Journal: Pain

Article Title: MicroRNA-mediated GABA Aα-1 receptor subunit down-regulation in adult spinal cord following neonatal cystitis-induced chronic visceral pain in rats.

doi: 10.1016/j.pain.2012.09.002

Figure Lengend Snippet: Fig. 4. (A) Real-time RT-PCR analysis of differentially expressed GABAAa-1 gene in spinal dorsal horns (L6–S1) from rats with neonatal cystitis. Two different treatment protocols were followed. Tissues were examined on P60 from the unchallenged protocol and on P30 from the re-challenged protocol. The ordinate value 2DCT corresponds to the mRNA expression relative to reference gene ribosomal RNA (rRNA); data are represented as mean ± SD (n = 5 per group). (B) Western blot analysis of GABAAa-1 protein in spinal dorsal horn samples (L6–S1) from rats with neonatal cystitis. The intensity of GABAAa-1 immunoreactivity for different tissues was normalized against the intensity of b-actin expression for the same tissue. Results are expressed as means ± SD (n = 5 per group). ⁄Significant difference at p < 0.05. (C) GABAAa-1 immunostaining of differentially expressed GABAAa-1 gene in spinal dorsal horns (L6–S1) from rats with neonatal cystitis. The tissues were examined on P60 after zymosan/saline treatment at P14 to P16. The scale bar is 50 lm. The intensity of staining for 10 individual cells from each group was determined; data are presented as means ± SD. ⁄Significant difference at p < 0.001.

Article Snippet: GABAAa-1 gene expression in spinal dorsal horn neurons (L6–S1) by real-time quantitative RT-PCR Total RNA was extracted from spinal dorsal horn samples from both the unchallenged (P60) and re-challenged (P30) groups of animals using Total RNA extraction kit from Bio-Rad (#732-6830).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunostaining, Saline, Staining

Journal: medRxiv

Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

doi: 10.1101/2023.01.24.23284890

Figure Lengend Snippet: a , b . In lung tissue obtained post-mortem from patients who died due to SARS-CoV-2 infection, immunohistochemistry showed increased total chondroitin sulfate compared to normal control lung tissue. c , d . In contrast, intensity and distribution of ARSB were markedly reduced in the Covid-19 lung tissue. Marked decline is evident in the membrane immunostaining, compared to the normal control. e , f . Carbohydrate sulfotransferase (CHST) 15 which is the sulfotransferase that adds a 6-sulfate group to chondroitin 4-sulfate to form chondroitin-4,6-sulfate (chondroitin sulfate E), has regions of marked intensity of immunostaining in both the Covid-19 lung and the normal lung. g , h . In vascular smooth muscle tissue of the Covid-19 lung, the CHST15 immunostaining is much more intense and less diffuse than in the normal lung vascular tissue. i . Treatment of the cultured cells with Interferon (IFN)β amplifies the impact of SPRBD by increasing the expression of ACE2 more than four times the control level (p<0.1×10 −5 , n=6). j . Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ∼2 ug/g protein (p=0.001, n=6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p=0.0005, n=6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFNβ (>5 µg/g protein for CS, n=6; 7.7 µg/g protein for sGAG, n=6). k . Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the airway cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFNβ (n=6, n=6) l . Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p=0.005, n=6), and declined over 50% by the combined exposure to SPRBD and IFNβ. m . The mRNA expression of ARSB also declined (p=2.6×10 −5 ; n=6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFNβ. n . Expression of both CHST15 and of CHST11, which transfers 4-sulfate groups to N-acetylgalactosamine residues to create chondroitin 4-sulfate, is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFNβ. All of the p-values were determined using unpaired t-test, two-tailed, with unequal variance, and error bars represent one standard deviation. [ACE2=angiotensin converting enzyme 2 receptor; AEC=normal, human small airway epithelial cells; ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; SPRBD=SARS CoV-2 spike protein receptor binding domain]

Article Snippet: Normal, primary, human small airway epithelial cells (PCS-301-010, ATCC, Manassas, VA) were grown in Airway Epithelial Cell Basal Medium (PCS-300-030, ATCC) supplemented with Bronchial Epithelial Cell Growth Kit (PCS-300-040) and Penicillin-Streptomycin (penicillin 10 U/ml; streptomycin 10 μg/ml), per recommendations.

Techniques: Infection, Immunohistochemistry, Control, Membrane, Immunostaining, Cell Culture, Expressing, Activity Assay, Two Tailed Test, Standard Deviation, Binding Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Laminin Receptors Basal Cell Adhesion Molecule/Lutheran and Integrin α7β1 on Human Hematopoietic Stem Cells

doi: 10.3389/fcell.2021.675240

Figure Lengend Snippet: Immunostaining of lineage negative HSPC with the anti-α7 integrin antibody. (A) The lineage-negative CBMNC fraction fixed by cytospin centrifugation was labeled with a mouse monoclonal anti-human integrin α7 chain antibody (red) and a rabbit monoclonal anti-human CD34 antibody (green). The cells were counterstained with DAPI (blue). The merged picture shows that all CD34 + HSPC express integrin α7. Representative of three independent experiments. (B) Lin- CD34+ cells were labeled with the unconjugated integrin antibody followed by an incubation with FITC-conjugated secondary antibody, and measured by flow cytometry. Representative plot of three independent experiments. (C) Two different preparations of lin- CD34+ cell extracts were analyzed by immunoblotting. An extract from HUVEC cells was used as a positive control. In all extracts a band of 117 kb can be detected.

Article Snippet: To obtain lineage-negative (lin-) stem and progenitor cells, the CBMNC fraction was magnetically labeled with the human lineage cell depletion kit (Miltenyi Biotec) containing biotin-conjugated antibodies against CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a followed by an incubation with anti-biotin microbeads followed by magnetic separation to obtain the unlabeled lineage-negative fraction.

Techniques: Immunostaining, Centrifugation, Labeling, Incubation, Flow Cytometry, Western Blot, Positive Control

Journal: Cell Death & Disease

Article Title: Zebularine regulates early stages of mESC differentiation: effect on cardiac commitment

doi: 10.1038/cddis.2013.88

Figure Lengend Snippet: Characterization of HS-181 cell line after treatment with zebularine. ( a ) RT-PCR of cardiac markers after treatment with zebularine. Myh7, Myh6, Actc, cTnI and Serca2 present more expression after treatment. ( b ) Immunostaining of treated cells to detect cardiac-specific proteins. Scale bars; 50 μ m. ( c ) Blots of Proteome Profiler Array and the resulting quantification histograms demonstrating inhibition of pluripotency marker expression and ( d ) increased levels of mesodermic proteins after zebularine treatment (black arrows)

Article Snippet: Protein expression profiles were assayed using the specific human pluripotent Stem Cell array kit ‘Proteome Profiler Array' (R&D Systems Europe, Abingdon, UK; ARY010) following the manufacturer's instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunostaining, Inhibition, Marker

Journal: PLoS ONE

Article Title: Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5

doi: 10.1371/journal.pone.0231064

Figure Lengend Snippet: Characteristics of sIBM patients.

Article Snippet: The levels of GDF15 and FGF21 were measured using a Quantikine Human GDF15 ELISA Kit and Quantikine Human FGF21 ELISA Kit (R&D Systems, Minneapolis, MN), respectively.

Techniques: Biomarker Discovery

Journal: PLoS ONE

Article Title: Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5

doi: 10.1371/journal.pone.0231064

Figure Lengend Snippet: A. Pathological images of the muscle tissue from sIBM patient (patient 4). (a) Hematoxylin-eosin staining of muscle biopsy tissue from a patient with sIBM revealed endomysial inflammatory infiltrates invading nonnecrotic fibers (arrow). (b) Modified Gomori trichrome staining of muscle biopsy tissue from a patient with sIBM revealed rimmed vacuoles (arrow). ( c ) Modified Gomori trichrome staining of muscle biopsy tissue from a patient with sIBM revealed ragged-red fibers (arrow). (d) The absence of cytochrome c oxidase staining in muscle biopsy tissue from a patient with sIBM (arrow). B. Pathological image of muscle tissue from an sIBM patient (patient 4). (a) Abnormal mitochondria with concentric cristae were observed by electron microscopy. Scale bar = 5 μm. (b) Enlarged mitochondria with abnormal cristae and dense granules. Scale bar = 5 μm. C. Measurement of the serum levels of GDF15 and FGF21 in sIBM patients and controls. The data represent the mean ± SEM. * p < 0.05 ( p = 0.015, nonparametric Wilcoxon rank sum test; n = 9 patients and n = 4 controls). D. Representative immunostaining of sIBM patient tissue and normal myoblasts with desmin and MF20. Scale bars = 50 μm. E. Bioenergetic assay of normal and sIBM patient myoblasts (sIBM1) in comparison with the OCR. The data represent the mean ± SD. ** p < 0.01, *** p < 0.001 (unpaired two-tailed Student’s t -test versus normal myoblasts). F. Bioenergetic assay of normal and sIBM patient myoblasts (sIBM1) in comparison with the ECAR. The data represent the mean ± SD.

Article Snippet: The levels of GDF15 and FGF21 were measured using a Quantikine Human GDF15 ELISA Kit and Quantikine Human FGF21 ELISA Kit (R&D Systems, Minneapolis, MN), respectively.

