Journal: Journal of Bacteriology

Article Title: PPAD Activity Promotes Outer Membrane Vesicle Biogenesis and Surface Translocation by Porphyromonas gingivalis

doi: 10.1128/JB.00343-20

Figure Lengend Snippet: Time-lapse microscopy of surface translocation by P. gingivalis strain 381 and the Δ ppad mutant. (a) Schematic illustration representing sequential stages of surface translocation by reference strain 381 as recorded for more than 160 h of incubation using the chamber slide system. (b) Frames 1a to e show the chronology of surface translocation by reference strain 381, including the hydrating stage and the formation of moving pseudofilaments, outward spreading of the colony, and subdiffusive cell-driven motility by individual cells that progresses to surface translocation and the formation of distal biofilms over time. Frames 2a to f represent the recordings of disordered stages of surface translocation by strain 381Δ ppad . The absence of ppad resulted in the formation of a limited hydration zone but very long pseudofilaments whose aggregation resulted in the observation of scattered biofilms and the extension of the time required to see outward spreading of the cells and subdiffusive cell-driven motility. Scale bars: 10 μm.

Article Snippet: To determine if PPAD activity alters gene expression, we performed transcriptomic analysis on RNA extracted from cells of strains 381 and 381Δ ppad grown as colony biofilms on BAPHK (Northeast Laboratory Services) for 24 h (the same growth conditions as used for cryo-SEM imaging).

Techniques: Time-lapse Microscopy, Translocation Assay, Mutagenesis, Incubation

Journal: Journal of Bacteriology

Article Title: PPAD Activity Promotes Outer Membrane Vesicle Biogenesis and Surface Translocation by Porphyromonas gingivalis

doi: 10.1128/JB.00343-20

Figure Lengend Snippet: Deletion of ppad inhibits OMV biogenesis and release. (a) NanoSight analysis of crude OMV preparations from 381 and 381Δ ppad planktonic cultures. The 381Δ ppad mutant produced OMVs that were fewer and smaller in size than those produced by strain 381. (b) Cryo-SEM imaging of colony biofilms showed that the 381 ppad mutant produced few to no OMVs within the biofilm matrix compared to the parent strain. (Inlay) Magnified section of micrograph. Scale bar: 2 μm. (c) Formation of biopearling assemblies by strain 381. SEM imaging of fixed content of the hydration zone showed that strain 381 formed biopearling assemblies (yellow arrows) via connecting membrane protrusions and large-size OMVs (red arrows) under surface translocation conditions. Deletion of ppad resulted in production of few small-sized OMVs (pink arrows) and in inhibition of biogenesis of large-size OMVs and biopearling assemblies. (Inlay) Magnified section of micrograph. Scale bar: 3 μm.

Article Snippet: To determine if PPAD activity alters gene expression, we performed transcriptomic analysis on RNA extracted from cells of strains 381 and 381Δ ppad grown as colony biofilms on BAPHK (Northeast Laboratory Services) for 24 h (the same growth conditions as used for cryo-SEM imaging).

Techniques: Mutagenesis, Produced, Imaging, Membrane, Translocation Assay, Inhibition

Journal: Nature Communications

Article Title: Efferent feedback controls bilateral auditory spontaneous activity

