Journal: Infection and Immunity

Article Title: Broadly Reactive Human Monoclonal Antibodies Targeting the Pneumococcal Histidine Triad Protein Protect against Fatal Pneumococcal Infection

doi: 10.1128/IAI.00747-20

Figure Lengend Snippet: Protective efficacy of anti-PhtD MAbs. (A) ELISA binding curve of MAb PhtD3, the isotype-switched MAb PhtD-IgG2a, and an IgG2a isotype control. Data points are averages of four replicates from one of at least two independent experiments. Error bars indicate 95% confidence intervals. (B) Prophylactic efficacy of MAb PhtD3 in an intranasal infection model of pneumococcal serotype 3 (strain WU2) in C57BL/6 mice. **, P = 0.0012; ns, not significant via log-rank (Mantel-Cox) test. n = 10 mice/group. (C) Prophylactic efficacy of MAb PhtD8 in an intranasal infection model of pneumococcal serotype 3 (strain WU2) in C57BL/6 mice. ***, P = 0.0009; ns, not significant via log-rank (Mantel-Cox) test. n = 10 mice/group. (C) Prophylactic efficacy of MAb PhtD3 in an intranasal infection model of pneumococcal serotype 4 (strain TIGR4) in CBA/N mice. **, P = 0.0045 via log-rank (Mantel-Cox) test. n = 15 mice/group. (D) Prophylactic efficacy of MAb PhtD3 in an intravenous infection model of pneumococcal serotype 4 (strain TIGR4) in C57BL/6 mice. * * , P = 0.0101 via log-rank (Mantel-Cox) test. n = 13 to 15 mice/group. (E) Treatment efficacy of MAb PhtD3 in an intranasal infection model of pneumococcal serotype 3 (strain WU2) in C57BL/6 mice. ** * , P = 0.0002; ns, not significant via log-rank (Mantel-Cox) test. n = 20 mice/group.

Article Snippet: Isotype-specific antibodies obtained from Southern Biotech (goat anti-human kappa–alkaline phosphatase [AP] [catalog number 100244-340], goat anti-human lambda–AP [catalog number 100244-376], mouse anti-human IgG1 [Fc]–AP [catalog number 100245714], mouse anti-human IgG2 [Fc]–AP [catalog number 100245-734], mouse anti-human IgG3 [hinge]–AP [catalog number 100245-824], and mouse anti-human IgG4 [Fc]–AP [catalog number 100245-812]) were diluted 1:1,000 in blocking buffer, and 50 μl of each solution was added to the respective wells.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Control, Infection

Journal: Cell and Tissue Research

Article Title: Histological study on the postnatal development of the nerve network in the rat ileal mucosa and submucosa

doi: 10.1007/s00441-025-03949-3

Figure Lengend Snippet: Postnatal change in the expressions of Sox10, GFAP and S100β in the submucosa of the rat ileum. a–c Immunofluorescence against Sox10 (red)/α-SMA (green, marker for smooth muscle cells). Oval nuclei immunopositive for Sox10 (white arrowheads) are observed in the ileal submucosa of a postnatal day 0 (P0) rat ( a ), a 2-week-old (2wk) rat ( b ), and a 4-week-old (4wk) rat ( c ). d Whole view of a transverse section with immunofluorescence against Sox10 (red)/α-SMA (green) from a 2wk rat. Arrowheads, Sox10 + cells in the submucosa. Red asterisk, mesentery. e The number of Sox10 + cells in the submucosa per a transverse section on the mesenteric (M) or antimesenteric (AM) side of the rat ileum. Results are shown as the means ± SD. Comparisons were performed by a paired-sample t -test. f–h Immunohistochemistry for GFAP in the rat ileal submucosa at P0 ( f ), 2wk ( g ), and 4wk ( h ). Immunopositivity for GFAP in the submucosa is not observed at P0 ( f ) or 2wk ( g ) but is weakly observed at 4wk ( h , arrowhead). i–k Immunohistochemistry for S100β in the rat ileal submucosa at P0 ( i ), 2wk ( j ), and 4wk ( k ). Immunopositivity for S100β in the ileal submucosa is observed at all stages, although it is weak at P0 and 2wk. Arrowheads, S100β + cells. Bars = 10 µm ( a–c, f–k ) or 100 µm ( d ). l The area immunopositive for S100β (S100β-IP) per a transverse section on the M or AM side of the rat ileum. Each median value is represented by a red horizontal bar. Comparisons were performed by a Wilcoxon signed-rank test. The numbers in parenthesis are the sample sizes ( e , l )

