Journal: Epilepsia

Article Title: Olig2-specific loss-of-function Slc35a2 results in hypomyelination and spontaneous seizures

doi: 10.1111/epi.18697

Figure Lengend Snippet: Slc35a2 conditional knockout (cKO) mice are smaller, fail to thrive, and have a shorter lifespan. (A) Representative photos of control and cKO mice at postnatal day (P) 15, P21, and P60 to visualize the disparity in size. (B) Graphical representation of female control ( n = 18) versus cKO mice ( n = 18). Female heterozygous and homozygous groups were combined due to low number of homozygous loss-of-function Slc35a2 females. Graphical representation of male control ( n = 17) and cKO mice ( n = 16). A three-way analysis of variance with Tukey post hoc test supports significant differences between genotypes, sex, and postnatal days ( F 2, 81 = 3.114, p = .0498). (C) Male cKO mice have a shorter lifespan in comparison to all other genotypes (Mantal–Cox, χ 2 = 34.19). * p < .05, *** p < .001, **** p < .0001. Error bars represent mean ± SEM.

Article Snippet: Slc35a2 -floxed mice were custom-generated for Cleveland Clinic by Cyagen Biosciences.

Techniques: Knock-Out, Control, Comparison

Journal: Epilepsia

Article Title: Olig2-specific loss-of-function Slc35a2 results in hypomyelination and spontaneous seizures

doi: 10.1111/epi.18697

Figure Lengend Snippet: Slc35a2 conditional knockout (cKO) mice suffer from motor deficits indicated by open field and locomotor habituation tests. (A) Postnatal day 30–60 randomly selected mice were subjected to a weekly 28-point NeuroScore test to measure overall motor function. Male cKO ( n = 5–6) mice exhibited severe ataxic gait and failed to climb a 45-degree slope when compared to their littermates ( n = 15–17; Friedman test, p = .037). There was no detectable difference between female cKO ( n = 5–6) and control mice ( n = 12–16). All other comparisons are not significant (ns). (B) Open field test supported motor deficits in both male and female cKO. In distance traveled (cm), there are significant differences in male cKO versus all other genotypes (two-way analysis of variance [ANOVA], F 1, 36 = 11.36, p = .0018). Sex differences were significantly different (two-way ANOVA, F 1, 36 = 9.905, p = .0033). In velocity (cm/s), a similar trend was found as the distance traveled where male cKO mice were overall much slower than the other genotypes (two-way ANOVA, F 1, 36 = 11.37, p = .0018) and sex differences were observed (two-way ANOVA, F 1, 36 = 9.975, p = .0032). No significant difference was observed in time spent in the center. (C) The locomotor habituation test further supports the presence of a motor deficit. For visualization purposes, males and females were graphed separately. All genotypes habituated to their novel environment similarly, but both male and female cKO mice had overall less beam horizontal and vertical breaks compared to their controls (three-way ANOVA, epoch vs. genotype, F 23, 828 = 3.719, p = .0001; F 23, 828 = 6.340, p = .0001, respectively). * p < .05, ** p < .01, *** p < .001, **** p < .0001. Error bars represent mean ± SEM.

Article Snippet: Slc35a2 -floxed mice were custom-generated for Cleveland Clinic by Cyagen Biosciences.

Techniques: Knock-Out, Control

Journal: Epilepsia

Article Title: Olig2-specific loss-of-function Slc35a2 results in hypomyelination and spontaneous seizures

doi: 10.1111/epi.18697

Figure Lengend Snippet: Slc35a2 conditional knockout (cKO) mice have lower seizure induction and develop severe spontaneous seizures. (A) Adult (postnatal day [P] 90–150) female and male mice were injected with 45 mg/kg ip pentylenetetrazole, and seizures were graded according to a modified Racine scale for mice. Female control mice ( n = 5) developed tonic–clonic seizures (Racine = 5–6). One control mouse had status epilepticus defined by multiple seizures lasting >5 min. In contrast, female cKO mice ( n = 5) displayed severe tonic–clonic seizures followed by death caused by respiratory arrest (Racine = 6–7; two-way analysis of variance [ANOVA] with Tukey post hoc test, F 1, 17 = 13, p = .0022). Male control mice ( n = 6) had neck jerking and mild brief clonic seizures (grade 3–4) with no mortality due to seizures. Conversely, male cKO mice ( n = 5) suffered severe tonic–clonic seizures with tonic extension and respiratory arrest followed by death (two-way ANOVA with Tukey post hoc test, F 1, 17 = 13, p < .0001). (B) Adult (P90–P100) female and male mice were implanted with electroencephalographic (EEG) electrodes. Six of eight EEG-implanted cKO ( n = 8; two male and six female) mice had seizures compared to no seizures in control mice ( n = 8; four male and four female). The average Racine score for cKO mice was 3.75 ± .84 (two-tailed t -test, t = 4.465, df = 7.0, p < .01). The average number of seizures for cKO mice per session was 7.5 ± 2.73 (two-tailed t -test, t = 2.752, df = 7.0, p < .05). “X” symbolizes males and “O” symbolizes females. (C) Representative EEG with spectral analysis of control male mouse spanning 2 min. (D) Representative EEG seizure of female cKO mouse demonstrating a 2-min seizure with spectral analysis. (E) Representative interictal epileptiform spiking in female cKO with spectral analysis over 15 s. * p < .05, ** p < .01, *** p < .001, **** p < .0001. Error bars represent mean ± SEM.

Article Snippet: Slc35a2 -floxed mice were custom-generated for Cleveland Clinic by Cyagen Biosciences.

Techniques: Knock-Out, Injection, Modification, Control, Two Tailed Test

Journal: Epilepsia

Article Title: Olig2-specific loss-of-function Slc35a2 results in hypomyelination and spontaneous seizures

doi: 10.1111/epi.18697

Figure Lengend Snippet: Slc35a2 conditional knockout (cKO) mice have oligodendroglial hypoplasia in the corpus callosum and decreased Slc35a2 protein levels. (A) Representative Olig2 staining showing regions of interests (ROIs) in postnatal day (P) 60 male and female cohorts ( n = 3 per genotype per sex). ROIs include the gray–white matter junction with the lateral corpus callosum (LCC), medial corpus callosum (mCC), motor cortex (MO), and caudate/putamen (CPu). Scale bars = 200 μm. (B) Representative high magnification images of the gray–white matter junction with the LCC in female control and cKO mice. Arrow in insets shows Olig2+ cells without Slc35a2 staining. Scale bars = 50 μm and 5 μm for insets. (C) Representative images from male control and cKO mice. Arrow points to oligodendrocyte without Slc35a2 staining in insets. Scale bars = 50 μm and 5 μm for inset. (D) All staining was carried out on adjacent sections per mouse ( n = 3 per group), and 2–3 sections per mouse were used as technical replicates. (E) Slc35a2 protein levels are reduced in male cKO ( n = 3) mice compared to littermate ( n = 3) controls (two-tailed t -test, t = 15.22, df = 4, p < .0001). (F) Slc35a2 (42-kDa) protein levels are not statistically different in female control ( n = 3 per genotype) and cKO mice (two-tailed t -test, t = 1.181, df = 4, p > .05). Western blots were performed in triplicate. * p < .05, ** p < .01, *** p < .001. Error bars represent mean ± SEM. Brightness was increased for better visibility. DAPI, 4,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, not significant.

Article Snippet: Slc35a2 -floxed mice were custom-generated for Cleveland Clinic by Cyagen Biosciences.

Techniques: Knock-Out, Staining, Control, Two Tailed Test, Western Blot

Journal: Epilepsia

Article Title: Olig2-specific loss-of-function Slc35a2 results in hypomyelination and spontaneous seizures

doi: 10.1111/epi.18697

Figure Lengend Snippet: Slc35a2 conditional knockout (cKO) mice do not have neuronal architecture abnormalities and have gliosis. Neuronal nuclei (NeuN) and glial fibrillary actin protein (GFAP) immunohistochemistry was conducted on paraformaldehyde-fixed tissue at the genu of corpus callosum (gCC) level. (A) Representative low magnification images of postnatal day (P) 60 female and male brains ( n = 3 per group) of the motor cortex (MO) stained with an anti-NeuN antibody. Scale bars = 100 μm. (B) Representative high-magnification images of motor cortex stained with NeuN and Slc35a2 . Slc35a2 staining is positive in the perinuclear space of neurons. Scale bars = 50 μm. (C) Semiquantification of NeuN+ cells in the motor cortex shows no difference between groups (two-way analysis of variance [ANOVA] with Tukey post hoc testing, F 1, 8 = .3021, p > .05). Female and male controls are represented by circles. Female and male cKO mice are represented by squares. (D) Representative GFAP+ area coverage of P60 female and male groups ( n = 3 per group). Scale bars = 2 mm. (E) Semiquantification of area percentage covered by GFAP staining shows statistically significant differences in female control and female cKO mice in gCC (two-way ANOVA with Tukey post hoc testing, F 1, 8 = 14.27, p < .05). There is a significant interaction effect between sex and genotype (two-way ANOVA, F 1, 8 = 28.01, p = .0007). In the caudate/putamen (CPu) region, there is higher GFAP reactivity in the male cKO mice compared to their male littermate controls (two-way ANOVA, F 1, 8 = 5.394, p = .0487). The interaction between sex and genotype is significant (two-way ANOVA, F 1, 8 = 38.79, p = .0003). All other comparisons are not significant (ns). * p < .05, ** p < .01, *** p < .001, **** p < .0001. Error bars represent mean ± SEM. Brightness was increased for better visibility. AC, anterior commissure; DAPI, 4,6-diamidino-2-phenylindole; LS, lateral septal.

Article Snippet: Slc35a2 -floxed mice were custom-generated for Cleveland Clinic by Cyagen Biosciences.

Techniques: Knock-Out, Immunohistochemistry, Staining, Control

Journal: Epilepsia

Article Title: Olig2-specific loss-of-function Slc35a2 results in hypomyelination and spontaneous seizures

doi: 10.1111/epi.18697

Figure Lengend Snippet: Slc35a2 conditional knockout (cKO) mice express hypomyelination patterns throughout several timepoints in myelination. 2′,3′-Cyclic nucleotide-3′-phosphodiesterase (CNPase) immunohistochemistry was performed using 30-μm-thick, paraformaldehyde-fixed brain tissue. Two to three sections per animal were used as technical replicates, and one section was used as a secondary antibody control by omitting the primary antibody. (A–C) CNPase, an enzyme found in the myelin sheath, is abundant and widespread at postnatal day (P) 15, P21, and P60 in littermate control mice ( n = 3 per timepoint per sex). cKO mice ( n = 3 per timepoint per sex) had patches of hypomyelination throughout the motor cortex as well as corpus callosum (CC), anterior commissure (AC), lateral septal (LS), and caudate/putamen (CPu) regions of the brain, indicated by black squares and high magnification images of each region of interest (ROI). This hypomyelination pattern progressed throughout P21 and P60. (D) Semiquantitative analysis using automated thresholding to determine area covered by stain was performed in ImageJ and averaged across all brain sections per animal. A two-way analysis of variance with Tukey post hoc testing was conducted on all ROIs to detect differences between all groups. Scale bars = 2 mm and 50 μm (A–C). * p < .05, ** p < .01, *** p < .001, **** p < .0001. Error bars represent mean ± SEM. Brightness was increased for better visibility. ns, not signifcant.

Article Snippet: Slc35a2 -floxed mice were custom-generated for Cleveland Clinic by Cyagen Biosciences.

Techniques: Knock-Out, Immunohistochemistry, Control, Staining

Journal:

Article Title: Hypoxia induces adhesion molecules on cancer cells: A missing link between Warburg effect and induction of selectin-ligand carbohydrates

doi: 10.1073/pnas.0402088101

Figure Lengend Snippet: Effect of hypoxia on FUT7 (A), ST3O (B), UGT1 (C), and SDC4 (D) promoter activity. The reporter construct of each gene was transfected to SW480 cells and cultured for 12, 24, or 48 h under hypoxic (1% O2) or normoxic (20% O2) conditions. (Lower) Effect of cotransfection of the dominant-negative form of HIF (DN-HIF). The same amount of empty vector served as negative control, and nonmutated HIF-1 and HIF-2 constructs in the same vector served as positive controls.

Article Snippet: Real-time quantitative RT-PCR analysis was performed by using TaqMan PCR detector Prism7000 (Perkin–Elmer Applied Biosystems) with TaqMan probes: Hs00161688_m1 (for ST3O ), Hs00194644_m1 (for UGT1 ), Hs00197884_ml (for GLUT1 ), Hs00233743_m1 (for α5-integrin, ITGA5 ), and Hs00161617_m1 (for syndecan-4, SDC4 ) as provided by the manufacturer.

Techniques: Activity Assay, Construct, Transfection, Cell Culture, Cotransfection, Dominant Negative Mutation, Plasmid Preparation, Negative Control

Journal:

Article Title: Hypoxia induces adhesion molecules on cancer cells: A missing link between Warburg effect and induction of selectin-ligand carbohydrates

doi: 10.1073/pnas.0402088101

Figure Lengend Snippet: Real-time RT-PCR analyses of expression of genes for fucosyltransferase VII (FUT7), sialyltransferase ST3Gal-I (ST3O), UDP-galactose transporter-1 (UGT1), GLUT1, syndecan-4 (SDC4), and α5-integrin (ITGA5) in cancer tissues prepared from the patients with colon cancer. Ca, cancer tissues; N, nonmalignant mucosa prepared from the same patient. A paired t test was performed to ascertain statistical significance between the amount in cancer tissue and in nonmalignant mucosa.

Article Snippet: Real-time quantitative RT-PCR analysis was performed by using TaqMan PCR detector Prism7000 (Perkin–Elmer Applied Biosystems) with TaqMan probes: Hs00161688_m1 (for ST3O ), Hs00194644_m1 (for UGT1 ), Hs00197884_ml (for GLUT1 ), Hs00233743_m1 (for α5-integrin, ITGA5 ), and Hs00161617_m1 (for syndecan-4, SDC4 ) as provided by the manufacturer.

Techniques: Quantitative RT-PCR, Expressing