Journal: Current Research in Microbial Sciences

Article Title: Molecular and phenotypic reidentification of Sporothrix schenckii clinical isolates preserved under mineral oil for 34 to 64 years in a culture collection in Brazil

doi: 10.1016/j.crmicr.2022.100128

Figure Lengend Snippet: Summary of the characteristics (morphological and physiological) of Sporothrix spp. recovered from the Culture Collection of Filamentous Fungi of Oswaldo Cruz Institute, Fiocruz.

Article Snippet: Several published BT2 sequences from Sporothrix species were retrieved from NCBI GenBank S. brasiliensis CBS120339 (formerly IPEC 16,490), S. globosa FMR 8600, S. luriei ATCC 18616T, S. palida CBS302.73, S. chilensis CBS 139,891, S. mexicana CBS 120,341, S . schenckii (FMR 8604; FMR 8605; FMR 8606; FMR 8608; FMR 8609; FMR 8677; FMR 8678; FMR 8679; IHEM3774; IHEM 3787; IHEM 15,502; IHEM 15,503; IHEM 15,508; URM4291; URM1013; URM4861; IHEM 15,511; IHEM 15,477; IHEM 15,486; IHEM 15,489; CMW7612; CBS359.36; NBRC8158) and sequences from both DNA strands were generated, edited with the Sequencher ver.

Techniques:

Journal: Current Research in Microbial Sciences

Article Title: Molecular and phenotypic reidentification of Sporothrix schenckii clinical isolates preserved under mineral oil for 34 to 64 years in a culture collection in Brazil

doi: 10.1016/j.crmicr.2022.100128

Figure Lengend Snippet: Macromorphological analysis of Sporothrix spp. after 21 days of incubation on PDA at 30 °C. A – IOC 1275; B – IOC 2993; C – IOC 1799; D – IOC 1835; E – IOC 1912; F – IOC 2547; G – IOC 2835.

Article Snippet: Several published BT2 sequences from Sporothrix species were retrieved from NCBI GenBank S. brasiliensis CBS120339 (formerly IPEC 16,490), S. globosa FMR 8600, S. luriei ATCC 18616T, S. palida CBS302.73, S. chilensis CBS 139,891, S. mexicana CBS 120,341, S . schenckii (FMR 8604; FMR 8605; FMR 8606; FMR 8608; FMR 8609; FMR 8677; FMR 8678; FMR 8679; IHEM3774; IHEM 3787; IHEM 15,502; IHEM 15,503; IHEM 15,508; URM4291; URM1013; URM4861; IHEM 15,511; IHEM 15,477; IHEM 15,486; IHEM 15,489; CMW7612; CBS359.36; NBRC8158) and sequences from both DNA strands were generated, edited with the Sequencher ver.

Techniques: Incubation

Journal: Current Research in Microbial Sciences

Article Title: Molecular and phenotypic reidentification of Sporothrix schenckii clinical isolates preserved under mineral oil for 34 to 64 years in a culture collection in Brazil

doi: 10.1016/j.crmicr.2022.100128

Figure Lengend Snippet: Micromorphological analysis of Sporothrix spp. after microculture for 12 days on PDA at 30 °C. The isolates IOC 1275, IOC 1799, IOC 1835, IOC 1912 and IOC 2835 showed sessile conidia (arrowhead) and septate hyaline hyphae (black arrow) containing conidiophores with conidia arranged in the shape of a daisy (red arrow). Isolate IOC 2993 presented only septate hyaline hyphae (black arrows). (1000X magnification).

Article Snippet: Several published BT2 sequences from Sporothrix species were retrieved from NCBI GenBank S. brasiliensis CBS120339 (formerly IPEC 16,490), S. globosa FMR 8600, S. luriei ATCC 18616T, S. palida CBS302.73, S. chilensis CBS 139,891, S. mexicana CBS 120,341, S . schenckii (FMR 8604; FMR 8605; FMR 8606; FMR 8608; FMR 8609; FMR 8677; FMR 8678; FMR 8679; IHEM3774; IHEM 3787; IHEM 15,502; IHEM 15,503; IHEM 15,508; URM4291; URM1013; URM4861; IHEM 15,511; IHEM 15,477; IHEM 15,486; IHEM 15,489; CMW7612; CBS359.36; NBRC8158) and sequences from both DNA strands were generated, edited with the Sequencher ver.

