-based differentiation protocol80. ALS-iPSCs were seeded on Geltrex-coated plates and maintained in mTeSR1 medium until confluent. Differentiation was initiated using neuron differentiation medium composed of DMEM/F12 and Neurobasal (1:1; Thermo Fisher Scientific, 11330057 and 21103049), supplemented with non-essential amino acids, GlutaMAX (Thermo Fisher Scientific, 35050038), B27 (Thermo Fisher Scientific, 12587010) and N2 (Thermo Fisher Scientific, 17502048).
From day 0 to day 6, the medium was further supplemented with 1 μM retinoic acid (Sigma-Aldrich, R2625), 1 μM smoothened agonist (SAG; Sigma-Aldrich, 566661), 0.1 μM LDN-193189 (Miltenyi Biotec, 130-103-925) and 10 μM SB-431542 (Miltenyi Biotec, 130-105-336). From day 7 to day 14, the neuron differentiation media were supplemented with 1 μM retinoic acid, 1 μM SAG, 4 μM SU-5402 (Sigma-Aldrich, SML0443) and 5 μM DAPT (Sigma-Aldrich, D5942). After day 14, differentiated motor neurons were dissociated and replated on poly-l-ornithine (Sigma-Aldrich, P3655) and laminin-coated (Thermo Fisher Scientific, 23017015) plates in Neurobasal medium, supplemented with non-essential amino acids, GlutaMAX, N2, B27 and neurotrophic factors (10 ng ml−1 BDNF (BIOZOL, 450-02) and 10 ng ml−1 GDNF (BIOZOL, 450-10)).
Lentiviral infection of human cells
Lentivirus (LV)-non-targeting short hairpin RNA (shRNA), LV-EPS8 shRNA 1 (TRCN0000061544), LV-EPS8 shRNA 2 (TRCN0000061545), LV-USP4 shRNA 1 (TRCN0000004039) and LV-USP4 shRNA 2 (TRCN0000004040) in the pLKO.1-puro backbone were obtained from Mission shRNA (Sigma-Aldrich). Supplementary Table 2 contains the target sequences of each shRNA construct.
To generate stable shRNA-expressing HEK293 cell lines, cells were transduced with 5 µl of concentrated lentivirus and selected with 2 µg ml−1 puromycin (Thermo Fisher Scientific, A1113803). For lentiviral infection of iPSCs, cells were dissociated using Accutase (Thermo Fisher Scientific, A1110501), and 100,000 cells were seeded on Geltrex-coated plates in mTeSR1 medium supplemented with 10 μM ROCK inhibitor for 1 day. The next day, cells were infected with 5 µl of concentrated lentivirus. Medium was replaced the following day to remove residual virus. Selection for lentiviral integration was performed using 2 µg ml−1 puromycin for 2 days.
Transfection of HEK293 cells
HEK293 cells (ATCC, CRL-11268) were seeded on 0.1% gelatin-coated plates. When cells reached approximately 40% confluency, they were transfected with 1 μg of one of the following plasmids using FuGENE HD (Promega), according to the manufacturer’s instructions: pARIS-mCherry-httQ23-GFP, pARIS-mCherry-httQ100-GFP, pLVX-Puro-TDP-43-WT, pLVX-Puro-TDP-43-A382T, pcDNA3.1-FUS-HA-WT or pcDNA3.1-FUS-HA-P525L. In the indicated experiments, cells were co-transfected with an additional 1 μg of the pCMV3-EPS8-HA plasmid. The cells were collected after 72 hours of incubation in standard medium. The pARIS-mCherry-httQ23-GFP and pARIS-mCherry-httQ100-GFP plasmids were generously provided by Frédéric Saudou81. The FUS-HA-WT and FUS-HA-P525L plasmids were a gift from Dorothee Dormann82. The pLVX-Puro-TDP-43-WT and pLVX-Puro-TDP-43-A382T plasmids were provided by Shawn Ferguson (Addgene, 133753 and 133756)83. The pCMV3-EPS8-HA plasmid was obtained from Sino Biological (HG11153-CY).
Filter trap and western blot
For filter trap assays, synchronized adult C. elegans were collected and washed with M9 buffer, and worm pellets were snap frozen in liquid nitrogen. Frozen pellets were thawed on ice and lysed in non-denaturing buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM sodium orthovanadate, 1 mM PMSF, protease inhibitor cocktail (Roche)) using a Precellys 24 homogenizer. Lysates were cleared of worm debris by centrifugation (8,000g, 5 minutes, 4 °C), and protein concentrations were determined using the BCA assay (Thermo Fisher Scientific). To assess protein levels by western blot, 30 μg of total protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). To assess aggregated proteins by filter trap, 100 μg of total protein was supplemented with SDS to a final concentration of 0.5% and loaded onto a cellulose acetate membrane assembled in a slot-blot apparatus (Bio-Rad). Then, the membrane was washed with 0.2% SDS, and SDS-resistant aggregates were detected by immunoblotting.
