Journal: PLoS ONE

Article Title: Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy

doi: 10.1371/journal.pone.0127174

Figure Lengend Snippet: Target organs or systems for NMs in the present study: immune system (HMDM, RAW264.7 and MH-S), respiratory system (Calu-3, 16HBE and RLE-6TN), male reproductive system (TM3 and TM4), gastrointestinal system (Caco-2), kidneys (NRK-52E) and embryo (NIH-3T3 and mES/D3).

Article Snippet: Mouse NIH/3T3 cells and D3 mouse Embryonic Stem cells (mES) were purchased from ATCC (Manassas, VA, USA).

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Molecular modeling-based predictions of protein stability and surface charge distribution in SPN-ARR domains of the Shank3 protein harboring Q321R and other ASD-risk mutations. (A) Structure of the SPN and ARR domains of the Shank3 protein (PDB ID: 5G4X), with the locations of amino acid residues mutated in autistic individuals (R12C, L68P, A198G, R300C, and Q321R) indicated. The SPN domain, the linker connecting the SPN and ARR domains, and the ARR domain of Shank3 are indicated in gray, green, and orange colors, respectively, as a ribbon diagram. The ASD-risk residues are indicated by ball-and-stick models. (B) Stability predictions of SPN and ARR domains of the Shank3 protein containing the indicated ASD-risk mutations, obtained using I-Mutant 2.0 software. A negative predicted free energy change (ΔΔG, in kcal/mol) indicates a decrease in the stability of the mutant protein. (C) Electrostatic surface charge distribution patterns in WT and mutant (Q321R) SPN and ARR domains of the Shank3 protein. Negative and positive surface charges are indicated in red and blue, respectively. Hydrophobic surfaces are indicated in white. Surface areas with the Shank3 Q321R mutation are indicated by dotted orange circles.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Mutagenesis, Software

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Generation and characterization of Shank3 Q321R mice. (A) A schematic diagram showing the location of the Shank3 Q321R mutation in the Shank3 protein. Ank, ankyrin repeat region; SH3, Src homology 3 domain; PDZ, PSD-95/Dlg/ZO-1 domain; Proline-rich, proline-rich region; SAM, sterile alpha motif domain. (B) A schematic diagram showing the gene-targeting strategy used to generate Shank3 Q321R mice. Note that the mutated codon corresponding to the Q321R mutation is located in exon 8 of the Shank3 gene. Primers (forward and reverse) for PCR genotyping are indicated. Neo, neomycin resistance; DTA, diphtheria toxin. (C) Confirmation of the Shank3 Q321R mutation by genomic DNA sequencing of Shank3 Q321R/Q321R mice. Results from a wild-type (WT) mouse are shown for comparison. (D) PCR genotyping of Shank3 Q321R/Q321R (HM/homozygous) and Shank3 +/Q321R (HT/heterozygous) mice. (E) Known Shank3 protein variants. The target region (aa 1431–1590) of the Shank3 antibody used in the immunoblot analysis (F) is indicated. (F) The levels of Shank3a, but not Shank3c/d or Shank3e, protein variants, are substantially decreased in whole-brain lysates from WT and Shank3 Q321R/Q321R and Shank3 +/Q321R mice (2 months; female), as determined by immunoblot analysis using Shank3-specific antibodies targeting amino acids 1431–1590 in the proline-rich region of the protein that can detect all three protein splice variants. n = 3 mice (WT, HT, and HM), ∗ P < 0.05, ∗∗ P < 0.01, ns, not significant, One sample t -test. (G) Normal gross morphology of the brains of WT and homozygous Shank3 Q321R/Q321R mice (2 months; female), as shown by immunostaining of coronal sections for the neuronal marker, NeuN. Scale bar, 1 mm.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Mutagenesis, Sterility, DNA Sequencing, Comparison, Western Blot, Immunostaining, Marker

