Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: TSP-receptor α2δ-1 is synaptically expressed in the retina, and its expression is reduced in RCS rats. A, Representative images of the retina stained for α2δ-1 from LE (healthy) and RCS (degenerative) retinas at P14 and (B) P30. C, Quantitative staining intensity analysis demonstrated that α2δ-1 expression was reduced in RCS rat as early as P14. D, α2δ-1 was enriched in both the OPL and IPL, and the expression gap became more distinct at P30. Representative images of the (E) OPL and (F and G) IPL with the synapses labeled for Bassoon (F, green), VGluT1 (G, green), α2δ-1 (red), and NR1 (blue) from LE retina on P21 demonstrated postsynaptic expression of α2δ-1. Representative images of the (H) OPL and (J) IPL synapses labeled for Bassoon (green) and α2δ-1 (red) from LE (healthy) and RCS (degenerative) retinas on P21. Quantification of α2δ-1-containing synapses formed in the (I) OPL and (K) IPL reveals that the number of α2δ-1 synapse is already reduced in RCS rat by P21. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. ***p < 0.0001.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Expressing, Staining, Labeling

Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: Subretinal hUTC transplantation preserves OPL synapses in RCS rats. Representative images of the OPL with the PR ribbon synapses labeled for (A) Bassoon (green) and mGluR6 (red) and (B) Bassoon (green) and α2δ-1 (red) from LE (control), RCS + BSS, and RCS + hUTC P21 and P60 retinas on P95. Quantification of the number of synapses in the OPL revealed that hUTC transplantation protected (C) ribbon synapses. D, Particularly, α2δ-1-containing synapses were specifically preserved following hUTC treatment. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. ***p < 0.0001.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Transplantation Assay, Labeling

Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: Subretinal hUTC transplantation preserves α2δ-1-containing synapses in the IPL of RCS rats. A, Representative images of the IPL labeled for VGluT1 (green) and PSD95 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. B, Quantification of excitatory synapses in the IPL revealed that synapse numbers did not differ between RCS+BSS and RCS+hUTC P21 and P60. C, Representative images of the IPL labeled for Bassoon (green) and α2δ-1 (red) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. D, The α2δ-1-containing synapses were specifically preserved with hUTC transplantation. E, Representative images of the IPL stained for Bassoon (green) and Gephyrin (blue) from LE (control), RCS+BSS, and RCS+hUTC P21 and P60 retinas on P95. F, hUTC transplantation did not preserve inhibitory synapses. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *p < 0.05.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: Transplantation Assay, Labeling, Staining

Journal: The Journal of Neuroscience

Article Title: Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity

doi: 10.1523/JNEUROSCI.1532-17.2018

Figure Lengend Snippet: KO of MERTK in MG results in impaired synapse development in vivo. A, Representative images of the OPL synapses labeled by Bassoon (green) and mGluR6 (red) from Control (CTR, right eyeballs) and MERTK KO (KO, left eyeballs) at P21 and P45. B, Quantification of synapses in the OPL revealed that synapse development is impaired in MG-specific MERTK-KO at both P21 and P45. C, IPL excitatory synapses are visualized by VGluT1 (green) and PSD95 (red). D, Quantification of synapse number demonstrates that early synaptic development at P21 is significantly affected by MERTK KO but not at P45. Representative images of the (E) OPL and (G) IPL synapses labeled for Bassoon (green) and α2δ-1 (red). Quantification of the number of α2δ-1-containing synapses in both (F) OPL and (H) IPL revealed that KO of MERTK in MG induces severely reduced number of α2δ-1-containing synapses at P21 and P45. Data were obtained from a minimum of 3 animals of either sex and expressed as mean ± SEM. *p < 0.05.

Article Snippet: Primary antibodies (mouse anti-Bassoon 1:500 [RRID: AB_10618753 , ADI-VAM-PS003-F, Enzo], rabbit anti-mGluR6 1:150 [RRID:not applicable, RA13105, Neuromics], guinea pig anti-VGlut1 1:750 [AB5905, Millipore], rabbit anti-PSD95 1:500 [RRID: AB_87705 , 51-6900, Invitrogen], mouse anti-Gephyrin 1:250 [RRID: AB_1279448 , 147-021, Synaptic Systems], goat anti-TSP1 1:200 [RRID: AB_2201958 , AF3074, R&D Systems], goat-anti-TSP2 1:200 [RRID: AB_2202068 , AF1635, R&D Systems], mouse anti-glutamine synthetase 1:1000 [RRID: AB_397879 , 610517, BD Biosciences], rabbit anti-SOX9 1:4000 [RRID: AB_2239761 , AB5535, Millipore], goat anti-ChAT[RRID: AB_11214092 , AB144P, Millipore], rabbit anti-α2δ-1 [RRID: AB_258885 , C5105, Sigma-Aldrich]), rabbit anti-α2δ-1 [RRID: AB_2039785 , ACC-015, Alomone Labs], and rabbit anti-GFAP [RRID: AB_10013382 , Z033429-2, Dako] were diluted in 5% NGS or 5% BSA containing PBST.

Techniques: In Vivo, Labeling

Journal: Frontiers in Microbiology

Article Title: Pathogenic Leptospira Evolved a Unique Gene Family Comprised of Ricin B-Like Lectin Domain-Containing Cytotoxins

doi: 10.3389/fmicb.2022.859680

Figure Lengend Snippet: Leptospira PF07598 gene family members, represented by LA3490 here, are predicted with high confidence to have two tandemly repeated, N-terminal ricin B-like (RBL) lectin domains. (A) Visualization of an AlphaFold 3D-generated model of full-length LA3490 ( ; ; ) showing four globular domains N-terminal to C-terminal (blue to red color) residues visualized in PyMOL 2.4.0 https://pymol.org/2/ . Phyre2 (Protein Fold Prediction Server; http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index ) had first predicted, with high (> 94%) confidence, that LA3490, as well as all other virulence-modifying (VM) proteins encoded by the PF07598 gene family, contains N-terminal β-trefoil folds identified as ricin B domains. (B) Ricin B domain (PBD; 2AAI-B, 7 aa to 129 aa). (C) Superimposition of 2AAI-B and N-terminal region of LA3490 (i.e., amino acid positions 40–150) was performed using PyMOL (TM) 2.4.0 showing structural conservation of RBL1 and the B chain of ricin (RMSD = 1.796 Å).

Article Snippet: For competitive binding assays, precoated with asialofetuin (2.5 ng/μl) plates were preincubated with either 25 or 50 nM recombinant ricin B chain (Vector Laboratories, United States) for 2 h prior to the addition of 50 nM of rLA3490/t3490 and a final 2-h incubation.

Techniques: Generated

Journal: Frontiers in Microbiology

Article Title: Pathogenic Leptospira Evolved a Unique Gene Family Comprised of Ricin B-Like Lectin Domain-Containing Cytotoxins

doi: 10.3389/fmicb.2022.859680

Figure Lengend Snippet: Three-dimensional metric multidimensional scaling (3DMMDS/Galaxy) plots depicting (orthologous) VM protein clusters. Clusters were identified among 940 PF07598 family VM proteins analyzed using bios2mds and visualized using principal component analysis in R. In addition to typical PF07598 paralogs, 42 natural deletion mutants lacking both ricin B-like lectin, RBL, subdomains (i.e., containing amino terminal signal sequence and toxin domain only) were included. (A) Carbohydrate-binding region (CBR), containing two unidentical tandem RBL subdomains. (B) Carboxy terminal toxin domain (CTD) encompassing discrete trafficking and DNase subdomains. For both, initial renderings were edited (cosmetic changes only) to aid visualization by enhancing the 3D effect. No coordinates were altered. Clusters containing VM protein variants found in Leptospira interrogans are highlighted (large spheres) and named using the reference L. interrogans serovar Copenhageni strain (PMID 15028702), L1-130 (UniProtKB) protein IDs. Orthologous clusters were grouped into three superclusters comprising VM protein paralogs (A, n = 2; B, n = 7; and C, n = 4) based upon percent identity (PID) ( – ). The color key uses the following convention: for species, L. i nterroga ns (ins), L. k irschne ri (kri), L. n oguch ii (nii), etc.; for serovar, e.g., C anico la (CLA), Lai (LAI), H ard jo (HJO), etc.; and for strains originating from Sri Lanka, e.g., L. interrogans serovar Unknown strain KW1 (KW1), etc. (C) Schematic showing a theoretical evolutionary history of the VM protein family, involving lateral transfer (LGT), gene duplication (purple arrows, II) and erosion (solid black arrows), and recombination (blue arrows = donor acquired via lateral gene transfer, I; broken arrows indicate intragenomic donor from closely related paralog). Circles represent theoretical evolving VM proteins over time; squares represent final evolved form at the current time. (D) Domain organization and junctions of chimeric Leptospira VM proteins resulting from CBR and CTD domain fusions of paralogs belonging to closely related CBR clusters, such as those related to Q72NW3 (e.g., WP.017856587.1) and Q72TZ4 (e.g., QHH71994.1) (∼99.1% PID, , ). These natural VM protein variants occur infrequently (∼2%) in L. interrogans and its sister species, L. kirschneri and L. noguchii. Chimeric VM proteins generally share a common junction regardless of the paralogs represented.

Article Snippet: For competitive binding assays, precoated with asialofetuin (2.5 ng/μl) plates were preincubated with either 25 or 50 nM recombinant ricin B chain (Vector Laboratories, United States) for 2 h prior to the addition of 50 nM of rLA3490/t3490 and a final 2-h incubation.

Techniques: Sequencing, Binding Assay

Journal: Frontiers in Microbiology

Article Title: Pathogenic Leptospira Evolved a Unique Gene Family Comprised of Ricin B-Like Lectin Domain-Containing Cytotoxins

doi: 10.3389/fmicb.2022.859680

Figure Lengend Snippet: VM Protein LA3490 is a bona fide R-type lectin. (A) Schematic depicting the organization of the recombinant mCherry (mC) fusion proteins used in the current study; t3490, amino acid positions 40–147 aa (minus SS, signal sequence); and rLA3490, 19–639 aa, also lacking SS. Recombinant fusions also include a glycine–serine (GGGGSGGGGSGGGGS) linker and C-terminal His 6 tag (purification), and N-terminal thioredoxin. RBL and CTD denominates ricin B-like lectin and carboxy terminal domain, respectively. (B) Asialofetuin-binding assay demonstrating that truncated (t3490) and full-length (rLA3490) VM proteins bind to asialofetuin in a dose-dependent manner similar to commercially available ricin B chain. (C) Competition assay showing that truncated (t3490), the ricin B domain of another VM protein, LA0620 (t0620), and full-length (rLA3490) compete for the same binding site as recombinant ricin B chain (25 nM and 50 nM). Assays were performed in microtiter plates using an ELISA format. Mouse polyclonal anti-LA3490 and anti-LA0620 antibodies (1:1,000 dilution) were used as primary and anti-mouse IgG as secondary antibody (used alone as a specificity control, labeled as 2 Ab control). (D) Native LA3490 (70.29 kDa) secreted by L. interrogans serovar Lai into EMJH culture supernatant in the presence of 120 mM NaCl binds to asialofetuin-coupled Sepharose beads (AFS). Proteins were eluted with 0.5 M lactose. Unconjugated Sepharose beads incubated with L. interrogans serovar Lai-conditioned medium, and AFS beads with PBS served as controls. Assays were run in triplicate, and experiments were repeated twice. The mean absorbance (± SEM) was visualized in GraphPad Prism 8 and considered statistically significant at p < 0.05. The blot shown in panel (D) was cropped from the full blot shown in .

Article Snippet: For competitive binding assays, precoated with asialofetuin (2.5 ng/μl) plates were preincubated with either 25 or 50 nM recombinant ricin B chain (Vector Laboratories, United States) for 2 h prior to the addition of 50 nM of rLA3490/t3490 and a final 2-h incubation.

Techniques: Recombinant, Sequencing, Purification, Binding Assay, Competitive Binding Assay, Enzyme-linked Immunosorbent Assay, Labeling, Incubation