If lysates were used solely for western blot, worms were lysed with a Precellys 24 homogenizer in buffer containing 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EDTA, 25 mM N-ethylmaleimide, 2 mM sodium orthovanadate, 1 mM PMSF and protease inhibitor cocktail. Lysates were cleared at 10,600g for 10 minutes at 4 °C, and 30 μg of protein was used for western blot experiments. For analysis of polyQ monomers and SDS-insoluble polyQ aggregates, age-synchronized worms were lysed by sonication in native buffer (50 mM Tris (pH 8), 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, protease inhibitor cocktail). Then, 30 μg of total protein was mixed with SDS to a final concentration of 0.4% and resolved by 12.5% SDS-PAGE.
For both filter trap and western blot analyses of C. elegans, immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000). Additionally, for western blot experiments, immunoblotting was conducted with anti-EPS8L2 (Abcam, ab85960, 1:1,000), anti-LGG-1 (ref. 84, 1:2,000) and α-tubulin (Sigma-Aldrich, T6199, 1:5,000).
For filter trap and western blot analysis of HEK293 cell lines, the cells were collected in lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM sodium orthovanadate, 1 mM PMSF, protease inhibitor cocktail), followed by homogenization through a 27-gauge syringe needle. Lysates from cells expressing pARIS-mCherry-httQ23-GFP, pARIS-mCherry-httQ100-GFP or without any overexpression were centrifuged at 8,000g for 5 minutes at 4 °C. Lysates from cells expressing FUS-HA-WT, FUS-HA-P525L, pLVX-Puro-TDP-43-WT or pLVX-Puro-TDP-43-A382T were centrifuged at 1,000g for 5 minutes at 4 °C. The supernatants were collected, and protein concentrations were measured with the BCA assay. For western blot, 30 μg of protein was analyzed as above. For filter trap analysis, 100 μg of total protein was supplemented with SDS to a final concentration of 0.5% and loaded onto a cellulose acetate membrane assembled in a slot-blot apparatus as described above. The membrane was then washed with 0.2% SDS, and SDS-resistant protein aggregates were evaluated by immunoblotting. For filter trap analysis, immunoblotting was conducted with antibodies against GFP (AMSBIO, TP401, 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000). For western blot, immunoblotting was conducted with anti-EPS8 (Proteintech, 12455-1-AP, 1:1,000), anti-β-actin (Abcam, 8226, 1:5,000), anti-HTT (Cell Signaling Technology, 5656, 1:1,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000), TDP-43 (Abcam, ab225710, 1:1,000), anti-LC3B (Cell Signaling Technology, 2775, 1:1,000) and anti-USP-4 (Abcam, ab181105, 1:1,000).
For necroptosis analysis, iPSC-derived motor neurons were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PMSF, protease inhibitor cocktail). Immunoblotting was performed using anti-phospho-RIP (Ser166) (Cell Signaling Technology, 65746, clone D1L3S, 1:1,000) and anti-RIP (Cell Signaling Technology, 3493, clone D94C12, 1:1,000). Densitometry of filter trap and western blot assays was performed using ImageJ software (version 1.51).
Protein immunoprecipitation for interaction analysis
HEK293 cells were collected and lysed in a protein lysis buffer containing 50 mM Tris-HCl (pH 6.7) 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF and protease inhibitor cocktail. Lysates were homogenized through a 27-gauge syringe needle and centrifuged at 13,000g for 15 minutes at 4 °C. Supernatants were incubated on ice for 1 hour with anti-USP-4 antibody (Abcam, ab181105, 1:100). As a negative control, the same amount of protein was incubated with anti-normal rabbit IgG (Cell Signaling Technology, 2729, 1:378). Samples were then incubated with 50 µl of µMACS MicroBeads for 1 hour at 4 °C with overhead shaking. Then, the samples were loaded onto pre-cleared µMACS columns (Miltenyi Biotec, 130-042-701). The beads were washed three times with a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 5% glycerol and 0.05% Triton, followed by five washes with 50 mM Tris (pH 7.5) and 150 mM NaCl. The samples were eluted with 75 μl of boiled 2× Laemmli buffer, boiled for 5 minutes at 95 °C and analyzed by western blotting.
Native gels analysis
HEK293 cells expressing CMV:mRFP-Q74 (ref. 30) were lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP-40, 2 mM EDTA, 1 mM EGTA, 10% glycerol, 2 mM sodium orthovanadate, 1 mM PMSF and protease inhibitor cocktail. Lysates were homogenized using a 27-gauge syringe needle and centrifuged at 12,000g for 15 minutes at 4 °C. Supernatants were collected, and protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific). Equal amounts of protein lysates were mixed 1:1 with sample buffer (50 mM Tris-HCl (pH 6.8), 10% glycerol, 0.01% bromophenol blue). Then, 20 μg of total protein was separated using 4–15% Tris-Glycine eXtended protein gels (Bio-Rad) and imaged via fluorescence using LICOR Odyssey M.