Article Snippet: The sections to be used for immunofluorescence analysis were reacted for 18 h at 6 °C with either of two pairs of antibodies: anti-mouse HuD rabbit polyclonal IgG (diluted 1:3,200, Cat# GTX134099, RRID: AB_2887214; GeneTex, Irvine, CA, USA)/anti-rat Tuj1 mouse monoclonal IgG (diluted 1:6,400, Cat# MAB1195, RRID: AB_357520; R&D Systems, Minneapolis, MN, USA) or anti-human Sox10 rabbit monoclonal IgG (diluted 1:400, Cat# NBP2-67,812; RRID: AB_3086734; Novus Biologicals)/anti-human α-smooth muscle actin (α-SMA) mouse monoclonal IgG (diluted 1:400, Cat# ab7817, RRID: AB_262054; Abcam).

Techniques: Immunofluorescence, Marker, Immunohistochemistry

Journal: Insects

Article Title: Suitability of Raycell MK2 Blood X-ray Irradiator for the Use in the Sterile Insect Technique: Dose Response in Fruit Flies, Tsetse Flies and Mosquitoes

doi: 10.3390/insects14010092

Figure Lengend Snippet: Dose response data of Ae. aegypti male pupae and adults irradiated with MK2 (X-ray) irradiator compared to the same strain irradiated in a GC220 instrument (gamma ray, (Yamada et al., 2022 )).

Article Snippet: Only the dose of 80 Gy that is used in operational programs for the induction of reproductive sterility in this species was assessed for both the gamma ray (Foss Model 812) and X-ray (MK2) treatments.

Techniques: Irradiation

Journal: Insects

Article Title: Suitability of Raycell MK2 Blood X-ray Irradiator for the Use in the Sterile Insect Technique: Dose Response in Fruit Flies, Tsetse Flies and Mosquitoes

doi: 10.3390/insects14010092

Figure Lengend Snippet: Dose response data of An. arabiensis male pupae and adults irradiated with MK2 (X-ray) irradiator compared to the same strain irradiated in GC220 irradiator (gamma ray).

Article Snippet: Only the dose of 80 Gy that is used in operational programs for the induction of reproductive sterility in this species was assessed for both the gamma ray (Foss Model 812) and X-ray (MK2) treatments.

Techniques: Irradiation

Journal: bioRxiv

Article Title: Tau accelerates tubulin exchange in the microtubule lattice

doi: 10.1101/2024.10.05.616777

Figure Lengend Snippet: a. Experimental setup to assess tubulin incorporation into the microtubule lattice in the presence of tau. First, dynamic microtubules were grown from GMPCPP-stabilized, surface-attached seeds in the presence of green-labeled GTP-tubulin (step I). Then, microtubules were capped with GMPCPP-tubulin to prevent further microtubule tip dynamics (step II). The microtubules were then incubated with 0 nM, 0.5 nM, or 20 nM unlabeled tau (black-white circles) and red-labeled GTP-tubulin for 15 min or 30 min (step III). The solution was replaced with buffer supplemented with taxol (step IV) to remove excessive background fluorescence, reveal tubulin incorporation sites (yellow star), and keep microtubules stable for imaging. b. 20 nM fluorescently labeled tau (magenta) homogeneously coat the microtubule lattice (green) before capping, with the exception of the GMPCPP-stabilized seed. Scale bar: 3 µm. c. Example images of microtubules (green) showing incorporation stretches after 15 min (red, stars) in the presence of 0 nM, 0.5 nM and 20 nM tau. The graphs show profile plots of the red channel along microtubules. Scale bars: 5 µm. d. Top left: The lengths of tubulin incorporation stretches increase in the presence of tau (> 2,200 µm microtubule length analyzed per condition; N=3 experiments per condition). Top right: The distances between incorporation stretches decrease in the presence of tau. Broken lines represent the median. Bottom: Tubulin incorporation is observed along 4.86% of the microtubule lattice at 0 nM tau, 11.38% at 0.5 nM tau and 19.14% at 20 nM tau. e. Example images with profile plots showing tubulin incorporation after 30 min in red (stars) and the lattice in green. Scale bars: 5 µm. f. Incorporation length (left) and distance between incorporation stretches (center) after 15 min vs. 30 min of incubation with red tubulin, for 0 nM and 20 nM tau. Broken lines represent the median. > 2,200 µm of microtubule length was analyzed in three independent experiments per condition. Tubulin incorporation was observed along 18.13% of the microtubule lattice for 0 nM tau and 30.59% for 20 nM tau (right).

Article Snippet: GMPcPP caps were grown by substituting in the before-mentioned buffer GTP with 0.5 mM GMPcPP (Jena Bioscience) and using 3 μM tubulin (100% labeled with ATTO-488) at 37°C.

