Journal: NMR in biomedicine

Article Title: Longitudinal manganese-enhanced magnetic resonance imaging of neural projections and activity

doi: 10.1002/nbm.4675

Figure Lengend Snippet: Longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) experimental design for the effects of acute threat over time. The top panel shows a timeline diagram of the longitudinal procedure. The procedure lasts about 30 days. Pairs of behavioral video recordings (green boxes) and MR images (blue boxes) are obtained across the timeline. At day 0, a baseline behavior and a pre-Mn(II) injection image are captured. Systemic Mn(II) is delivered (0.3 mmol/kg in buffered saline) by IP injection. On day 1, 24 h after injection, another behavior-MRI pair is captured, the mouse is awakened from the imaging anesthesia and returned to the arena, where it is first exposed to neutral then to predator odor, a naturalistic threat. At day 8, another behavior-MRI pair are captured, Mn(II) is reinjected, and a final image is obtained 24 h later, on day 9. Thus five images are obtained across a 10-day period. Different genotypes can be compared, adding an additional condition. Adapted from Uselman et al.18 The bottom panel depicts the complete strategy: paired video recording of behavior and MR scans are captured. After data collection, video recordings of behavior are analyzed with Noldus Ethovision software, and MR images are processed by skull-stripping, spatial alignment, and intensity normalization. Statistical parametric mapping with pairwise t-tests comparing each time point with preinjection images yields unbiased brain-wide information about activity-dependent Mn(II) accumulations. Differing volumes of segmental activity can be obtained using automated application of a digital atlas based on Allen Brain Atlas anatomy and identifies specific segments whose volume of statistically significant Mn(II) enhancement changes between time points. This analysis allows brain-wide data-driven unbiased quantitative comparisons of segmental activity between time points, here displayed as a column graph (lower right panel). Segments are gouped according to larger regions, indicated from left to right by alternating gray-white backgrounds and ordered from anterior to posterior. For more details, please see the original publication, Uselman et al.18

Article Snippet: 249 , 255 , 289 , 322 - 329 An atlas based on a high-resolution MEMRI image (56-μm isotropic voxels), handdrawn and annotated with reference to the histological atlas from the Allen Institute for Brain Science, Mouse Brain Reference Atlas, 330 is optimal for alignment with MEMRI data.

Techniques: Magnetic Resonance Imaging, Injection, Saline, Imaging, Software, Stripping Membranes, Activity Assay

Journal: PLoS ONE

Article Title: IL-12p40 Deficiency Leads to Uncontrolled Trypanosoma cruzi Dissemination in the Spinal Cord Resulting in Neuronal Death and Motor Dysfunction

doi: 10.1371/journal.pone.0049022

Figure Lengend Snippet: A) After WT and IL-12p40KO mouse transcardiac perfusion, spinal cord upper lumbar intumescences were submitted to histopathological analysis. While WT spinal cord displayed preserved morphology (left picture), with few inflammatory cells (inset, arrow), IL-12p40KO spinal cord presented intense morphological disarrangement (right picture), due to the large amount of inflammatory foci (inset, arrows). Representative pictures of HE-stained spinal cord sections of WT and IL-12p40KO mice in the sixth week after infection. Scale bars: 100 µm; insets, 20 µm. B) Once a week during seven weeks, cDNA was obtained from the spinal cord lower lumbar segments of WT and IL-12p40KO mice and submitted to real time RT-PCR procedures, using primers for CD3, TNF-α, IFN-γ, iNOS, IL-10, arginase I and HPRT, β-actin and GAPDH as endogenous control. After the fourth week of infection, the transcription ratio of all genes analyzed tended to remission in the WT tissue, while they continued increasing in the IL-12p40KO spinal cord. Of note, also around the fourth week of infection, a switch in the transcription level of the analyzed genes was observed between the two mouse strains, when IL-12p40KO mice started expressing more RNA than WT ones. Mean ± SEM. **p<0.01; ***p<0.001 when comparing paired WT and IL-12p40KO groups according to Bonferroni post-tests. n = 4 for each strain in each time point.

Article Snippet: All samples were analyzed in duplicate and relative mRNA levels were obtained by normalizing the target gene to an endogenous reference (Actb (Mouse ACTB Endogenous Control, ABI) and Gapdh (Mouse GAPDH Endogenous Control, ABI) were used in multiplex PCR, the former with iNOS and arginase I and the latter with IL-10, or HPRT for the others) and then relative to a calibrator sample (uninfected mice), by applying the 2 −ΔΔCt formula, being mRNA levels expressed as n -fold difference relative to the calibrator.

Techniques: Staining, Infection, Quantitative RT-PCR, Control, Expressing

Journal: PLoS ONE

Article Title: IL-12p40 Deficiency Leads to Uncontrolled Trypanosoma cruzi Dissemination in the Spinal Cord Resulting in Neuronal Death and Motor Dysfunction

doi: 10.1371/journal.pone.0049022

Figure Lengend Snippet: After transcardiac perfusion, spinal cord lower lumbar segments were processed for RNA extraction and cDNA production. Real time RT-PCR was performed using primers for T. cruzi 18S rRNA and HPRT as endogenous control. For the absolute quantification, sample values were fitted into a standard curve, constructed from a pattern sample with known number of copies of T. cruzi 18S rRNA. IL-12p40KO mice presented an ascending parasitism while WT ones managed to constrain the infection by the third week, thus decreasing the parasitic load after the fifth week. Representative pictures of HE-stained upper lumbar spinal cord sections, obtained from WT (upper square) and IL-12p40KO mice (lower square) in the sixth week after the infection. Note the high amount of amastigote nests (arrows) in the IL-12p40KO tissue. Scale bars: 20 µm. Mean ± SEM. ***p<0.001 when comparing paired WT and IL-12p40KO groups according to Bonferroni post-tests. n = 4 for each strain in each time point.

Article Snippet: All samples were analyzed in duplicate and relative mRNA levels were obtained by normalizing the target gene to an endogenous reference (Actb (Mouse ACTB Endogenous Control, ABI) and Gapdh (Mouse GAPDH Endogenous Control, ABI) were used in multiplex PCR, the former with iNOS and arginase I and the latter with IL-10, or HPRT for the others) and then relative to a calibrator sample (uninfected mice), by applying the 2 −ΔΔCt formula, being mRNA levels expressed as n -fold difference relative to the calibrator.

Techniques: RNA Extraction, Quantitative RT-PCR, Control, Quantitative Proteomics, Construct, Infection, Staining

Journal: bioRxiv

Article Title: The protein phosphatases MoPtc1 and MoPtc2 are induced during pathogen-host interactions and play synergistic roles in regulating MAPK pathways in Magnaporthe oryzae

doi: 10.1101/2022.09.09.507255

Figure Lengend Snippet: MoPtc1 and MoPtc3 regulate MAPK signaling cascades in M. oryzae . (A) Immunoblot assay showing the phosphorylation level of Mps1 in Guy11, Δ Moptc1 , Δ Moptc3 and Δ Moptc1 Δ Moptc3 strains. The strains were shaken in complete media (CM) and protein extracted for western blot analyses. Equal amounts of protein were loaded into each well and the phosphorylation level was detected using phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E) rabbit mAb and p44/42 MAPK (Erk1/2) antibodies respectively. (B) The band intensity ratio for Phosphorylated Mps1 (P-Mps1) compared with the control (Mps1). These ratios were calculated by dividing p-Mps1/Mps1. (C) Western blot assay showing the Pmk1 phosphorylation level in Guy11, Δ Moptc1, and Δ Moptc3 and Δ Moptc1 Δ Moptc3 strains. The strains were shaken in complete media (CM) and protein extracted for western blot analyses. Equal amounts of protein were loaded into each well and the phosphorylation level was detected using phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E) rabbit mAb and p44/42 MAPK (Erk1/2) antibodies respectively. (D) The band intensity ratio for Phosphorylated Pmk1 (P-Pmk1) compared with the control (Pmk1). These ratios were calculated by dividing p-Pmk1/Pmk1. (E) Western blot image quantifying the Osm1-MAPK phosphorylation in Guy11 and Δ MoPtc1 , Δ MoPtc3 Δ MoPtc1 Δ MoPtc3 strains. The strains were shaken in complete media (CM) and protein extracted for western blot analyses. The phosphorylation level of MoOsm1 in all strains was detected using phosphop38 MAPK (Thr180/Tyr182) (D3F9) XP rabbit mAb while β-actin was detected using actin (2P2) mouse mAb. Equal amounts of protein were loaded into each well. (F) The band intensity ratio between phosphorylated Osm1 (P-Osm1) and β-actin. The single asterisk (*) represent statistical significance with adjusted P Value of 0.0338.

Article Snippet: The protein samples were also detected with actin (2P2) mouse mAb as reference (Abmart, Shanghai, China).

Techniques: Western Blot, Phospho-proteomics, Control