Immunocytochemistry
Cells were fixed with 4% paraformaldehyde in PBS for 20 minutes, followed by permeabilization with 0.2% Triton X-100 in PBS (10 minutes) and blocking with 3% BSA in 0.2% Triton X-100 in PBS (10 minutes). The cells were then incubated with anti-MAP2 (Sigma-Aldrich, M1406, 1:300) and rabbit anti-cleaved caspase-3 (Cell Signalling Technology, 9661S, 1:300) for 2 hours at room temperature. After washing with PBS, cells were incubated with secondary antibodies (Alexa Fluor 488 goat anti-mouse (Thermo Fisher Scientific, A-11029, 1:500) and Alexa Fluor 568 F(ab′)2 fragment of goat anti-rabbit IgG (H + L) (Thermo Fisher Scientific, A-21069, 1:500)) and Hoechst 33342 (Life Technologies, 1656104) for 1 hour at room temperature. Finally, the coverslips were rinsed in PBS, followed by a distilled water wash, and then mounted onto microscope slides with FluorSave Reagent (Merck, 345789).
CytoD, RAC activator and DUB inhibitor treatment
For CytoD treatment, worms were collected and randomly divided equally into M9 solutions containing either 10 μM CytoD (STEMCELL Technologies, 100-0557) or DMSO as a vehicle control. The worms were incubated with CytoD or DMSO for 6 hours on a shaker. For DUB inhibitor experiments, worms were collected and randomly transferred onto plates with OP50 bacteria covered with a final concentration of 13.7 μg ml−1 PR-619 (Merck, 662141) or vehicle control (DMSO) for either 4 hours or 1 day as indicated in the corresponding figures.
HEK293 cells were treated with 2 μM CytoD or DMSO for 4 hours before lysis. For RAC activation, cells were treated with 2 U ml−1 Rac/Cdc42 Activator II (Cytoskeleton, CN02-A) for 6 hours.
Proteasome activity
Day 5 adult worms and HEK293 cells were lysed in proteasome activity assay buffer (50 mM Tris-HCl (pH 7.5), 10% glycerol, 5 mM MgCl2, 0.5 mM EDTA, 2 mM ATP, 1 mM DTT) using a Precellys 24 or a 27-gauge syringe, respectively. The samples were centrifuged at 10,000g for 10 minutes at 4 °C, and the supernatants were collected. Protein concentrations were determined using the BCA protein assay kit.
To measure chymotrypsin-like proteasome activity, 25 μg of total protein was incubated with the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (Enzo Life Sciences, BML-P802) in 96-well plates (BD Falcon). Fluorescence was measured every 5 minutes for 2 hours at 20 °C (C. elegans) or 37 °C (human cells) using a microplate fluorometer (PerkinElmer, EnSpire) at 380-nm excitation and 460-nm emission.
Statistics and reproducibility
For quantification of filter trap and western blot data, we presented the results as relative changes compared with the corresponding control conditions. To average and statistically analyze independent experiments for these assays, we normalized test conditions to their corresponding control groups measured concurrently in each replicate experiment. Given that all the control groups were set to 100, we used a non-parametric Wilcoxon test when comparing two conditions to assess changes in protein aggregation and protein levels. For all other assays, we used parametric tests. Data distribution was assumed to be normal, but this was not formally tested. When more than two conditions or two independent variables were compared, we used one-way or two-way ANOVA followed by multiple comparisons tests. All statistical analyses were performed using GraphPad Prism (version 10.4.1).
For lifespan experiments, we used GraphPad Prism (version 10.4.1) and OASIS (version 1)85 to determine median and mean lifespan, respectively. The P values were calculated using the log-rank (Mantel–Cox) method and refer to comparisons between experimental and control animals within a single lifespan experiment. Each lifespan graph represents a single, representative experiment. Supplementary Table 1 contains the number of total/censored worms as well as detailed statistical analyses for each replicate lifespan experiment.
No statistical methods were used to predetermine sample size, but our sample sizes are similar to, or greater than, those reported in previous publications using the same procedures9,14,16,26,30,33,44,46,50,73,75,76,78,86,87,88. For motility assays, worms were excluded from analysis if they showed fewer than 0.1 body bends per second or were not recognized by the ImageJ software. No animals or data points were excluded from other analyses. For lifespan assays, worms were randomly picked and transferred from the synchronized population to the different experimental conditions. For all other experiments, worms were randomly distributed into the various experimental groups from single pulls of synchronized populations. Human cells were distributed to the various groups of all experiments from single pulls. Data collection was not randomized. Data collection and analysis were not performed blinded to the conditions of the experiments.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.