Techniques: Labeling, Incubation, Fluorescence, Imaging

Journal: bioRxiv

Article Title: Tau accelerates tubulin exchange in the microtubule lattice

doi: 10.1101/2024.10.05.616777

Figure Lengend Snippet: a. Experimental setup to confirm that tau reduces tubulin loss in the absence of free tubulin. Microtubules were grown with green labeled tubulin (step I) and capped (step II). They were then incubated with or without 20 nM tau in the absence of free tubulin for 5 min (step III), followed by a 15 min incubation step with red-labeled tubulin in the absence of tau (step IV). Microtubules were imaged after washout of free tubulin as before (step V). b. Example images showing tubulin incorporation (red, stars) into microtubules (green) after a 5 min incubation step without free tubulin. The profile plots show the fluorescence intensity along the microtubules in the red channel. Scale bars: 5 µm. c. Tubulin incorporation sites are shorter (top left) and more spaced (top right) in the presence of 20 nM tau compared to the control, leading to overall less tubulin incorporation (bottom). More than 1,700 µm of microtubule length was analyzed in three independent experiments per condition. d. Experimental setup to quantify microtubule fracture in the absence of free tubulin. Microtubules were grown with GTP-tubulin (step I) and capped with GMPCPP-tubulin (step II). Then, they were incubated with or without 20 nM tau in the absence of free tubulin (step III) and immediately imaged for 50 min (step IV). e. Image sequence showing a microtubule developing a damaged region, which is visible due to reduced green fluorescence (arrow) resulting from loss of tubulin from the lattice. The microtubule eventually broke along the softened region and disassembled. Scale bar: 3 µm. f. Survival curves showing that microtubule survival increases in the presence of tau. For 0 nM tau, 83 microtubules were analyzed in five independent experiments. For 20 nM tau, 65 microtubules were analyzed in four independent experiments. The differences in survival are statistically significant (p < 0.0001), based on a log-rank (Mantel-Cox) test. g. Lengths of the damaged lattice regions prior to fracture showing reduced fluorescence increased in the presence of tau. 19 and 22 fracture events were analyzed for 0 nM and 20 nM tau from five and four independent experiments, respectively.

Article Snippet: GMPcPP caps were grown by substituting in the before-mentioned buffer GTP with 0.5 mM GMPcPP (Jena Bioscience) and using 3 μM tubulin (100% labeled with ATTO-488) at 37°C.

Techniques: Labeling, Incubation, Fluorescence, Control, Sequencing

Journal: bioRxiv

Article Title: Tau accelerates tubulin exchange in the microtubule lattice

doi: 10.1101/2024.10.05.616777

Figure Lengend Snippet: a. Tau stabilizes longitudinal dimer–dimer contacts. Free energy profiles for tubulin dissociation in the presence (dark blue curve) and absence (light blue curve) of tau (magenta) as a function of the center-of-mass distance between adjacent monomers. In the presence of tau, the binding affinity along longitudinal dissociation of tubulin dimer and monomer is stronger than in the absence of tau. Shaded areas represent the standard error of the mean. The inset shows the structure of three tubulin monomers (green, top view) with a single monomer depicted in both the associated and dissociated states from the microtubule-tau complex. b. Schematic representation of the kinetic Monte Carlo model for the microtubule lattice. The microtubule is modeled as the canonical 13 protofilament three-start helix structure on the scale of the monomer, such that the dynamics of monomer vacancies (labeled V 1 , V 2+ , and V 2- ) can be investigated. V 2+ and V 2- refer to the orientation of the newly created seams towards the microtubule plus-end and minus-end, respectively. Shown are also the three possible kinetic transitions with their respective rate constants (k on : GTP-dimer attachment, k off GTP or GDP dimer detachment, k hy GTP dimer hydrolysis) and the relevant energetic contributions which govern the kinetics of dimer detachment within Kramer’s theory. c. Simulation of microtubule fracture of end-stabilized microtubules. Kymographs of a representative lattice configuration with one V 1 - and one V 2+ -type defect marked by → and ↑ arrows, respectively, along a 10 µm long microtubule. Scale bars: 2 µm vertical, 1 min horizontal. The color code indicates the number of intact protofilaments. Parameters are ΔG S =0.5 kT, and ΔG tot =-36 kT, A=1.5 (control) and ΔG tot =-36.2 kT, A=2.1 (tau) and as indicated in Table S2. d. Left: Simulated survival curves of end-stabilized microtubules in the absence of free tubulin, in the presence (dark blue) and absence (light blue) of tau. The curves show the survival of 1000 microtubules with randomly placed defects (frequency 0.15 𝜇m -1 ). Right: Simulated longitudinal damage size, corresponding to the loss of all 13 protofilaments. Dark blue denotes the simulations with tau, light blue the control case without tau. e. Schematic representation of defect motion via dimer detachment and attachment. f. Simulation of dimer incorporation dynamics into end-stabilized microtubules. Kymographs of a representative lattice configuration with V 1 - as well as V 2+ - and V 2- -type defects marked by →, ↑ and ↓ arrows, respectively, along a 10 µm long microtubule. Scale bars: 2 µm vertical, 1 min horizontal. The color code indicates the number of exchanged protofilaments. Parameters are ΔG S =0.5 kT, and ΔG tot =-36 kT, A=1.5 (control) and ΔG tot =-36.2 kT, A=2.1 (tau) with ΔG V =1.25 kT and as indicated in Table S2. To compare the simulated results with experiments, the longitudinal positions of incorporated dimers were convoluted with a point spread function as described in the SI. g. Length and frequency of incorporation stretches. Left: Incorporation length depending on the defect type (V 1 , V 2+ , V 2- ) in the presence of tau (dark blue) compared to the control (light blue) after 15 min. Right top: Simulated distribution of (visible) incorporation sizes after 15 min in the presence (dark blue) and absence (light blue) of tau for 10 µm long microtubule with randomly placed defects (frequency 0.15 𝜇m -1 ) showing a bimodal distribution in the presence of tau. The frequency of visible incorporations are 0.056 𝜇m -1 (control) and 0.076 𝜇m -1 (with tau). To compare the simulated results with experiments, the longitudinal positions of incorporated dimers were convoluted with a point spread function as described in the SI. Right bottom: Experimental distribution of visible incorporation sizes. The data correspond to the data shown in . h. Exemplary representation of the elimination of two V 1 lattice defects via annealing (top) and a V 2+ defect via motion up to the microtubule extremity (bottom). i. Experimental setup to visualize tubulin incorporation after an initial 30 min tubulin incorporation step in the presence or absence of tau. Microtubules were grown with green-labeled tubulin (step I) and capped (step II) before incubation with unlabeled tubulin in the presence or absence of 20 nM tau for 30 min (step III). This initial incorporation step was followed by an additional 15 min incorporation step in the presence of red-labeled tubulin and in the absence of tau (step IV) before washout and imaging (step V). j. Incorporation lengths (left) and distances between incorporation stretches (right). Broken lines represent the median. Orange lines represent the median values from (incorporation length and distance between incorporations for 0 nM tau) as reference, respectively. The resulting tubulin incorporation lengths are comparable between the samples, but the control sample shows slightly more frequent tubulin incorporations (> 3,000 µm of microtubule length was analyzed from three independent experiments per condition). k. Distances between tubulin incorporation stretches from the graph on the left were binned according to microtubule length, showing a marked increase for short (0-10 µm) microtubules in the presence of tau. Black lines represent the median. The orange line represents the median from (distance between incorporation stretches for 0 nM tau) as reference.

Article Snippet: GMPcPP caps were grown by substituting in the before-mentioned buffer GTP with 0.5 mM GMPcPP (Jena Bioscience) and using 3 μM tubulin (100% labeled with ATTO-488) at 37°C.

Techniques: Binding Assay, Labeling, Control, Incubation, Imaging

Journal: Insects

Article Title: Suitability of Raycell MK2 Blood X-ray Irradiator for the Use in the Sterile Insect Technique: Dose Response in Fruit Flies, Tsetse Flies and Mosquitoes

doi: 10.3390/insects14010092

Figure Lengend Snippet: Dose response data of Ae. aegypti male pupae and adults irradiated with MK2 (X-ray) irradiator compared to the same strain irradiated in a GC220 instrument (gamma ray, (Yamada et al., 2022 )).

Article Snippet: Anastrepha ludens irradiated as pupae in hypoxic conditions with 80 Gy with both X-rays (MK2) and gamma rays (FOSS 812) resulted in 100% sterility.

Techniques: Irradiation

Journal: Insects

Article Title: Suitability of Raycell MK2 Blood X-ray Irradiator for the Use in the Sterile Insect Technique: Dose Response in Fruit Flies, Tsetse Flies and Mosquitoes

doi: 10.3390/insects14010092

Figure Lengend Snippet: Dose response data of An. arabiensis male pupae and adults irradiated with MK2 (X-ray) irradiator compared to the same strain irradiated in GC220 irradiator (gamma ray).

Article Snippet: Anastrepha ludens irradiated as pupae in hypoxic conditions with 80 Gy with both X-rays (MK2) and gamma rays (FOSS 812) resulted in 100% sterility.

Techniques: Irradiation