EMBO/soda-nel huggingface dataset:
Polyglutamine-expanded androgen receptor interferes with TFEB to elicit autophagy defects in SBMA (2014)
figf7

(a) We performed filter-trap assays to determine the extent of accumulation of insoluble AR protein in NPC lines from three different controls and three different SBMA patients. Significantly higher levels of insoluble AR protein were present in the SBMA NPC lines, as based on densitometry; n = 3 independent experiments, P 0.05, t-test, t(4) = 4.026. AU, arbitrary units. (b) NPCs from three different controls and three different SBMA patients were cultured in the presence of JC-1 dye. Results are presented as a Tukey box plots with box corresponding to first to third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(4) = 3.983. (c) We transfected NPC lines derived from SBMA patients and controls (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector. Autophagic vesicles were detected as colored puncta, as shown. Scale bar, 10 μm. (d) Quantification of autophagic vesicle type from c. SBMA NPCs exhibit a significant increase in autophagosomes; n = 18 different samples: 3 clones per line, 3 lines per group, *P 0.05, t-test; t(18) = 2.3799 (autophagosomes). (e) We isolated RNAs from SBMA and control NPC lines (three different NPC lines per human subject) and then measured the expression levels of TFEB target genes including CYCS (cytochrome c), t(4) = 2.844; MCOLN1, t(4) = 3.246; GLA, t(4) = 3.174; ACADM (acyl-CoA dehydrogenase), t(4) = 4.405; COX6A1 (cytochrome oxidase 6A1), t(4) = 5.584; and the TFEB gene itself, t(4) = 0.593. Significant expression reductions were noted in SBMA NPC lines for all tested TFEB target genes. *P 0.05, **P 0.01. Data in d,e are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.
Polyglutamine-expanded androgen receptor interferes with TFEB to elicit autophagy defects in SBMA (2014)
figf8

(a) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone (DHT) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation (IP). Immunoblotting (IB) for AR confirmed expression of AR in control (Ctrl) and SBMA NPCs, and immunoblotting for TFEB confirmed the interaction of AR with TFEB in both control and SBMA NPCs. (b) We transfected SBMA NPC lines (three different NPC lines per patient) with either the BFP-empty vector or BFP-TFEB expression construct, treated the NPCs with JC-1 dye and counted the percentage of cells with depolarized mitochondria. Results are presented as Tukey box plots with boxes corresponding to first and third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(6) = 3.217. (c) Representative images from transfection of SBMA NPCs with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct. BFP-empty vector–expressing SBMA NPCs display a preponderance of yellow puncta (autophagosomes), while BFP-TFEB–expressing SBMA NPCs exhibit a shift toward almost entirely red puncta (autolysosomes), consistent with rescue of the autophagic flux defect. Scale bars, 20 μm. (d) We transfected SBMA NPC lines (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct, counted the number of autophagosomes and autolysosomes in BFP-expressing NPCs and determined the number of autophagic vesicles per cell. TFEB overexpression markedly reduced autophagosome formation in SBMA NPCs. ***P 0.001; t-test, t(30) = 4.373. Data in d are presented as mean ± s.e.m. Individual P values are available in the Supplementary Methods Checklist.
BAG3 mediates chaperone‐based aggresome‐targeting and selective autophagy of misfolded proteins (2010)
Figure 1