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Suppressed neuronal excitability but normal mEPSCs and mIPSCs in hippocampal CA1 pyramidal neurons from male Shank3 Q321R/Q321R mice. (A) Normal mEPSC frequency and amplitude in CA1 pyramidal neurons in the hippocampus of male Shank3 Q321R/Q321R mice (P21–25). The indicated values represent means ± SEM. n = 14 neurons from 3 mice (WT) and 15 neurons from 3 mice (Q321R), ns, not significant, Mann–Whitney U -test (for frequency) and Student’s t -test (for amplitude). (B) Normal mIPSC frequency and amplitude in CA1 pyramidal neurons in the hippocampus of male Shank3 Q321R/Q321R mice (P21–25). n = 12 neurons from 5 mice (WT) and 13 neurons from 3 mice (Q321R), ns, not significant, Student’s t -test (for frequency) and Student’s t -test (for amplitude). (C–F) Suppressed neuronal excitability in hippocampal CA1 pyramidal neurons from male Shank3 Q321R/Q321R mice (P22–26), as shown by the number of action potential fired plotted against injected currents (F) . Note that the current-voltage relationship (C) , input resistance (D) , and Sag ratio (E) were not changed. n = 14 neurons from 5 mice (WT) and 16 neurons from 4 mice (Q321R), ∗ P < 0.05, ∗∗∗ P < 0.001, ns, not significant, repeated measures two-way ANOVA (for current-membrane potential and current-firing curves) and Mann–Whitney U -test (for input resistance and sag ratio). (G) Normal mEPSC frequency and amplitude in dorsolateral striatal neurons from male Shank3 Q321R/Q321R mice (P28–43). n = 16 neurons from 4 mice (WT) and 16 neurons from 5 mice (Q321R), ns, not significant, Mann–Whitney U -test (for frequency) and Student’s t -test (for amplitude). (H) Normal mIPSC frequency and amplitude in dorsolateral striatal neurons from male Shank3 Q321R/Q321R mice (P28–43). n = 16 neurons from 6 mice (WT) and 17 neurons from 7 mice (Q321R), ns, not significant, Student’s t -test (for frequency) and Mann–Whitney U -test (for amplitude).

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: MANN-WHITNEY, Injection, Membrane

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Normal locomotor activity and moderate anxiolytic-like behavior in heterozygous Shank3 +/Q321R mice. (A,B) Normal locomotor activity in Shank3 +/Q321R mice (2–3 months; male) in Laboras cages, where locomotor activity was measured together with other movements for four consecutive days in the absence of habituation. OFF/ON, light-off/on. n = 13 mice (WT) and 13 mice (HT), ns, not significant, two-way ANOVA (genotype main effect p -value = 0.1813 in A ), and Student’s t -test (B) . (C,D) Normal locomotor activity in Shank3 +/Q321R mice (2–3 months; male) in the open-field test, as shown by the distance moved. n = 13 mice (WT) and 13 mice (HT), ns, not significant, repeated measures two-way ANOVA (C) and Student’s t -test (D) . (E) Normal anxiety-like behavior in Shank3 +/Q321R mice (2–3 months; male) in the open-field test, as shown by the time spent in the center region of the open-field arena. n = 13 mice (WT) and 13 mice (HT), ns, not significant, Mann–Whitney U -test. (F) Normal anxiety-like behavior in Shank3 +/Q321R mice (2–3 months; male) in the elevated plus-maze test, as shown by the time spent in the open arms of the maze. n = 13 mice (WT) and 13 mice (HT), ns, not significant, Welch’s t -test. (G) Anxiolytic-like behavior in Shank3 +/Q321R mice (2–3 months; male) in the light-dark test, as shown by the time spent in the light chamber of the light-dark apparatus. n = 13 mice (WT) and 12 mice (HT), ∗∗ P < 0.01, Student’s t -test.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Activity Assay, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Normal social interaction, moderately enhanced social communication and self-grooming, and suppressed digging in heterozygous Shank3 +/Q321R mice. (A) Normal social approach in Shank3 +/Q321R mice (2–3 months; male) in the three-chamber test, as shown by time spent sniffing. S1, a stranger; O, object; S2, new stranger. Social novelty recognition, measured by the preference for new stranger (S2) over old stranger (S1), could not be determined due the lack of normal social novelty recognition in WT mice. n = 10 mice (WT), 13 mice (HT), ∗∗∗ P < 0.001, ns, not significant, Welch’s t -test, Mann–Whitney U -test, and Student’s t -test. (B) Moderately increased courtship USVs emitted by Shank3 +/Q321R mice (2–3 months; male) upon encounter with a novel female stranger, as shown by the normal number of USVs but increased mean duration of each USV calls. n = 12 mice (WT), 12 mice (HT), ∗ P < 0.05, ns, not significant, Student’s t -test. (C) Enhanced self-grooming in Shank3 +/Q321R mice (2–3 months; male) in home cages with bedding (10 min), as shown by total self-grooming time. n = 12 mice (WT), 13 mice (HT), ∗ P < 0.05, Student’s t -test. (D) Suppressed digging in Shank3 +/Q321R mice (2–3 months; male) in home cages with bedding (10 min), as shown by total digging time. n = 12 mice (WT), 13 mice (HT), ∗ P < 0.05, Welch’s t -test. (E–I) Normal self-grooming in Shank3 +/Q321R mice (2–3 months; male) in Laboras cages, as shown by total self-grooming duration. n = 13 mice (WT), 13 mice (HT), ns, not significant, two-way ANOVA (genotype main effect p -value = 0.4087 in E ), Mann-Whitney test (F,H,I) , Student’s t -test (G) .

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Normal locomotor activity and moderate anxiolytic-like behavior in homozygous Shank3 Q321R/Q321R mice. (A,B) Normal locomotor activity in Shank3 Q321R/Q321R mice (2–4 months; male), in Laboras cages, where locomotor activity was measured together with other movements for four consecutive days in the absence of habituation. Note that the WT data in this panel and other panels in this figure are identical to those shown in because WT, heterozygous, and homozygous mice were tested together in the same behavioral tests. OFF/ON, light-off/on. n = 13 mice (WT) and 14 mice (Q321R), ns, not significant, two-way ANOVA ( A , genotype main effect p -value = 0.9356) and Student’s t -test (B) . (C,D) Normal locomotor activity in Shank3 Q321R/Q321R mice (2 months; male) in the open-field test, as shown by the distance moved. Note that Shank3 Q321R mice spent a normal amount of time in the center region of the open-field arena, suggestive of normal anxiety-like behavior. n = 13 mice (WT) and 14 mice (Q321R), ∗∗∗ P < 0.001, ns, not significant, repeated measures two-way ANOVA (C) and Student’s t -test (D) . (E) Normal anxiety-like behavior in Shank3 Q321R/Q321R mice (2 months; male) in the open-field test, as shown by the time spent in the center region of the open-field arena. n = 13 mice (WT) and 14 mice (Q321R), ns, not significant, Student’s t -test. (F) Anxiolytic-like behavior in Shank3 Q321R/Q321R mice (2 months; male) in the elevated plus-maze test, as shown by the increased time spent in the open arms of the maze. n = 13 mice (WT) and 14 mice (Q321R), ∗ P < 0.05, Mann–Whitney U -test. (G) Anxiolytic-like behavior in Shank3 Q321R/Q321R mice (2 months; male) in the light-dark test, as shown by the increased time spent in the light chamber of the light-dark apparatus. n = 13 mice (WT) and 14 mice (Q321R), ∗ P < 0.05, Welch’s t -test.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Activity Assay, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Normal social interaction and communication, moderately enhanced stress-induced self-grooming, and suppressed digging in homozygous Shank3 Q321R/Q321R mice. (A) Normal social approach in Shank3 Q321R/Q321R mice (3 months; male) in the three-chamber test, as shown by time spent sniffing social (S1) or object (O) target. Social novelty recognition, measured by the preference for new stranger (S2) over old stranger (S1), could not be determined due the lack of normal social novelty recognition in WT mice. Note that the WT data in this panel and other panels in this figure are identical to those shown in because WT, heterozygous, and homozygous mice were tested together in the same behavioral tests. n = 10 mice (WT), 14 mice (Q321R), ∗∗∗ P < 0.001, ns, not significant, Welch’s t -test (for S1 vs. O) and Student’s t -test (for S1 vs. S2). (B) Normal social interaction in Shank3 Q321R/Q321R mice (3 months; male) in bidirectional direct social-interaction tests, as shown by total time spent in social interaction. n = 10 pairs (WT), 13 pairs (Q321R), ns, not significant, Student’s t -test. (C) Normal USVs emitted by Shank3 Q321R/Q321R mice (4 months; male) upon encounter with a novel female stranger (courtship USVs), as shown by the total number of USVs and the mean duration of each USV calls. n = 12 mice (WT), 14 mice (Q321R), ns, not significant, Student’s t -test. (D) Enhanced self-grooming in Shank3 Q321R/Q321R mice (2 months; male) in home cages with bedding (10 min), as shown by total self-grooming time. n = 12 mice (WT), 13 mice (Q321R), ∗∗∗ P < 0.001, Student’s t -test. (E) Suppressed digging in Shank3 Q321R/Q321R mice (2 months; male) in home cages with bedding (10 min), as shown by total digging time. n = 12 mice (WT), 13 mice (Q321R), ∗ P < 0.05, Welch’s t -test. (F–J) Normal self-grooming in Shank3 Q321R/Q321R mice (2 months; male) in Laboras cages, where self-grooming was measured together with other movements for four consecutive days in the absence of habituation. OFF/ON, light-off/on. Note that there are no genotype differences during the first 10 min, 1 or 12 h, the entire session (96 h), or during light-off and light-on periods (48 h each). n = 13 mice (WT) and 14 mice (Q321R), ns, not significant, two-way ANOVA (genotype main effect p -value = 0.9754 in F ), Student’s t -test (H) and Mann–Whitney U -test (G,I,J) .