Techniques:

Journal: Current Research in Microbial Sciences

Article Title: Molecular and phenotypic reidentification of Sporothrix schenckii clinical isolates preserved under mineral oil for 34 to 64 years in a culture collection in Brazil

doi: 10.1016/j.crmicr.2022.100128

Figure Lengend Snippet: Microculture of Sporothrix spp. IOC 2993 for 12 days at 30 °C. A and B show the isolate cultured on PDA producing only thin, hyaline hypha. C and D show the isolates cultured on MEA with rose bush branches presenting sessile conidia and thin, septate hyaline hypha containing conidia arranged in the shape of a daisy at the end of conidiophores (Arrows) (1000X magnification).

Article Snippet: Several published BT2 sequences from Sporothrix species were retrieved from NCBI GenBank S. brasiliensis CBS120339 (formerly IPEC 16,490), S. globosa FMR 8600, S. luriei ATCC 18616T, S. palida CBS302.73, S. chilensis CBS 139,891, S. mexicana CBS 120,341, S . schenckii (FMR 8604; FMR 8605; FMR 8606; FMR 8608; FMR 8609; FMR 8677; FMR 8678; FMR 8679; IHEM3774; IHEM 3787; IHEM 15,502; IHEM 15,503; IHEM 15,508; URM4291; URM1013; URM4861; IHEM 15,511; IHEM 15,477; IHEM 15,486; IHEM 15,489; CMW7612; CBS359.36; NBRC8158) and sequences from both DNA strands were generated, edited with the Sequencher ver.

Techniques: Cell Culture

Journal: Current Research in Microbial Sciences

Article Title: Molecular and phenotypic reidentification of Sporothrix schenckii clinical isolates preserved under mineral oil for 34 to 64 years in a culture collection in Brazil

doi: 10.1016/j.crmicr.2022.100128

Figure Lengend Snippet: Phylogenetic relationships between the isolates IOC 2547, IOC 1835, IOC 1912, IOC 1799 IOC 1275, IOC 2993 and IOC 2835 with reference strains of the Sporothrix schenckii complex inferred from β-tubulin sequences by Neighbor-Joining method . The optimal tree is shown. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches . The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. This analysis involved 31 nucleotide sequences. There were a total of 428 positions in the final dataset. Evolutionary analyses were conducted in MEGA X . Bootstrap support values above 80% are indicated at the nodes.

Article Snippet: Several published BT2 sequences from Sporothrix species were retrieved from NCBI GenBank S. brasiliensis CBS120339 (formerly IPEC 16,490), S. globosa FMR 8600, S. luriei ATCC 18616T, S. palida CBS302.73, S. chilensis CBS 139,891, S. mexicana CBS 120,341, S . schenckii (FMR 8604; FMR 8605; FMR 8606; FMR 8608; FMR 8609; FMR 8677; FMR 8678; FMR 8679; IHEM3774; IHEM 3787; IHEM 15,502; IHEM 15,503; IHEM 15,508; URM4291; URM1013; URM4861; IHEM 15,511; IHEM 15,477; IHEM 15,486; IHEM 15,489; CMW7612; CBS359.36; NBRC8158) and sequences from both DNA strands were generated, edited with the Sequencher ver.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: C-terminal and full length TDP-43 specie differ according to FTLD-TDP lesion type but not genetic mutation

doi: 10.1186/s40478-019-0755-x

Figure Lengend Snippet: The amino acid sequence of TDP-43 from N terminus (1) to C terminus (414). The highlighted regions are the peptides used to generate the polyclonal antibodies (MC2079, MC2085 and pTDP 409/410)

Article Snippet: In all 24 cases we performed serial sectioning and TDP-43 immunohistochemistry with four different TDP-43 antibodies: phosphorylated TDP-43 (pTDP-43) antibody (pS409/410, 1:5000 mouse monoclonal, Cosmo Bio Co., LTD) that recognizes TDP-43 with phosphorylated epitopes; anti TDP-43 antibody that recognizes a neoepitope in the C terminal fragment of cleaved TDP-43(cTDP-43) (MC2085, 1:2500 rabbit polyclonal, gift from Leonard Petrucelli, Mayo Clinic) [ ]; anti TDP-43 antibody that recognizes an epitope in the amino terminus (nTDP-43) (MC1079; 1:2500 rabbit polyclonal, gift from Leonard Petrucelli, Mayo Clinic) [ ] and a rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group; Chicago IL; dilution 1:3000) which is a commercially available antibody.

Techniques: Sequencing

Journal: The Journal of Clinical Investigation

Article Title: Virus-mediated delivery of antibody targeting TAR DNA-binding protein-43 mitigates associated neuropathology

doi: 10.1172/JCI123931

Figure Lengend Snippet: (A) Binding of p65 to TDP-43 was measured in the presence of PBS or anti–TDP-43 N-terminal antibody (Proteintech), monoclonal antibodies against RRM1 TDP-43 (named C10, G8, and E6), or BSA. n = 8 wells per condition (dots). One-way ANOVA P < 0.0001; *P < 0.05 and ***P < 0.001 versus PBS, by Tukey’s multiple comparisons test. Data represent the mean ± SEM. (B) Schematic representation of the pscFv9 plasmid used for scFv production and expression. (C) Representative Western blot of cytoplasmic and nuclear fractions of Hek293 cells. Anti-Myc antibody revealed scFv and laminin A/C or actin in the different fractions. (D) Representative image of media from transfected Hek293 cells probed with anti-Myc antibody. Ponceau staining was used as a reference. (E) Different concentrations of TDP-43 (1–206 aa, Proteintech) or BSA were loaded onto a dot blot membrane. Immunoblots were performed with media containing pscFv9-transfected Hek293 cells and E6 monoclonal antibody. Signals were revealed with anti–Myc-HRP antibody for scFv conditions or anti–mouse HRP for E6. Ponceau staining was used as a reference. (F) Representative blot of TDP-43 immunoprecipitation in pscFv9-transfected Hek293 cells. Experiments in C, D, and F were conducted more than 3 times. Empty, no scFv; CTR, control D1.3 scFv.

Article Snippet: Therefore, we derived scFv antibodies from this monoclonal antibody. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 E6-derived scFv antibodies are able to recognize TDP-43. ( A ) Binding of p65 to TDP-43 was measured in the presence of PBS or anti–TDP-43 N-terminal antibody (Proteintech), monoclonal antibodies against RRM1 TDP-43 (named C10, G8, and E6), or BSA. n = 8 wells per condition (dots).

Techniques: Binding Assay, Plasmid Preparation, Expressing, Western Blot, Transfection, Staining, Dot Blot, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: Virus-mediated delivery of antibody targeting TAR DNA-binding protein-43 mitigates associated neuropathology

doi: 10.1172/JCI123931

Figure Lengend Snippet: (A) Binding of p65 to TDP-43 was measured in the presence of equal volumes of PBS or media from pscFv9-transfected Hek293cells. n = 5–8 wells per condition (dots). One-way ANOVA P = 0.002; *P < 0.05 versus PBS; #P < 0.05 or ##P < 0.001 versus control scFv by Tukey’s multiple comparisons test. (B) BV2 cells were transfected with pscFv9, stimulated with LPS or PBS, and lysated for the luciferase assay. n = 4–5 individual experiments (dots). Two-way ANOVA P = 0.0002; §§§P < 0.001 versus LPS; ***P < 0.001 versus empty; and ###P < 0.001 versus control scFv, by Tukey’s multiple comparisons test. (C and D) BV2 p65-luc cells were treated or not for 6 hours or 10 hours with scFv antibodies, together with 4 hours of LPS treatment. (C) Representative Western blots of total lysates of Myc-tag scFv inside the cells and actin (n = 3 pooled experiments). The blot of the untreated cells at 6 hours was run on the same gel but was noncontiguous. (D) NF-κB activity assessed by luciferase assay. n = 4–6 replicates (dots) from 2 independent experiments (2–3 wells each). Two-way ANOVA P < 0.0001; *P < 0.05 and ***P < 0.001 versus untreated cells; ###P < 0.001 versus control scFv, by Tukey’s multiple comparisons test. Data represent the mean ± SEM. RLU, relative luminescence units; CTR, control D1.3 scFv.

Article Snippet: Therefore, we derived scFv antibodies from this monoclonal antibody. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 E6-derived scFv antibodies are able to recognize TDP-43. ( A ) Binding of p65 to TDP-43 was measured in the presence of PBS or anti–TDP-43 N-terminal antibody (Proteintech), monoclonal antibodies against RRM1 TDP-43 (named C10, G8, and E6), or BSA. n = 8 wells per condition (dots).

Techniques: Binding Assay, Transfection, Luciferase, Western Blot, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Virus-mediated delivery of antibody targeting TAR DNA-binding protein-43 mitigates associated neuropathology

doi: 10.1172/JCI123931

Figure Lengend Snippet: (A) Representative blot and quantification of TDP-43 levels in Hek293 cells transfected with pscFv9 plasmid. Graph shows quantification of TDP-43 normalized to actin and expressed as the fold change versus empty vector. n = 7–9 independent experiments (dots). One-way ANOVA P = 0.0003; *P < 0.05 and ***P < 0.0001 versus empty transfected, by Tukey’s multiple comparisons test. (B and C) Representative merged images of colocalization between scFv (Myc, green) and ubiquitin or LC3 (red). Arrows show cells with colocalization; yellow arrows indicate cells enlarged 2.5 times in the insets. Scale bars: 20 μm. The experiment was performed twice. (D) Representative blots and quantification of immunoprecipitation for all K48-polyubiquitinated (proteasome) or K63-polyubiquitinated (autophagy) proteins and Western blot for TDP-43. Graphs indicate the quantification of TDP-43 signal in immunoprecipitation experiments, expressed as the fold change versus empty vector. n = 3 independent experiment (dots). K63-ubiquitinated TDP-43: P = 0.0082, by 1-way ANOVA; **P < 0.01 versus empty vector–transfected cells, by Dunnett’s multiple comparisons test. K48-ubiquitinated TDP-43: P = 0.0128, by 1-way ANOVA; *P < 0.05 versus empty vector–transfected cells, by Dunnett’s multiple comparisons test. Data represent the mean ± SEM. Empty, no scFv (A and D); CTR, D1.3 scFv (B and C) or 8H11 anti-GFP scFv (A and D).

Article Snippet: Therefore, we derived scFv antibodies from this monoclonal antibody. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 E6-derived scFv antibodies are able to recognize TDP-43. ( A ) Binding of p65 to TDP-43 was measured in the presence of PBS or anti–TDP-43 N-terminal antibody (Proteintech), monoclonal antibodies against RRM1 TDP-43 (named C10, G8, and E6), or BSA. n = 8 wells per condition (dots).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Virus-mediated delivery of antibody targeting TAR DNA-binding protein-43 mitigates associated neuropathology

doi: 10.1172/JCI123931

Figure Lengend Snippet: (A) Representative enlarged images and TDP-43 mislocalization determined by quantification of the integrated density of TDP-43 immunoreactivity in nuclear and cytoplasmic compartments. Images show merged scFv (Myc, green), hTDP-43 (red), and Hoechst (nuclei, blue) channels, and lines circumscribe the perimeter of the representative cells. Scale bars: 5 μm. Data in the graph represent the cytoplasmic to nuclear ratio quantified in at least 200 cells per frame. n = 5–10 different frames (dots) analyzed from 2 independent experiments. One-way ANOVA P < 0.0001; ***P < 0.001 versus control-transfected cells; #P < 0.05 and ###P < 0.001 versus empty vector–transfected cells, by Tukey’s multiple comparisons test. (B) Representative image and quantification of dot blot analysis for soluble or insoluble TDP-43 in transfected Hek293 cells treated with 50 μM EA. Data represent TDP-43 immunoreactivity normalized to Ponceau from 5 to 9 replicates (dots) from 3 independent experiments. Two-way ANOVA interaction P = 0.249, scFv P = 0.0014, and soluble/insoluble P < 0.0001; ***P < 0.001 versus empty vector–transfected cells, by Tukey’s multiple comparisons test. Data represent the mean ± SEM. Empty, no scFv, CTR, control D1.3 scFv.

Article Snippet: Therefore, we derived scFv antibodies from this monoclonal antibody. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 E6-derived scFv antibodies are able to recognize TDP-43. ( A ) Binding of p65 to TDP-43 was measured in the presence of PBS or anti–TDP-43 N-terminal antibody (Proteintech), monoclonal antibodies against RRM1 TDP-43 (named C10, G8, and E6), or BSA. n = 8 wells per condition (dots).