BAG3 facilitates aggresome formation. (A) Upper panel: immunoblot analysis of COS7 cells treated for 12 h with MG132 (25 μM) and vinblastine (25 μM). Lower panel: real‐time PCR analysis showing gene expression changes in COS7 cells following MG132 exposure (25 μM, 12 h). (B,C) Immunofluorescence staining of indicated proteins in COS7 cells treated as in (A). Scale bar, 20 μm. (D) COS7 cells were transfected for 24 h with nonsense or bag3 siRNA and treated with MG132 (25 μM, 12 h). Aggresomes were visualized by ubiquitin immunostaining (supplementary Fig S1D online). Diagram shows the percentage of aggresome‐positive cells. (E) Aggresome quantification as in supplementary Fig S1D online from cells analysed in (H). Images are shown in supplementary Fig S1G online. Diagram shows the percentage of aggresome‐positive cells at 4, 24 and 48 h after MG132 wash out. (F) Cells were treated as in (H); however, 4 h after MG132 wash out, BafA1 (5 μM) was added for 20 h to inhibit aggresome clearance. Diagrams show the percentage of aggresome‐positive cells and the mean size of aggresomes (supplementary Fig S1H online). Images are shown in supplementary Fig S1G online. (G) Dot‐blot (upper panel, quantification in supplementary Fig S1B online) and western blot (lower panel) analyses of 0.5% NP40‐soluble and ‐insoluble proteins from cells shown in Fig 1D; supplementary Fig S1D online. (H) COS7 cells transfected with BAG3 or empty vector were treated with either MG132 (25 μM, 18 h) or DMSO as a control. After MG132 wash out, cells were allowed to recover for 4, 24 and 48 h and 0.5% NP40‐soluble and ‐insoluble proteins were analysed by immunoblotting. (I) COS7 cells were transfected for 24 h with GST or GST fused to DIC (GST‐DIC) and treated with MG132 (25 μM, 12 h). Indicated proteins in whole‐cell lysates and in precipitates obtained by GST pull‐down with glutathione beads were analysed by immunoblotting. BafA1, bafilomycin A1; ctrl, control; DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; Hsp70, heat‐shock protein 70; nons, nonsense; siRNA, short‐interfering RNA; vinblast, vinblastine.
BAG3 mediates chaperone‐based aggresome‐targeting and selective autophagy of misfolded proteins (2010)
Figure 2

BAG3 specifically directs Hsp70 substrates to the aggresome by loading misfolded proteins on to dynein. (A) GST pull‐down analysis as in Fig 1I, but performed in cells transfected for 36 h with full‐length FLAG‐BAG3 (I) or the BAG3 deletion mutants FLAG–(Δ421–498)–BAG3 (II), FLAG–BAG‐domain (III), and FLAG–PxxP–BAG‐domain (IV). Asterisk, nonspecific band. (B) HEK cells were transfected for 18 h with SODG85R‐GFP, along with the indicated FLAG‐tagged BAG3 constructs (shown in A) and HA‐tagged Hsp70 (HA‐Hsp70). SODG85R‐GFP showed three distinct localization patterns: cytoplasmic, pre‐aggresomal and aggresomal (see representative pictures on the right and supplementary Fig S2F online). The percentages of cells bearing pre‐aggresomal (upper panel) and aggresomal (lower panel) SODG85R‐GFP are shown. Scale bar, 10 μm. (C) HEK cells transfected with SODG85R‐GFP, BAG3 and Hsp70 were additionally transfected with FLAG‐p50, as indicated. Diagrams show the percentages of cells with pre‐aggresomal and aggresomal SODG85R‐GFP. Right panel: representative image showing pre‐aggresomal localization of FLAG‐p50 and SODG85R‐GFP. Scale bar, 5 μm. (D) GST pull‐down analysis as in (A), but in HEK cells transfected for 18 h with SODWT‐GFP or mutant SODG85R‐GFP, along with FLAG‐BAG3. (E) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA. At 6 h after transfection, lactacystin (10 μM) or DMSO as vehicle control was added for 12 h and GST pull‐down analysis was performed as in (D). (F) Upper panel: sucrose‐density gradient analysis of extracts from HEK cells transfected with SODG85R‐GFP and GST‐DIC, along with FLAG‐BAG3 or vector control. Note the immunoreactive bands in high‐density fraction 14 on BAG3 overexpression (arrow). Migration of a 20S marker protein is indicated. Lower panel: GST‐DIC pull‐down analyses of dynein complexes present in sucrose fraction 6 and 14. (G) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells transfected with FLAG‐tagged BAG3 constructs (I–IV; indicated as in A). (H) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells treated with PYR41 (10 μM; 12 h), as indicated. DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; HA, haemagglutinin; Hsp70, heat‐shock protein 70; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short‐interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.
BAG3 mediates chaperone‐based aggresome‐targeting and selective autophagy of misfolded proteins (2010)
Figure 3