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Normal object-recognition and fear memory in homozygous male Shank3 Q321R/Q321R mice. (A) Normal levels of object recognition memory in Shank3 Q321R/Q321R mice (2–4 months; male) in the novel object recognition test, as shown by the discrimination index over a familiar object and a novel object (see Materials and Methods for details) presented 24 h after exploring two identical objects on the first day. n = 11 mice (WT), 13 mice (Q321R), ns, not significant, Student’s t -test. (B) Normal acquisition, retrieval, and extinction of contextual fear memory Shank3 Q321R/Q321R mice (3–6 months; male), as shown by freezing levels. Mice were given 5 foot shocks (2-min intervals) during the 12-min fear memory acquisition phase (day 1), and were consecutively exposed to the same context 24 h after the training (day 2) and also during days 3–9 for fear extinction. n = 13 mice (WT), 17 mice (Q321R), repeated measures two-way ANOVA. (C) Normal somatosensory function in Shank3 Q321R/Q321R mice (6 months; male) in the hot plate test, as shown by latency to first licking/jumping. n = 17 mice (WT) and 20 mice (Q321R), ns, not significant, Student’s t -test (for latency to first licking), and Mann–Whitney U -test (for latency to first jumping). (D) Normal somatosensory function in Shank3 Q321R/Q321R mice (2 months; male) in the von Frey test, as shown by threshold for response to stimulation. n = 10 mice (WT) and 11 mice (Q321R), ns, not significant, Mann–Whitney U -test.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Hot Plate Test, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Abnormal EEG patterns in Shank3 Q321R/Q321R mice. (A,B) Abnormal EEG patterns in frontal and parietal lobes of the Shank3 Q321R/Q321R brain (3 months; male). EEG power was calculated by combining data from both left and right hemispheres in the frontal or parietal lobe. Note that the EEG power in the delta frequency range is decreased, whereas that in the alpha frequency range is increased. The vertical dotted lines in the power spectral density (PSD) diagrams indicate the boundaries between different frequency ranges (delta, 0–4 Hz; theta, 4–12 Hz; alpha, 12–30 Hz; low gamma, 30–80 Hz; high gamma, 80–130 Hz). n = 10 mice (WT) and 10 mice (Q321R) for frontal lobe and 9 mice (WT) and 10 mice (Q321R) for parietal lobe, ∗ P < 0.05, ns, not significant, Student’s t -test, Mann–Whitney U -test, Welch’s t -test.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Decreased susceptibility to PTZ-induced seizures in Shank3 Q321R/Q321R mice. (A) Percentage of animals that can reach stage 1 (behavioral arrest) seizures. Seizures were induced in WT and Shank3 Q321R/Q321R mice (3–4 months; male) by intraperitoneal injection of PTZ (50 mg/kg). n = 10 mice (WT), 13 mice (Q321R), ∗∗ P < 0.01, log-rank (Mantel-Cox) test ( p = 0.0047). (B) Latency to reach stage 1 (behavioral arrest) seizures. ∗ P < 0.05, Mann–Whitney U -test. (C) Percentage of animals that can reach stage 2 (myoclonic) seizures. ns, not significant, log-rank (Mantel-Cox) test ( p = 0.1881). (D) Latency to reach stage 2 (myoclonic) seizures. ns, not significant, Mann–Whitney U -test. (E) Seizure susceptibility based on the latencies to reach stage 1, 2, or 3 seizure (see Materials and Methods for details on the definition of seizure susceptibility). ∗∗ P < 0.01, Student’s t -test. (F) Duration of seizure stages 1, 2, and 3. ∗∗ P < 0.01, ∗∗∗ P < 0.001, ns, not significant, one-way ANOVA with Kruskal–Wallis/Holm–Sidak test (within-genotype stage 1/2/3 comparison) and Mann–Whitney U -test (between-genotype stage 1/2/3 comparison). (G) Final seizure stages that animals reached, expressed as a percentage of animals. ∗ P < 0.05, Chi-square test.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Injection, MANN-WHITNEY, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Summary of electrophysiological, behavioral, and brain function phenotypes in Shank3 Q321R/Q321R mice.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Activity Assay