Techniques: Staining, Modification, Electron Microscopy, Immunostaining, Comparison, Two Tailed Test

Journal: PLoS ONE

Article Title: Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5

doi: 10.1371/journal.pone.0231064

Figure Lengend Snippet: A. Sensitivity of normal control myoblasts to BSO. The data represent the mean ± SE. *** p < 0.001 (two-way ANOVA and Tukey-Kramer test versus DDW). B. Sensitivity of sIBM patient myoblasts to BSO, as measured by the cell viability assay and the level of LDH in the culture medium. The data represent the mean ± SEM. * p < 0.05, *** p < 0.001 (two-way ANOVA and Tukey-Kramer test versus BSO + DDW). The red square indicates a significant increase or decreased compared with DDW. C. Cell protective effect of MA-5 on sIBM myoblasts, as measured by the cell viability assay and the level of LDH in the culture medium. The data represent the mean ± SEM. # p < 0.05, ### p < 0.001 (two-way ANOVA and Tukey post hoc test versus control); ** p < 0.01, *** p < 0.001 (two-way ANOVA and Tukey-Kramer test versus BSO + DMSO). The red square indicates a significant increase or decrease compared with DMSO. D. Measurement of sIBM patient myoblasts by the cell viability assay (left panel) after 72-h DMSO application as a control or 72-h 10 μM MA-5 treatment under oxidative stress conditions induced by 24-h BSO treatment. The levels of GDF15 (middle panel) in the medium of cultured myoblasts were measured in the same manner. The levels of FGF21 (right panel) in the medium of cultured myoblasts were measured in the same manner. The data represent the mean ± SE. ## p < 0.01, ### p < 0.001 (two-way ANOVA and Tukey-Kramer test versus DMSO); ** p < 0.01, *** p < 0.001 (two-way ANOVA and Tukey-Kramer test versus BSO + DMSO).

Article Snippet: The levels of GDF15 and FGF21 were measured using a Quantikine Human GDF15 ELISA Kit and Quantikine Human FGF21 ELISA Kit (R&D Systems, Minneapolis, MN), respectively.

Techniques: Control, Viability Assay, Cell Culture

Journal: bioRxiv

Article Title: Safety and efficacy study of CRISPR/Cas9 treatment of sickle cell disease in clinically relevant conditions highlights disease-specific responses

doi: 10.1101/2024.01.14.575586

Figure Lengend Snippet: ( A ) Time course analysis of InDel frequency measured in CB-derived HD HSPCs treated with three different Cas9 RNPs (Cas9×2NLS_GFP, Cas9×2NLS, Cas9×3NLS) in the presence (+E, red line) or in the absence (-E, blue line) of the electroporation enhancer. We used the −197 sgRNA. ( B ) Time course analysis of CDKN1A mRNA expression in CB-derived HD HSPCs treated with three different Cas9 RNP nucleases (Cas9×2NLS_GFP, Cas9×2NLS, Cas9×3NLS) in the presence (+E, red line) or in the absence (-E, blue line) of the electroporation enhancer. Data are reported as mean ± SEM of n=2 run in triplicate. **p<0.01; paired t-test. ( C ) GE efficiency in cord blood-derived (HD) and non-mobilized (SCD) CD34+ cells measured by Sanger sequencing followed by TIDE analysis in samples edited with two different HBG -targeting sgRNA (the −197 sgRNA or the −115 sgRNA targeting the −115 region of the HBG promoters ). Each colored dot indicates a different culture condition. Data are reported as mean ± SEM of 4 replicates. ( D ) Time course analysis of the percentage of dead cells (measured as 7AAD + cells) in edited (ED, green line) and control samples (UT, grey line) in HD- (filled dots) vs SCD (empty dots) patient-derived HSPCs cultured in the presence (+) or in the absence (−) of SR1 and/or UM171. Data are reported as mean ± SEM of 2 replicates. ( E ) Bar plots showing the cell composition of HD- (top panel) and SCD (bottom panel) patient-derived HSPCs cultured in the presence (+) or in the absence (−) of SR1 and/or UM171. We defined 5 populations with increasing stemness properties (CD34-cells, CD34+/CD38high cells, CD34+/CD38low−/CD133-cells, CD34+/CD38low−/CD133+/CD90-cells, and CD34+/CD38low−/CD133+/CD90+ cells). Starting from day 4, compared to HD samples, SCD cells had a larger fraction of more differentiated CD34 − cells and the CD34 + /CD38low − /CD133 + /CD90 + cell population (more enriched in HSCs) disappeared in SCD samples. In fact, this latter fraction is still present in HD cells at day 4, although it is smaller compared to the beginning of the culture. However, it is worth to notice that since the beginning SCD samples have a higher proportion of CD34 − cells and a lower percentage of CD34 + /CD38low − /CD133 + /CD90 + cells, which could be ascribed to the cell origin (patient adult vs HD cord blood HSPCs). Data are reported as mean ± SEM of 2 replicates.