doi: 10.1038/s41467-021-22796-8

Figure Lengend Snippet: a Experimental setup for wide-field calcium imaging showing a typical field of view in mice. Superior colliculi (SC), inferior colliculi (IC), and part of the cerebellum (Cb) are delineated in dashed orange, magenta, yellow lines, respectively. All movies are acquired at 10 Hz. b Example Δ F / F 0 images showing spontaneous events at different postnatal ages. Superior colliculi (SC), inferior colliculi (IC) are delineated in dashed orange and magenta lines, respectively. White arrowheads: spontaneous bands in the IC. White arrows: spontaneous (retinal) waves in the SC. Colormap: parula (MATLAB). c Example montages (Δ F / F 0 ) of spontaneous activity bands in the IC. Two magenta arrows represent major axes of the intensity profile in the left and right hemispheres. d Average peak amplitude of spontaneous activity bands across age groups. Defined as the mean Δ F / F 0 amplitude at peak. e Event frequency across age groups. Quantified as the number of individual peaks (bands) per minute as identified in the line-scan analysis. f Average normalized bandwidth across age groups. Defined as the mean spatial half-width of the fluorescence peaks normalized by the width of the IC. g Schematics for analysis of global bilateral correlations. The left hemisphere and its corresponding mean fluorescent trace are colored in blue. The right hemisphere and its mean fluorescent trace are colored in orange. A partial correlation between the two traces was computed while regressing out the effect of the mean activity overall pixels outside the IC ( r = 0.52139). h Example seed-based correlation maps at P6. Dashed magenta lines delineate symmetrical regions of interest (ROIs) in the contralateral hemisphere with respect to the reference seeds. Magenta dots denote maximum correlation in the ROIs (max r ). White dots in the left hemisphere indicates seed locations. i Example seed-based correlation maps at P12. j Average global bilateral correlation across age groups (P0–P12). k Summary quantification of seed-based bilateral correlation grouped by future frequency regions. “Low”, “mid”, and “high” corresponds to maps where reference seeds located in the putative low-, mid-, and high-frequency regions. “Averaged” correlation is defined as the mean correlation averaged over the three regions. Postnatal ages are shown on the x- axis. Box plots in Fig. 1: hinges: 25 percentile (top), 75 percentile (bottom). Box whiskers (bars): Max value (top), Min value (bottom). The line in the middle of the box is plotted at the median. Significance marks: n.s. p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, **** p < 0.0001, two-tailed unpaired t test with Welch’s correction. A number of animals: P0–P1 ( N = 8); P3–P4 ( N = 11); P6–P7 ( N = 10); P9–P10 ( N = 9); P11–P12 ( N = 11); P13 ( N = 9). Scale bar indicates 1 mm. Source data and exact p values are provided as a Source Data file.

Article Snippet: Acoustic stimuli were generated with customized open-source software in MATLAB (“Baphy”, Neural Systems Laboratory, University of Maryland College Park).

Techniques: Imaging, Activity Assay, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Efferent feedback controls bilateral auditory spontaneous activity

doi: 10.1038/s41467-021-22796-8

Figure Lengend Snippet: a Snapshots of example auditory brainstem response (ABR) curves. White arrowheads indicate ABR waves. Scale bar indicates 2 µV. The White dashed line represents the detection threshold. b Auditory thresholds measured by auditory brainstem response (ABR) in control and α9/α10 nAChR knockout animals at P14.0. A number of animals: Control P14.0 = 9 (GCaMP6s negative littermates of animals used in ( e )). α9/α10 knockout P14.0 = 6. Magenta arrowheads indicate that part of the data is not available in the knockout group (two animals did not respond to 4 kHz tones.). n.s., two-way ANOVA, F = 1.409, p = 0.0604 on the column (genotype) factor. c Auditory thresholds measured by wide-field imaging. A number of animals: Control = 8 (wide-type SNAP25-G6s = 6; α9/α10 double heterozygous SNAP25-G6s = 2). Knockout = 8 (mix of single knockout of either α9 or α10 subunit and double knockout). Two-way ANOVA, F = 31.98, **** p < 0.0001 on the column (genotype) factor. d Example Δ F / F 0 response images to different acoustic stimuli. Row direction: Sound level. Column direction: frequency. Solid magenta lines denote the auditory thresholds at different frequencies. Dashed red rectangles highlight columns of responses to different frequencies at the same SPL level (tonotopy). Het heterozygous, KO knockout. Colormap: parula (MATLAB). Top image array: an example from a P14.0 α9/α10 double heterozygous control. Bottom image array: an example from its P14.0 α9 knockout α10 heterozygous littermate. Schematics in the middle: major axes and tonotopic reversals in the two hemispheres of the IC. Box plots in Fig. 5: hinges: 25 percentile (top), 75 percentile (bottom). Box whiskers (bars): Max value (top), Min value (bottom). Source data and exact p values are provided as a Source Data file.

Article Snippet: Acoustic stimuli were generated with customized open-source software in MATLAB (“Baphy”, Neural Systems Laboratory, University of Maryland College Park).

Techniques: Control, Knock-Out, Imaging, Double Knockout