Aggresome‐targeted proteins are degraded by autophagy. (A) Analysis of SODG85R‐GFP (green) localization with aggresome marker vimentin, the autophagic receptors p62 and NBR1, the autophagosome marker LC3 and the lysosomal marker LAMP2 (all in red) in BAG3/Hsp70‐transfected HEK cells. Representative merged images of cells with cytoplasmic, pre‐aggresomal and aggresomal SOD distribution are shown. Arrows indicate LC3‐positive autophagosomes. Scale bar, 5 μm. (B) Transmission electron microscope analysis of SODG85R‐GFP aggresome in cells transfected as in (A). Marked area in (a) is shown magnified in (b). Arrows, autophagosomes. Arrowheads, autolysosomes. Scale bar, 1 μm. (C) HEK cells transfected for 8 h with SODG85R‐GFP along with FLAG‐BAG3 and HA‐Hsp70, as indicated, were treated for 24 h with BafA1 (0.1 μM). Indicated proteins were analysed by immunoblotting. Diagram shows the ratio of SODG85R‐GFP levels in BafA1‐treated cells to those in untreated cells. (D) Same analysis as in (C) but with SODWT‐GFP. (E) Similar analysis as in (C) but in cells transfected 24 h before with nonsense or atg7 siRNA. (F) HEK cells were transfected with SODG85R‐GFP and indicated FLAG‐tagged BAG3 constructs together with HA‐Hsp70, and lysosomal degradation of SODG85R‐GFP was examined as in (C). Corresponding immunoblots are shown in supplementary Fig S3F online. Ag, aggresome; BafA1, bafilomycin A1; GFP, green fluorescent protein; HA, haemagglutinin; Hsp70, heat‐shock protein 70; N, nucleus; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.
BAG3 mediates chaperone‐based aggresome‐targeting and selective autophagy of misfolded proteins (2010)
Figure 4

Conclusion

BAG3 associates with aggresomes in vivo. (A) Spinal cord sections of mice overexpressing SODWT and SODG85R (disease end‐stage) were immunostained for BAG3 and SOD1. Arrows indicate SOD1‐ and BAG3‐positive aggregates in the neuropil. Scale bar, 20 μm. (B) Localization analysis of BAG3 and SODG85R in surviving spinal cord motor neurons from SODG85R mice at the time of disease symptom onset. Arrows indicate perinuclear aggresomes containing BAG3 and SODG85R. Scale bar, 10 μm. Magnific., magnification; SOD, superoxide dismutase; SODWT, wild‐type SOD.
Shelterin and subtelomeric DNA sequences control nucleosome maintenance and genome stability (2018)
Figure 1

Figure 1 - CLRC promotes the shelterin-chromatin association.

A Scheme of the shelterin complex and CLRC at telomeres.

B Mass spectrometry analysis of proteins co-purified with Ccq1HA. Shown is a volcano plot for proteins significantly enriched in Ccq1HA relative to untagged Ccq1 (left panel: WT cells; right panel: rik1∆ cells). Members of the CLRC complex and shelterin are highlighted in red and blue, respectively. Bottom panel displays proteins enriched (log2 ≥ 5 or pval ≤ 0.01).

C-E ChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments).

F ChIP analysis of Ccq1HA and FLAG-TetR-Clr4* (see scheme). Expression and tethering of FLAG-TetR-Clr4* are controlled by thiamine/AHT addition (red dots indicate induced/tethered FLAG-TetR-Clr4*; empty circles indicate non-induced/non-bound controls) (n = 3 independent experiments).

Data information: In (C-F) data are normalized to input and represented as mean ± SEM.
Shelterin and subtelomeric DNA sequences control nucleosome maintenance and genome stability (2018)
Figure 2

Figure 2 - Ccq1 promotes nucleosome stability at native subtelomeres.

A Left: Schemes of CLRC and the shelterin complex. Middle and right: Scheme depicting the position of the ura4+ reporter gene and heterochromatin islands relative to telomeric repeats and TAS regions; note that the minichromosomesome Ch16 does not contain TAS.

B-C ChIP-qPCR analyses of H3K9me2 (B) and H3 (C) in WT and ccq1Δ cells (n = 3 independent experiments.

D Fold change of WT over ccq1Δ for H3K9me2 and H3 levels (see methods).

Data information: In (B, C), data are normalized to input and to the average of three euchromatic loci (EC) as internal control (see methods) and represented as mean ± SEM.
Shelterin and subtelomeric DNA sequences control nucleosome maintenance and genome stability (2018)
Figure 3

Figure 3 - Ccq1 maintains nucleosome stability through recruiting CLRC and SHREC.

A Scheme of shelterin, CLRC and SHREC complexes and Swi6HP1 bound to H3K9me2.

B-D ChIP-qPCR analyses of H3 and H3K9me2 in WT and mutants as indicated (n = 3 independent experiments).

Data information: In (B-D), data are represented as mean ± SEM (normalization as in Fig 2B and C).
Shelterin and subtelomeric DNA sequences control nucleosome maintenance and genome stability (2018)
Figure 5

Figure 5 - Ectopic insertion of TAS fragment is sufficient to cause nucleosome instability.

A ChIP-qPCR analysis of H3 at endogenous TAS and a fragment spanning the TAS region from 115-905 bp (relative to telomeric repeat) inserted into the leu1+ locus (see scheme). The TAS fragment was inserted into a strain that lacks endogenous TAS (see text) [12]. Shown are ChIP analyses for WT (left) and ccq1∆ (right; note different the scale of the y-axis) (n = 9-10 independent experiments except for ectopic TAS in ccq1∆ strain where n = 3).

B ChIP-qPCR analysis of H3 at reporter genes (ura4+ and his3+) at various chromosomal locations (see schemes) in WT cells. TAS1, TAS2, and TAS3 correspond to position 116 bp, 2851 bp and 6291 bp (relative to telomeric repeats), respectively (n = 3 independent experiments).

Data information: In (A, B), data are represented as mean ± SEM (normalization as in Fig 2B and C).
Shelterin and subtelomeric DNA sequences control nucleosome maintenance and genome stability (2018)
Figure 6

Figure 6 - Nucleosome stability at subtelomeres is linked to heterochromatic silencing.

A, B RT-qPCR analysis of transcript levels of TERRA (A) and tlh1+ (B) in WT strain and mutants as indicated (double mutants with ccq1Δ are indicated by blue dot).

Data information: In (A, B), data is represented as mean ± SEM from 3 independent experiments and shown relative to WT level (see methods).
Shelterin and subtelomeric DNA sequences control nucleosome maintenance and genome stability (2018)
Figure 7

Figure 7 - Subtelomeric DNA promotes recombination in the absence of Ccq1.

A Genomic copy number of his3+, ura4+ and TAS1 in indicated strains harboring the reporter genes tel1L::his3+ and tel2L::ura4+. Cultures from individual WT and freshly generated knockout clones (ccq1Δ, n = 16; clr4Δ, n = 5; ccq1Δ clr4Δ, n = 5) were pre-grown on selective media for several days and inoculated at day 0 to grow in liquid media with regular back-dilution (every 24 hours, approximately 7 generations). Samples were taken at indicated harvest times, and relative copy numbers of genomic regions were assessed by qPCR (normalization against intrachromosomal loci). Black and rainbow color lines indicate WT strains and individual ccq1+ deletion mutants (clones), respectively.

B qPCR analysis as in (A) but with strains harboring the minichromosome Ch16 m23::ura4+ (ccq1Δ, n = 14). Since Ch16 is unstable in ccq1Δ cells (see Appendix Fig S3A-B and [37], m23::ura4+ has been normalized to minichromosome levels (details on normalization, see methods). Note that Ch16 does not harbor TAS sequences.
Bronchioalveolar stem cells are a main source for regeneration of distal lung epithelia (2019)
Figure 1

Figure 1. Engineering of split-Cre and split-tTA effector molecules.

A. Schematic depiction of bronchioalveolar cell types and strategy for selective in vivo manipulation of dual-marker expressing target cells.

B. Split-effector construct design.

C. Quantification of Cre activity by dual luciferase assays (mean±SD, n=3).

D. Quantification of tTA activity by dual luciferase assays (mean±SD, n=3 (N and C) or n=7 (N/C)).

Data information: Npu: Nostoc punctiforme, Ssp: Synechocystis sp., CDS: coding sequence. BASCs: bronchioalveolar stem cells, BADJ: bronchioalveolar duct junction, NLS: nuclear localization signal.
Bronchioalveolar stem cells are a main source for regeneration of distal lung epithelia (2019)
Figure 2

Figure 2. Expression of split-effectors from Spc and Ccsp loci targets BASCs at BADJs.

A-B. Scheme of engineered Spc and Ccsp loci after integration of split-Cre and split-tTA effectors (red mark = stop codon) and comparison of endogenous SPC or CCSP expression and YFP or mCherry reporter expression in lung sections. Blue: DAPI. Scale bar: 100 µm.

C-D. β-galactosidase and H&E staining of lung sections from BASC tracer and BASC viewer animals. Scale bars: 500 µm (overview) and 20 µm (boxed magnification).

E. Localization of β-gal+ cells in adult BASC viewer animals. BADJ-associated and "alveolar" β-gal+ cells are expressed as percentage of all lineage-labeled cells (means±SD n=3).

F. Number of β-gal+ cells in adult BASC tracer and BASC viewer animals, normalized to the number of terminal bronchioles (TBs) (means±SD n=3).
Bronchioalveolar stem cells are a main source for regeneration of distal lung epithelia (2019)
Figure 3

Figure 3. Selective and reversible manipulation of CCSP and SPC co-expressing BASCs by split-tTA effectors.

A. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of adult BASC viewer animals. Arrow highlights labeled BASC at BADJ (magnified in box). Blue: DAPI. Scale bars: 100 µm (overview) and 20 µm (boxed magnification).

B. β-galactosidase staining of AT2 cells (YFP+), Club cells (mCherry+) and BASCs (mCherry+/YFP+) isolated from BASC viewer mice by fluorescence-activated cell sorting (FACS). Scale bar: 50 µm.

C. Doxycycline-dependent reversible labeling of BASCs in BASC viewer mice. Scale bar: 200 µm.
Bronchioalveolar stem cells are a main source for regeneration of distal lung epithelia (2019)
Figure 5

Figure 5. BASCs continuously contribute to homeostatic renewal of the bronchioalveolar epithelium.

A. Schematic depiction of the strategy to trace BASCs and descendants via the split-tTA system.

B. Quantification of β-gal+ cells in FACS-purified AT2 and Club cell fractions isolated from 3 months old BASC v-race mice (mean±SD, n=3) and example of β-galactosidase label distribution in lung sections. Scale bar: 100 µm.

C. BASCs emerge at early postnatal stages and contribute to epithelial turnover throughout life. β-galactosidase staining of lung sections derived from BASC v-race animals collected at indicated time points. Arrow highlights β-gal+ cell at P6. Scale bar: 200 µm.
Bronchioalveolar stem cells are a main source for regeneration of distal lung epithelia (2019)
Figure 6

Figure 6. BASCs contribute substantially to lung regeneration and display multi-lineage differentiation potential.

A. β-galactosidase staining of cleared whole lung preparations isolated from control and injured BASC v-race animals 21 days (bleomycin, naphthalene) or 35 days (influenza) following injury. Scale bar: 5 mm (whole lungs) and 1 mm (magnification).

B. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of control and injured BASC v-race mice. Clusters of BASC-derived AT2 (YFP+) and Club cells (mCherry+) are highlighted. Blue: DAPI. Scale bar: 200 µm.

C. Contribution of BASCs to different cellular compartments. Bronchiolar and alveolar accumulations of lineage-traced BASC derivatives are expressed as percentage of all clusters containing ≥ 10 β-gal+ cells. Data depicted as mean±SD, n=4 (bleomycin, naphthalene) or n=5 (influenza).
Bronchioalveolar stem cells are a main source for regeneration of distal lung epithelia (2019)
Figure 7

Figure 7. BASCs expand in response to bronchiolar injury and are removed by IV infection.

A. β-galactosidase and H&E staining of lung sections from control (non-infected) and injured BASC viewer mice 7 days post infection (7 dpi) with influenza virus. Scale bar: 200 µm.

B. Abundance of β-gal+ cells in control (non-infected) and injured (7 dpi) BASC viewer animals. Data depicted as mean±SD, n = 4, *p = 0.0286 (Mann-Whitney test), TB - terminal bronchiole.

C. Club cell depletion/recovery after naphthalene treatment. Microscopic imaging of epifluorescence in lung sections from control and injured BASC viewer animals isolated 3, 7 and 21 days post naphthalene (dpn). Blue: DAPI. Scale bar: 200 µm.

D. β-galactosidase staining of lung sections from control and injured BASC viewer animals 3, 7 and 21 days post naphthalene (dpn). Scale bar: 200 µm.

E. Abundance of β-gal+ cells in control and injured BASC viewer animals. The percentage of terminal bronchioles containing 0, 1, 2, 3, 4, ≥ 5 β-gal+ cells is shown. Data depicted as mean±SD, n=3.
Bronchioalveolar stem cells are a main source for regeneration of distal lung epithelia (2019)
Figure 8

Figure 8. BASCs are a main cellular source for distal airway regeneration.

A. Quantification of lineage-traced Club cells in distal TBs and β-galactosidase staining of cleared accessory lobe preparation (scale bar: 250 µm) isolated from injured BASC v-race mice at 21 dpn (mean±SD, n=4). Bottom panel: example of sequential microscopic imaging to quantify β-gal+ mCherry+ cells in distal TBs. Arrows denote last airway bifurcation before bronchioalveolar duct junction (BADJ). Scale bar: 200 µm.

B. β-galactosidase staining of FACS-purified Club cells from control and injured BASC v-race animals. Scale bar: 50 µm. The percentage of lineage-traced Club cells is shown (mean±SD, n=4 (oil ctrl.) or n=5 (21 dpn), *p = 0.0159 (Mann-Whitney test).

C. Combined β-galactosidase (blue) and acetylated tubulin staining (cilia, brown color) of lung sections from BASC v-race animals at 21 dpn. Red: DAPI (pseudocolor). Examples of lineage-traced ciliated cells (arrowheads) and Club cells (asterisks) are highlighted. Scale bar: 50 µm.

D. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and quantification of β-gal+ bronchioles in lung sections from adult BASC v-race (ctrl.) and BASC-ablated mice (DTA). Data depicted as mean±SD, n=3.

E. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and fluorescence microscopy of lung sections from uninjured and naphthalene-exposed BASC v-race (ctrl.) and BASC-ablated (DTA) mice 12 days post naphthalene (dpn). Dotted lines depict bronchiolar boundaries. Scale bar: 100 µm.

F. Quantification of Club cell recovery in injured BASC v-race (ctrl.) and BASC-ablated (DTA) animals, expressed as percentage of bronchiolar circumference re-populated by mCherry+ cells at 12 dpn. Data depicted as mean±SD, n=5, **p = 0.0079 (Mann-Whitney test). DTA - diphtheria toxin fragment A.

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