Article Snippet: HSPCs were purified by immunomagnetic selection after immunostaining using the CD34 MicroBead Kit (Miltenyi Biotec).

Techniques: Derivative Assay, Electroporation, Expressing, Sequencing, Control, Cell Culture

Journal: bioRxiv

Article Title: Safety and efficacy study of CRISPR/Cas9 treatment of sickle cell disease in clinically relevant conditions highlights disease-specific responses

doi: 10.1101/2024.01.14.575586

Figure Lengend Snippet: ( A ) InDel frequency at HBG1/2 promoters of Cas9-treated HD and SCD plerixafor-mobilized CD34 + cells, as evaluated by Sanger sequencing 6 days after transfection. ( B ) Unsupervised principal component analysis (PCA) of HD and SCD HSPCs two days after nucleofection. Different colors represent different donors. Shape symbols distinguish different sgRNA treatments (rhombuses: UT; triangles: −197, squares: −196; circles: AAVS1). ( C ) Venn diagrams showing the DEGs upregulated (upper panel) and downregulated (lower panel) upon treatment with the sgRNA −197, −196 and AAVS1 in HD (red) and SCD (yellow) treated HSPCs. ( D ) Functional enrichment analysis of up-regulated genes (FDR<0.05 and FC>2) in sgRNA-treated vs untreated (UT) samples from SCD patients and HD. The most enriched Hallmark gene sets are shown on the y-axis. The x-axis shows sample comparisons. The red color gradient indicates the statistical significance of the enrichment [expressed as –log 10 (FDR)]; the color scale values span from 0 to the 85th percentile of the dataset. The size of the circles reflects the quantity of genes associated with each Hallmark gene set.

Article Snippet: HSPCs were purified by immunomagnetic selection after immunostaining using the CD34 MicroBead Kit (Miltenyi Biotec).

Techniques: Sequencing, Transfection, Functional Assay

Journal: bioRxiv

Article Title: IL-11 neutralising therapies target hepatic stellate cell-induced liver inflammation and fibrosis in NASH

doi: 10.1101/470062

Figure Lengend Snippet: a , IL-11 is upregulated in hepatic stellate cells (HSCs) stimulated with TGFβ1 (n=3). b , IL-11 protein is secreted from HSCs stimulated with TGFβ1 (ELISA, n=3). c , Human precision cut liver slices were stimulated with TGFβ1 and IL-11 protein was measured in supernatant (ELISA, n=3). d , IL6R, gp130, and IL11RA expression in HSCs (TPM, transcripts per million). e, f , Representative fluorescence images (scale bars, 200 µm) of HSCs and automated fluorescence quantification for ( e ) ACTA2 +ve cells and ( f ) Collagen I immunostaining following incubation without stimulus (-), with TGFβ1, PDGF, or IL-11. g , Collagen secretion supernatants of HSC stimulated with TGFβ1, PDGF, or IL-11 (Sirius red assay. n≥7). h , Dose-dependent matrigel invasion of HSCs induced by IL-11(n=3). i , Hyper IL-11 induces IL-11 protein secretion from HSCs (ELISA, n=3). a-c, e-g, i , TGFβ1 (5 ng ml -1 ), Hyper IL-11 (0.2 ng ml -1 ), PDGF (20 ng ml -1 ), IL-11 (5 ng ml -1 ); 24 h stimulation; h , 48 h stimulation. j , Schematic of mice receiving daily subcutaneous injection of either saline (control) or rmIl-11 (100 µg kg -1 ). k , Relative liver hydroxyproline content, l , mRNA expression of pro-fibrotic markers, and m , serum ALT levels (k, l , control, n=7; rmIl-11, n=10; m , control n=8; rmIl-11, n=11). a, b, g, h, i, k, m Data are represented as mean ±s.d; c-f, l , Box-and-whisker plots show median (middle line), 25th–75th percentiles (box) and min-max percentiles (whiskers). a-d, i, k-m , Two-tailed Student’s t -test; e-h , two-tailed Dunnett’s test. FC: fold change; I/A: intensity/area.

Article Snippet: Total secreted collagen in the cell culture supernatant was quantified using Sirius red collagen detection kit (9062, Chondrex).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence, Immunostaining, Incubation, Injection, Whisker Assay, Two Tailed Test