Techniques: Transfection, Plasmid Preparation, Dot Blot

Journal: The Journal of Clinical Investigation

Article Title: Virus-mediated delivery of antibody targeting TAR DNA-binding protein-43 mitigates associated neuropathology

doi: 10.1172/JCI123931

Figure Lengend Snippet: NOR test 2 months (A) and 4 months (B) after injection. Graphs in A and B show the percentage of time spent with objects and the number of individual mice tested (dots). (A) Two-way ANOVA P = 0.0004; ***P < 0.001 and *P < 0.05, by Sidak’s multiple comparisons test. (B) Two-way ANOVA P < 0.0001; ***P < 0.001 for familiar versus novel object, by Sidak’s multiple comparisons test; *P < 0.05 and **P < 0.01 among groups, by uncorrected Fisher’s least significant difference (LSD) test. (C) Quantification of hTDP-43 mislocalization in cortical neurons and representative images. Data represent the cytoplasmic to nuclear ratio of TDP-43 integrated density. n = 3 individual mice (dots). Two-way ANOVA interaction P = 0.979, hemisphere P = 0.3747, scFv P < 0.0001; **P < 0.01 versus control scFv, by Sidak’s multiple comparisons test. Shown are hTDP-43 (green) and merged images of hTDP-43, NeuN (red), and Hoechst (nuclei, blue). Scale bars: 20 μm. Representative blots and quantification of nuclear and cytoplasmic (D) and insoluble (E) hTDP-43 in ipsilateral cortices. n = 3 individual mice (dots). hTDP-43 was normalized to p84 or GAPDH fraction markers (D) and to Ponceau (E). Values are expressed as the fold change versus control scFv. Statistical analysis was done by unpaired t test. Data represent the mean ± SEM. CTR, 8H11 scFv.

Article Snippet: Therefore, we derived scFv antibodies from this monoclonal antibody. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 E6-derived scFv antibodies are able to recognize TDP-43. ( A ) Binding of p65 to TDP-43 was measured in the presence of PBS or anti–TDP-43 N-terminal antibody (Proteintech), monoclonal antibodies against RRM1 TDP-43 (named C10, G8, and E6), or BSA. n = 8 wells per condition (dots).

Techniques: Injection

Journal: The Journal of Clinical Investigation

Article Title: Virus-mediated delivery of antibody targeting TAR DNA-binding protein-43 mitigates associated neuropathology

doi: 10.1172/JCI123931

Figure Lengend Snippet: (A) Grip strength analysis showing the percentage of maximum performance of each single mouse and the number of individual mice (dots) analyzed. Two-way ANOVA corrected for repeated-measures interaction P = 0.823, time P = 0.0512, scFv P < 0.0001; *P < 0.05 versus control scFv, by uncorrected Fisher’s LSD. (B) Percentage of total NMJs. n = 3 individual mice (dots). Two-way ANOVA P = 0.0063; *P < 0.05 versus control scFv, by uncorrected Fisher’s LSD test. (C) Representative image and quantification of hTDP-43 (green) in motor neurons (CHAT, red) of lumbar spinal cord. The cytoplasmic to nuclear ratio of the TDP-43 integrated density of each cell was plotted according to the area of the cell. Data represent single-cell ratios (dots) analyzed from 4 individual mice. Solid line indicates the linear regression and dotted lines the 95% CIs. Slope diversity P = 0.0033. Scale bars: 10 μm. Representative blots and quantification of nuclear and cytoplasmic (D) and insoluble (E) pan–TDP-43 in lumbar spinal cord from 3 individual mice (dots). TDP-43 was normalized to p84 or GAPDH fraction markers (D) or to Ponceau (E), and values are expressed as the fold change versus control scFv. (B–E) Statistical significance was determined by unpaired t test. Data represent the mean ± SEM. CTR, 8H11 scFv.

Article Snippet: Therefore, we derived scFv antibodies from this monoclonal antibody. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1 caption a7 caption a8 E6-derived scFv antibodies are able to recognize TDP-43. ( A ) Binding of p65 to TDP-43 was measured in the presence of PBS or anti–TDP-43 N-terminal antibody (Proteintech), monoclonal antibodies against RRM1 TDP-43 (named C10, G8, and E6), or BSA. n = 8 wells per condition (dots).

Techniques: