Journal: Materials

Article Title: The Effect of PEGylated Graphene Oxide Nanoparticles on the Th17-Polarization of Activated T Helpers

doi: 10.3390/ma16020877

Figure Lengend Snippet: Percentage of Th17/22 cells (CCR4+CCR6+) from ZA-CD3+CD4+ lymphocytes in helper T cell cultures supplemented with pegylated GO particles in two concentrations. Note: the x -axis indicates the type and concentration of nanoparticles; the y -axis is the percentage of cells. Control—culture without GO. Data are presented as median (Me) and quartiles (Q1–Q3).

Article Snippet: Antibodies used are listed as follows: mouse IgG1 against human CXCR3-PE-Vio615 (REA 232 clone), CD4-PerCP (VIT4 clone), CCR4-PE-Vio 770 (REA279 clone), CCR6-APC (REA190) (all Miltenyi Biotec, Bergisch Gladbach, Germany), and CD3-Pacific Blue TM (UCTH1 clone) (BioLegend, San Diego, CA, USA).

Techniques: Concentration Assay, Control

Journal: Materials

Article Title: The Effect of PEGylated Graphene Oxide Nanoparticles on the Th17-Polarization of Activated T Helpers

doi: 10.3390/ma16020877

Figure Lengend Snippet: Percentage of “classical” Th17 (CCR4+CXCR3-) and Th17.1 (CCR4-CXCR3+) from CD4+CCR6+ cells in T helper cultures supplemented with PEGylated GO particles in two concentrations. Note: the x -axis indicates the type and concentration of nanoparticles; the y -axis is the percentage of cells. Control—culture without GO. Data are presented as median (Me) and quartiles (Q1–Q3).

Article Snippet: Antibodies used are listed as follows: mouse IgG1 against human CXCR3-PE-Vio615 (REA 232 clone), CD4-PerCP (VIT4 clone), CCR4-PE-Vio 770 (REA279 clone), CCR6-APC (REA190) (all Miltenyi Biotec, Bergisch Gladbach, Germany), and CD3-Pacific Blue TM (UCTH1 clone) (BioLegend, San Diego, CA, USA).

Techniques: Concentration Assay, Control

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Generation and analysis of the Zic3 V5 and Zic3 ins5V mouse lines. (A) Schematic illustrating the site of CRISPR cleavage position relative to Zic3 and the homology between the donor template and genomic region, (A′) the desired V5 epitope tagged Zic3 mouse line ( Zic3 V5 ) and (A″) the arrangement of the insertion in the Zic3 ins5V mouse line. In A″, primers are shown as black half arrows and asterisk denotes the location of Kozak sequence. (B) The tail of the first Zic3 ins5V mouse and tail kinks are indicated with arrowheads. (C) PCR electrophoretogram displaying the amplicon produced by the insertion in Zic3 ins5V mouse using primers shown in A″. Note the amplicon is larger than the amplicons produced by either wild-type or Zic3 V5 /wt mice, indicating an undesired large insertion occurred. (D,E) Partial Sanger sequencing chromatogram of the PCR amplicons shown in C of a Zic3 ins5V/y mouse showing that (D) the right homology arm has been inserted into the 5′UTR and (E) the V5 tag was inserted within Zic3 exon 1. HA, homology arm; wt, wild-type; ntc, no template control.

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques: CRISPR, Sequencing, Amplification, Produced, Control

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Zic3 ins5V mice display reduced viability

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques:

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Neural tube defects observed in Zic3 LacZ and Zic3 ins5V embryos. (A) Lateral view illustration of a 14.5 dpc embryo showing regions of the neural tube (adapted from ). (B-B′) Lateral and (B″) frontal views of a wild-type embryo. White lines in B indicate regions sectioned in F and H. (C) Lateral view of Zic3 LacZ/LacZ null embryo with a neural tube defect in the cervical region. (D) Lateral and (D′) frontal views of a Zic3 LacZ/y null embryo with a neural tube defect in the cephalic region. (E) Lateral and frontal (E′) views of a Zic3 LacZ/LacZ null embryo with midline facial defects. White lines in E indicate regions sectioned in G and I. (F) Section of wild-type embryo forebrain with two separate lateral/telencephalic ventricles and the third ventricle at the midline of the diencephalon. (G) Section of Zic3 LacZ/LacZ embryo forebrain displaying a single fused ventricle and lack of the third ventricle in the diencephalon. (H) Section of wild-type embryo head with two eyes on surface of the embryo. (I) Section of Zic3 LacZ/LacZ embryo head indicating one of the eyes is dysmorphic and located abnormally close to the midline. (J) Lateral view of Zic3 ins5V / ins5V embryo with a neural tube defect in the cervical region. (K) Lateral and (K′) frontal views of a Zic3 ins5V/y embryo with an encephalocele in the cephalic region. (L) Lateral and frontal (L′) views of a Zic3 ins5V/y embryo with a neural tube defect in the cephalic region. (M-M′) Lateral and (M″) frontal views of a Zic3 ins5V/y embryo with craniorachischisis and several facial defects. (N) Transverse section of embryo shown in (M) taken from the head at the level of the eye and stained with Hematoxylin and Eosin. Arrows point to lateral ventricles, arrowheads indicate eyes, and asterisk (*) denotes diencephalon. (B-E′, J-L′ and M-M′) scale bar: 2 mm; (F-I and M″) scale bar: 1 mm.

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques: Staining

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Zic3 ins5V embryos exhibit heart defects. (A-D) Gross embryonic hearts at 13.5-14.5 dpc and (A′-D″) transverse sections stained with Hematoxylin and Eosin. (A-A″) Wild-type heart with proper orientation of ventricles. (B-D) Heart looping defects found in Zic3 ins5V embryos, including (B) dextrocardia, (C) incomplete looping and (D) dextrocardia with incomplete looping. In addition to looping defects, Zic3 ins5V heart sections also revealed (B′-B″) a double outlet right ventricle (DORV) defect. (C′-C″) Atrial isomerism with an atrioventricular canal defect. (D′-D″) Atrioventricular canal defect and thin wall. Arrowheads point to atrioventricular canal defects and asterisks (*) denote atrial isomerism. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; PA, pulmonary artery; Ao, aorta. Scale bars: 500 µm.

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques: Staining

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Zic3 is overexpressed and undertranslated in Zic3 ins5V embryos. (A) Relative expression levels of Zic3 in wild-type ( n =3; wt, blue bar) and Zic3 ins5V ( n =5; red bar) embryos at the headfold stage (7.75 dpc) were assessed using Taqman probes for qPCR. Tbp was used for data normalization. Bars display geometric means and error bars represent geometric standard deviation (SD). Dots represent individual samples. (B) Whole-mount in situ hybridization using a riboprobe for Zic3 on 7.75 dpc wild-type ( n =13) and Zic3 ins5V ( n =9) embryos; scale bar: 250 µm. (C) Whole-mount in situ hybridization using a riboprobe for Zic3 on 10.5 dpc wild-type ( n =7) and Zic3 ins5V ( n =6) embryos; scale bar: 2000 µm. Purple staining in (B) headfold and primitive streak and (C) somites, limb buds and brain. A and P indicate anterior and posterior of embryos, respectively. (D) Western blot images for V5 (ZIC3) from nuclear enriched lysates of wild-type ( n =2), Zic3 V5 ( n =4) and Zic3 ins5V ( n =4) 10.5 dpc embryos. LAMIN B served as nuclear loading control. Full blots are shown in Fig. S3 .

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques: Expressing, Standard Deviation, In Situ Hybridization, Staining, Western Blot, Control

Journal: Biology Open

Article Title: A CRISPR mis-insertion in the Zic3 5′UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin

doi: 10.1242/bio.061677

Figure Lengend Snippet: Zic3 ins5V mRNA is predicted to contain a stem-loop hairpin structure. Predicted secondary structure of (A) Zic3 w t and (B) Zic3 ins5V via RNAfold. Scale indicates the base-pair probability, with red bases having the highest probability. The novel secondary structure is indicated by the thick arrow and the asterisk denotes the start codon location. (C) Schematic of hairpin generated in Zic3 plasmids. (D) Representative western blot image for FLAG (ZIC3) from NIH3T3 nuclear lysates. LAMIN B served as nuclear loading control. For each plasmid, n =3 independent transfections and western blots. -, untransfected; wt, wild-type mouse Zic3 (FLAG tagged) plasmid; 25 hp and 40 hp are mouse Zic3 plasmids with 25 bp and 40 bp stem hairpins, respectively. Full blots shown in Fig. S3 .

Article Snippet: The wild-type mouse Zic3 plasmid containing a C-terminal myc-FLAG tag was obtained from OriGene (MR223858; OriGene Technologies, Rockville, MD, USA).

Techniques: Generated, Western Blot, Control, Plasmid Preparation, Transfection

Journal: Frontiers in Immunology

Article Title: Identification of novel blood-based extracellular vesicles biomarker candidates with potential specificity for traumatic brain injury in polytrauma patients

doi: 10.3389/fimmu.2024.1347767

Figure Lengend Snippet: CD13, CD196, and MOG-expressing EVs are significantly increased in the plasma of traumatic brain injury (TBI) patients. CD13+ and CD196+ EVs were increased in TBI patients at 48h and MOG (Myelin Oligodendrocyte Glycoprotein)+ EVs were increased in TBI patients at the ER time point. (A) CD9/CD63/CD81 normalized APC median intensity signal for the EV surface protein CD13 differed between TBI patients and PT patients/healthy controls in the MACSPlex analysis. (B) Western blot and normalized Western blot quantification for CD13+ EVs. CD13 was further evaluated by Western blot (20 μg protein was loaded in each lane) in plasma EVs from healthy, TBI 48h, and PT 48h patients (n = 3). CD13 was normalized to the CD81 expression of the same samples and the relative signal intensity values are shown in the figure. (C) CD196 EV surface expression differed between TBI and PT patients and healthy controls in the MACSPlex analysis. (D) The increased expression of CD196+ EVs was confirmed by Western blot analysis. (E) MOG+ EVs were significantly increased at the ER time point in the TBI group compared to healthy controls and polytrauma patients (ER and 48h). (F) The Western blot analysis and quantification further confirmed the results obtained in MACSPlex analysis. Results are shown as boxplots of the median. * p<0.05; ** p< 0.01; § p<0.05 among marked groups only.

Article Snippet: Antibodies against CD13 (Proteintech, 14553, 1:1000), CD196 (Thermofischer, 14-1969-80, 1:1000), MOG (BioLegend, 859901, 1: 1000), CD81 (Invitrogen, 10630D, 1:1000), and either anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody (Cell signaling Technology, #7074,1:2000), or anti-mouse IgG-HRP-linked antibody (Cell signaling Technology, #7076,1:2000) were used accordingly.

Techniques: Expressing, Clinical Proteomics, Western Blot

Journal: Frontiers in Immunology

Article Title: Identification of novel blood-based extracellular vesicles biomarker candidates with potential specificity for traumatic brain injury in polytrauma patients

doi: 10.3389/fimmu.2024.1347767

Figure Lengend Snippet: Correlation analysis of EVs’ markers and clinical parameters.

Article Snippet: Antibodies against CD13 (Proteintech, 14553, 1:1000), CD196 (Thermofischer, 14-1969-80, 1:1000), MOG (BioLegend, 859901, 1: 1000), CD81 (Invitrogen, 10630D, 1:1000), and either anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody (Cell signaling Technology, #7074,1:2000), or anti-mouse IgG-HRP-linked antibody (Cell signaling Technology, #7076,1:2000) were used accordingly.

Techniques: