Journal: Nature Communications

Article Title: Identification of scaffold proteins for improved endogenous engineering of extracellular vesicles

doi: 10.1038/s41467-023-40453-0

Figure Lengend Snippet: a Huh-7 cells were treated with EVs for 4 h and stained with LysoTracker to visualize lysosomes. Confocal microscopy images from representative regions from the whole well are shown. The experiment was performed once. b Huh-7 cells were treated with EVs for 8 h. Cellular mNG MFI was quantified using flow cytometry. Data are shown as mean ± standard deviation of three biological replicates. The degree of correlation was analyzed with linear regression and is shown as goodness-of-fit ( R 2 ) and significance of non-zero slope (P). c Biodistribution of EVs in mice. NMRI mice were intraperitoneally injected with d -luciferin substrate. Five minutes later, mice were intravenously injected with the same amount of engineered EVs (based on Tluc activity) and imaged with IVIS. Subsequently, major organs were collected for ex vivo bioluminescence measurements. Representative IVIS images are shown. N = 3. d Tluc activity in organs ex vivo after IVIS. Results are shown as mean ± standard deviation of three mice. e Scheme of generating albumin-binding EVs. EVs were collected from HEK-293T cells stably expressing the fusion proteins. f Albumin-binding EVs were injected intravenously and their concentration in plasma was determined. Data are shown as mean ± standard deviation. N = 7 (for TSPAN2-related) or 3 (for TSPAN3-related). g Scheme of sLeX display on EVs. EVs were collected from HEK-293T cells stably expressing the components. h HUVEC cells were activated by TNF-α for 2 h and treated with EVs for 6 h. Cellular mNG MFI was quantified using flow cytometry and is shown as fold-change over un-activated cells. c , e , g Created with BioRender.com. Data are shown as mean ± standard deviation of three biological replicates. Two-sided Student’s t test. Source data are provided as a Source Data file.

Article Snippet: Live animals (isoflurane sedated) were imaged every 5 min over 30 min by IVIS Spectrum (PerkinElmer, USA) with an exposure time of 30 s. Immediately after completion of IVIS session, the mouse was bled for collecting blood in EDTA-coated tubes and sacrificed for collecting major organs.

Techniques: Staining, Confocal Microscopy, Flow Cytometry, Standard Deviation, Injection, Activity Assay, Ex Vivo, Binding Assay, Stable Transfection, Expressing, Concentration Assay, Clinical Proteomics

Journal: PLoS Pathogens

Article Title: Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV

doi: 10.1371/journal.ppat.1008560

Figure Lengend Snippet: A) Experimental scheme of iDCgB immunization and HCMV infection in humanized mice. Mice were transplanted with CB-CD34 + cells and immunized with autologous iDCgB at weeks 6, 7, 10, 11 after HCT. HCMV infection was performed at week 17 post HCT with 1x10 6 HCMV/GLuc-infected MRC-5 cells (i.p.). From week 24 to 25, all mice were treated with granulocyte-colony stimulating factor (G-CSF, 2.5 μg/mouse/day) for 7 days in order to reactivate HCMV. B) Kinetics of human hematopoiesis in blood. The frequencies of human CD45 + (left) and CD3 + positive cells (right) were analyzed (at weeks 15, 17, 20, 24, 25) in blood of humanized mice comparing HCMV (blue) and iDCgB/HCMV cohorts (red). Human hematopoietic reconstitution was persistent and comparable for both cohorts. C) Mesenteric, axillary, inguinal, iliac and total detectable lymph nodes were quantified. Graph shows the averages with the standard deviation of the mean for HCMV (blue) and iDCgB/HCMV (red) cohorts. D) Optical imaging analysis of the bioluminescence signal detected in the torso and abdomen and displayed in photons/sec/cm 2 /sr for all mice (left panels: HCMV; right panels: iDCgB/HCMV). Data was obtained with three independent experiments using cord blood from three donors (D1, D2, D3). Signal intensity was measured with the same settings for all cohorts and mice with 300 sec exposure time, f-stop 1 and medium binning. E) Quantified bioluminescence signal displayed in photons/sec showing decrease of signal in iDCgB/HCMV cohort (red dots) compared with HCMV cohort (blue dots). ROI was set the same for all mice encompassing the whole mouse body. A significant protective effect caused by iDCgB immunization is noticeable. For statistical analysis t-Test with Welch’s correction was used and significances are indicated with ***p<0.001. Standard deviation of the mean is indicated. F) HCMV DNA viral copies were determined by quantitative PCR in LI (HCMV n = 12; iDCgB/HCMV n = 16 available) and BM (HCMV n = 8 available; iDCgB/HCMV n = 12 available). Viral copies are indicated in log 10 copies/μgDNA. For statistical analysis, t-Test with Welch’s correction was used and significances are indicated with **p<0.01 and ***p<0.001. The standard deviation of the mean is indicated. G) Total counts (# positive cells) for huCD45 + /CD4 + T cells (left) and huCD45 + /CD8 + T cells (right) measured for cells recovered from SPL (left panels) and BM (right panels) for different subtypes (N, CM, EM, TE) in the HCMV (blue dots) or the iDCgB/HCMV cohort (red dots). Representative gating example for CD45RA and CD62L staining is shown in . Each dot represents one mouse. H) Frequencies of PD-1 + CD3 + / CD4 + / CD8 + T cells (% positive cells) in LN of HCMV (blue dots) or iDCgB/HCMV cohort (red dots). I) In vitro analyses of antigen-specific T cell responses. mLN of the mice for each cohort were pooled, cohorts were analyzed separately and then the data was merged (HCMV: blue; n = 1 cohort; iDCgB/HCMV: red; n = 2 cohorts). The cells were supplemented with cytokines, stimulated with recombinant proteins (pp65, IE1 or gB) in vitro and analyzed by flow cytometry (Ø denotes no antigen stimulation). EM CD8 + or CD4 + cells were gated and IFN-γ + or TNF-α + cells were quantified. The schematic representation of the assay and exemplary gating approaches are shown in . Left panels: Detection of IFN-γ expression in CD8 + (top) or CD4 + (lower panel) T cells. Left panels: Detection of TNF-α expression in CD8 + (top) or CD4 + (lower panel) T cells. J) Total counts (# positive cells) of pan-B cells (CD19 + ), memory B cells (CD19 + CD27 + ), IgA + / IgG + / IgM + B cells, plasmablasts (PB) and plasma cells (PC) measured in HCMV (blue dots) or iDCgB/HCMV cohort (red dots). Exemplary gating approach is shown in . Each experiment was repeated at least twice with similar results. Negative binominal regression analyses were performed for all statistical determinations of the flow cytometry analysis for the total cells (# positive cells). Beta regression analysis was performed for frequencies (% positive cells). Significances are depicted with *p<0.05, **p<0.01, ***p<0.001. K) Total human IgG measured in plasma by ELISA (μg/ml) for HCMV (blue dots, n = 6 available) or iDCgB cohort (red dots, n = 9 available) (p = 0.0508 was determined with t-Test with Welch’s correction). L), M) Representative examples showing detection of IgM - IgG + positive B cells binding to gB recovered from SPL of HCMV or iDCgB/HCMV cohorts, respectively. Left panels: Gating of IgM - /IgG + cells. Right panels: Gating of gB + binding cells showing frequency within the IgM - /IgG + population. Exemplary gating approach with all markers used is shown in Supplementary Material. N) Plasma was used in strips immunoassays for the qualitative determination of IgG-reactivity against HCMV antigens (IE1, CM2, p150, pp65, gB1 and gB2). Three representative stripes are shown for iDCgB/HCMV (top, note broad cross-reactivity against multiple antigens or HCMV (bottom). Qualitative results for all the mice analyzed are shown in . O) Antinuclear antibodies (ANAs) that bind to contents of the cell nucleus were investigated. HEp-2 cells were used as a substrate to detect the auto-antibodies by indirect immunofluorescence. Human sera obtained from two subjects with autoimmunity disorders were used as reference showing bright signals in nucleus and occasionally in cytoplasm (left panels). Similar analyses performed with plasma obtained from mice in the HCMV cohort showed 6 out of 7 cases ANA positivity (middle panels). One single mouse of the 10 cases analyzed in the iDCgB/HCMV cohort showed faint immunofluorescence signals in the cytoplasm.

Article Snippet: In vivo optical imaging analyses were performed with an IVIS SpectrumCT (PerkinElmer, Waltham, MA) and all pictures were taken with a field of view C, f stop 1 and medium binning for each mouse.

Techniques: Infection, Standard Deviation, Optical Imaging, Real-time Polymerase Chain Reaction, Staining, In Vitro, Recombinant, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV

doi: 10.1371/journal.ppat.1008560

Figure Lengend Snippet: A) Kolmogorov-Smirnov (KS) test was performed on the cellular immunophenotypic biomarkers (without missing values) using all samples (incorporating both iDCgB/HCMV (n = 17); HCMV (n = 12) and in total n = 29). The sensitivity of the KS test on the sample size was evaluated by leave one-sample out (LOO), repeated for all samples. The thresholds for KS statistic and p-value were 0.3 and 0.1, respectively. The frequency of each biomarker satisfying the KS test criteria in LOO is shown. B) The sample distribution is shown for each single biomarker that satisfied the KS test thresholds and appeared with highest frequency in LOO. C) Four biomarker combination sets selected according to the pipeline (see ) and showed 73–87% accuracies to predict the iDCgB/HCMV group. D) Principal component analysis was performed for the four selected biomarkers sets (shown in c). The distribution of samples in the first principal component (PC1) is shown. E) Effects of anti-CD20 (B cell) depletion in the HCMV/GLuc infection signal. Top panels: Baseline optical imaging analyses of HCMV-infected mice (Reconstitution R1, HCMV, n = 3) or iDCgB immunized and then infected with HCMV (HCMV+iDCgB, n = 4; R1: iDCgB generated with 2 vectors; R2: iDCgB generated with a tricistronic lentiviral vector). Bottom panels: Optical imaging analyses 5 days after anti-CD20 (10 mg/kg) treatment for B cell depletion. All mice were analyzed with the same settings; the radiance of the bio-luminescence signals is indicated by the colored bar on the right side (p/sec/cm 2 /sr). F) Quantified total Flux (photons/ second, p/s) for HCMV and HCMV+iDC/gB cohorts, before and after anti-CD20 depletion. ROI was quantified for the frontal torso and abdomen and kept constant for all mice. The horizontal bars in black indicate the median values for each cohort and time of analyses. The P values were determined by t-Test. The % increase is relative to the baseline values prior to depletion (see and Tables ).

Article Snippet: In vivo optical imaging analyses were performed with an IVIS SpectrumCT (PerkinElmer, Waltham, MA) and all pictures were taken with a field of view C, f stop 1 and medium binning for each mouse.

Techniques: Biomarker Assay, Infection, Optical Imaging, Generated, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV

doi: 10.1371/journal.ppat.1008560

Figure Lengend Snippet: A) Scheme of ELISA assay using wells coated with recombinant gB protein. Monoclonal IgGs were serially diluted, transferred to wells and immune detection was performed with an HRP-conjugated antibody against human IgG. Assays were performed in duplicate independent experiments and the results were merged for plotting the data. B) OD450 measurements for reference SM5-1 monoclonal IgG (orange), Kiovig pooled IgG (green) and negative control (grey). C) OD450 measurements for monoclonal IgGs derived from iDCgB immunized mice (black) are shown. SM5-1 was included as a reference (orange). D) OD450 measurements for monoclonal IgGs derived from HCMV (blue) and iDCgB/HCMV (red) cohorts are shown. SM5-1 was included as a reference (orange). E) Scheme of ELISA assay using wells coated with protein lysates from 293T/gB cells. Monoclonal IgGs were serially diluted, transferred to wells and immune detection was performed with an HRP-conjugated antibody against human IgG. Assays were performed in duplicate independent experiments and the results were merged for plotting the data. F) OD450 measurements for reference SM5-1 monoclonal IgG against HCMV gB (orange), Kiovig pooled IgG (green) and negative control (grey). G) OD450 depicted for monoclonal antibodies derived from iDCgB immunized (black) cohort. SM5-1 (orange) measurement was included as a reference. H) ELISA (OD450) measurements for monoclonal IgG derived from HCMV (blue) and iDCgB/HCMV (red) cohort. SM5-1 (orange) was included as reference. For F) G) H) Wells coated with protein lysates obtained from control 293T/w.t. cells were included in the ELISA assay as negative control. No cross-reactivity was detectable. I) Scheme of the in vitro neutralization assay. Antibodies were serially diluted and incubated with HCMV prior to infection. TB40-GLuc viruses were pre-incubated with the antibodies for 1 h and MRC-5 cells were infected with the virus-antibody mixture. Spinoculation was performed and 1h later medium was exchanged. The catalytic activity of the secreted GLuc signal was measured in the supernatant 24h later by luminometry as relative light units (RLU). The experiment was performed as independent duplicates and the results were merged. The % neutralization (y-axis) was plotted against the log 10 μg/ml antibodies (x-axis), the neutralization curves were adjusted after non-linear regression analyses and the IC 50 values were calculated . J) % neutralization of HCMV by the reference SM5-1 IgG (light green) and Kiovig polyclonal antibodies (dark green). Infected MRC-5 cells not exposed to the mAbs were included for RLU measurements and used as 100% infection reference (Mock, grey). K) % neutralization of HCMV by mAbs DC06 and DC16 derived from the iDCgB cohort (black). SM5-1 IgG (light green) is shown as reference. L) % neutralization of HCMV by the mAb PR32 derived from a HCMV-protected mouse of the iDCgB/HCMV cohort (red). SM5-1 IgG (light green) is shown as reference. The other six cloned antibodies (DC14, DC17, CV03, CV04, PR17, PR28 and PR35) did not neutralize HCMV infection. All assays were performed in duplicate independent experiments. For ELISA and neutralization assays, measurements were taken for two and four wells, respectively. For all graphs error bars indicate standard deviation of the mean and corresponding EC 50 and IC 50 values are shown in . M) Schematic representation of the experiment to test the effects of passive immunization by adoptive antibody transfer against HCMV. For the control group, mice were infected with HCMV 16 weeks after HCT and G-CSF treatment was performed between weeks 19 and 20 after HCT. For the passive immunization cohort, mice were injected i.v. with the monoclonal antibodies (DC06 and PR32, 0.5 mg each in total 1.0 mg) on the day prior to HCMV infection. After infection, the mice were injected i.v. with the monoclonal antibodies (DC06 and PR32, 0.125 mg each in total 0.25 mg) additional seven times. N) Optical imaging analysis performed for the control HCMV cohort (left) or the cohort treated with mAbs (D06 and PR32) to test the effects of passive immunization against HCMV infection (top, “INFEC”) and after HCMV reactivation induced by G-CSF treatment (bottom, “REAC”). All mice were analyzed with the same settings; the range of the bio-luminescence signals is indicated by the colored bar on the right side (radiance: p/sec/cm 2 /sr). O) Quantified total Flux (phonons/second, p/s) for the control (CTR/HCMV, blue) or mAb-treated cohort (mABs/HCMV, red). ROI was quantified for the frontal torso and abdomen and kept constant for all mice. Data was obtained for the two time points to monitor INFEC (left) and REAC (right). P) Quantification of HCMV viral copies (log 10 copies/μg DNA) detected in liver (LI) and bone marrow (BM) comparing HCMV-infected mice (blue) and mice treated by with mAbs (red). The horizontal bars in black indicate the median values for each cohort and time of analyses. The P values were determined by t-Test. The % increase is relative to the CTR non-treated cohort (see ).

Article Snippet: In vivo optical imaging analyses were performed with an IVIS SpectrumCT (PerkinElmer, Waltham, MA) and all pictures were taken with a field of view C, f stop 1 and medium binning for each mouse.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Negative Control, Derivative Assay, Control, In Vitro, Neutralization, Incubation, Infection, Virus, Activity Assay, Clone Assay, Standard Deviation, Injection, Optical Imaging

Journal: Scientific Reports

Article Title: PI3K-targeting strategy using alpelisib to enhance the antitumor effect of paclitaxel in human gastric cancer

doi: 10.1038/s41598-020-68998-w

Figure Lengend Snippet: Combined effects of alpelisib and paclitaxel on cell proliferation and colony formation in vitro. ( A ) Five PIK3CA wild-type cells (SNU1, SNU16, SNU484, SNU638, and SNU668) and three PIK3CA -mutant cells (SNU601, AGS, and MKN1) were treated with paclitaxel (0, 0.125 nM, 0.25 nM, 0.5 nM, 1 nM, 2 nM, 4 nM, 8 nM, 10 nM, and 20 nM) for 72 h. The IC 50 values were calculated using CalcuSyn software. Data expressed as mean ± standard deviation of three replicates. ( B ) The eight GC cell lines were exposed to increasing concentration of alpelisib and paclitaxel at a fixed ratio. The synergistic potential of alpelisib combined with paclitaxel was determined by calculating the combination index (CI) using CalcuSyn software according to Chou-Talalay method. The CI values < 1, = 1, and > 1 indicate synergistic, additive, and antagonistic effects, respectively. ( C ) Colony formation assays were conducted in two PIK3CA wild-type (SNU638 and SNU668) and three PIK3CA -mutant (SNU601, AGS, and MKN1) cells. The Student’s t-test was used to compare two independent groups. * p < 0.05; ** p < 0.01; and *** p < 0.001.

Article Snippet: Paclitaxel was purchased from Selleckchem (Houston, TX, USA).

Techniques: In Vitro, Mutagenesis, Software, Standard Deviation, Concentration Assay

Journal: Scientific Reports

Article Title: PI3K-targeting strategy using alpelisib to enhance the antitumor effect of paclitaxel in human gastric cancer

doi: 10.1038/s41598-020-68998-w

Figure Lengend Snippet: Combined effects of alpelisib and paclitaxel on caspase 3/7 activity, apoptosis, PI3K downstream molecules, PI3K p110α activity, and the expression levels of γ-H2ax in gastric cancer cells. ( A ) Caspase 3/7 activity (RLU, relative luminescence units) was quantified 24 h after alpelisib, paclitaxel, or their combination treatment in SNU638, SNU668, SNU601, AGS, and MKN1 cells. The Student’s t -test was used to compare two independent groups. * p < 0.05; ** p < 0.01; and *** p < 0.001. ( B ) Apoptosis was evaluated by flow cytometry of Annexin V-propidium iodide (PI) double-stained gastric cancer cells after treatment with alpelisib and/or paclitaxel for 24 h. The Y-axis represents the PI-labeled population, whereas the X-axis represents the Annexin V positive cells. The left lower quadrant (Annexin V-, PI-) indicates normal cells, whereas the right lower quadrant (Annexin V+, PI−) and the right upper quadrant (Annexin V+, PI+) are the early and late apoptotic cells, respectively. The Student’s t -test was used to compare two independent groups. ** p < 0.01; and *** p < 0.001. ( C ) SNU638, SNU668, SNU601, AGS, and MKN1 cells were treated with 5 μM of alpelisib and/or 3 nM of paclitaxel for 30 min. Expression levels of PI3K p110α, p-AKT S473, p-AKT T308, AKT, p-S6K1, S6K1, p-4E-BP1, 4E-BP1, p-GSK3β, GSK3β, p-BAD, and BAD were determined by Western blot. β-actin was the control. Protein expression was analyzed by ImageJ software. ( D ) The biotinylated-PIP3 (B-PIP3) was set as 100%. The kinase reactions with or without alpelisib or wortmannin were referenced to the B-PIP3 signal to calculate the relative effects of PI3K inhibitors. The recombinant GRP-1 protein was used as the capture protein. GRP-1 bound to the glutathione-coated plate competitively captures either the PIP3 generated by the kinase reaction or the B-PIP3. The Student’s t -test was used to compare two independent groups. * p < 0.05 and ** p < 0.01. ( E ) SNU638, SNU668, SNU601, AGS, and MKN1 cells were treated with 5 μM of alpelisib and/or 3 nM of paclitaxel for 72 h. The expression levels of γ-H2ax was determined by Western blot. Vinculin was the control. Protein expression was analyzed by ImageJ software.

Article Snippet: Paclitaxel was purchased from Selleckchem (Houston, TX, USA).

Techniques: Activity Assay, Expressing, Flow Cytometry, Staining, Labeling, Western Blot, Control, Software, Recombinant, Generated

Journal: Scientific Reports

Article Title: PI3K-targeting strategy using alpelisib to enhance the antitumor effect of paclitaxel in human gastric cancer

doi: 10.1038/s41598-020-68998-w

Figure Lengend Snippet: Combined effects of alpelisib and paclitaxel on cell migration and epithelial–mesenchymal transition markers expression. ( A ) The migration of SNU638, SNU668, SNU601, AGS, and MKN1 cells was assessed by the wound healing assay after 16 h of treatment. Representative images of the scratched areas are shown. Cell migration was quantified with ImageJ software. The Student’s t -test was used to compare two independent groups. * p < 0.05; ** p < 0.01; and *** p < 0.001. ( B ) SNU638, SNU668, SNU601, AGS, and MKN1 cells were treated with alpelisib combined with paclitaxel for 16 h. Expression levels of E-cadherin, Snail, Slug, Twist, and vimentin were determined by Western blot. GAPDH was the control. Protein expression was analyzed by ImageJ software.

Article Snippet: Paclitaxel was purchased from Selleckchem (Houston, TX, USA).

Techniques: Migration, Expressing, Wound Healing Assay, Software, Western Blot, Control

Journal: Scientific Reports

Article Title: PI3K-targeting strategy using alpelisib to enhance the antitumor effect of paclitaxel in human gastric cancer

doi: 10.1038/s41598-020-68998-w

Figure Lengend Snippet: In vivo anti-tumor activity and tolerability of alpelisib and paclitaxel combination in the MKN1 xenograft model. ( A ) The survival of mice was analyzed by the Kaplan–Meier method. Five mice were assigned to each treatment group. ( B ) For 4 weeks of treatment period, the change in body weight was plotted according to the treatment groups. For the in vivo study, the values were presented as the mean ± standard error of the mean. The Student’s t -test was used to compare two independent groups. ** p < 0.01. ( C ) Tumor volumes were measured every week for 4 weeks. The Student’s t -test was used to compare two independent groups. * p < 0.05; ** p < 0.01; and *** p < 0.001. ( D ) The representative images of MKN1 luciferase tumor-bearing mice indicate the size of tumors at 5 weeks. d-luciferin was injected into mice for 10 min and was analyzed using in vivo optical imaging system 200 (IVIS 200). ( E ) Formalin-fixed paraffin-embedded tumor sections were stained with H&E, Ki-67, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) antibodies.

Article Snippet: Paclitaxel was purchased from Selleckchem (Houston, TX, USA).

Techniques: In Vivo, Activity Assay, Luciferase, Injection, Optical Imaging, Formalin-fixed Paraffin-Embedded, Staining, TUNEL Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: BET inhibitor nanotherapy halts kidney damage and reduces chronic kidney disease progression after ischemia-reperfusion injury.

doi: 10.1016/j.biopha.2024.116492

Figure Lengend Snippet: Fig. 1. Synthesis and characterisation of JQ1 liposomal nanoparticles. (A) Schematic representation of type I liposomes (I), type II liposomes (II), and type III li posomes (III) procedure of synthesis. (B) Viability of HK-2 cells determined using the MTT assay. Cells were treated with unencapsulated JQ1 (+) or its inactive enantiomer JQ1 (-), or with JQ1 (+) encapsulated in types I to III liposome nanoparticles (NPs). Empty NPs were used to assess NP- and JQ1-independent toxicities. Results are expressed as a percentage of the control cells (untreated, black bars). Data are presented as mean ± standard deviation of n = 4 replicates (wells) per group and are representative of three independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05, compared to each control (black bars). Ctrl, control. (C) Hydrodynamic diameter (HD) and surface ζ -potential at pH 7 of JQ1-NPs type III liposomes analysed by dynamic light scattering (DLS). PDI, polydispersity index. (D) Transmission electron microscope (TEM) micrograph of JQ1 type III liposomes (left) and average size distribution of type III JQ1-NPs analysed by TEM (n=200) (right). (E) Fluorescence emission spectrum after excitation with DsRed ex/em filter and T1- weighted MRI phantom images. OI, optical imaging. MRI, magnetic resonance imaging. (F) Plots of T1 and T2 relaxation times as a function of gadolinium (Gd) concentration.

Article Snippet: The inhibitor JQ1 (+) (supplied by James Bradner’s laboratory from Dana-Farber Cancer Institute, Boston, MA, USA) or its inactive enantiomer JQ1 (-) (Selleckchem, Houston, TX, USA) were used for 24 h at concentrations raging 50–500 nM.

Techniques: Liposomes, MTT Assay, Control, Standard Deviation, Comparison, Transmission Assay, Microscopy, Fluorescence, Optical Imaging, Magnetic Resonance Imaging, Concentration Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: BET inhibitor nanotherapy halts kidney damage and reduces chronic kidney disease progression after ischemia-reperfusion injury.

doi: 10.1016/j.biopha.2024.116492

Figure Lengend Snippet: Fig. 2. In vitro effectivity of JQ1-loaded nanoparticles (NPs). (A) Representative flow cytometry histograms showing the fluorescent intensity of rhodamine red (RdR) NPs-JQ1 uptake by HK-2 cells (blue) compared to control untreated cells (red) at 1, 3, 6, 18, and 24 hours. (B) Mean fluorescence intensity (MFI) of RdR-NPs-JQ1 treated (blue line) and untreated cells (red line) along with the percentage of positive cells (right Y axis, black dotted line) are represented at the indicated times. Gene expression levels of IL6 (C) and CCL2 (D) were analysed by qPCR in HK-2 cells treated with empty NPs, NPs-JQ1, JQ1 (+), or JQ1 (-) for 24 hours at different doses and TNF-α stimulation (10 ng/mL) for the last 3 hours. L1, L2, and L3 represent the three lipid concentrations (0.5, 2.5, and 25 mM, respectively) used in the NPs- JQ1 treatments for the corresponding 10, 50, and 250 nM doses of JQ1. GAPDH was used as a housekeeping gene. For all experiments, error bars represent the mean ± standard deviation of n = 3 per condition and data are representative of three independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparison tests. * p<0.05, compared to each control (empty vs NPs-JQ1 doses and JQ1 (-) vs JQ1 (+) doses); # p<0.05 compared to unstimulated cells.

Article Snippet: The inhibitor JQ1 (+) (supplied by James Bradner’s laboratory from Dana-Farber Cancer Institute, Boston, MA, USA) or its inactive enantiomer JQ1 (-) (Selleckchem, Houston, TX, USA) were used for 24 h at concentrations raging 50–500 nM.

Techniques: In Vitro, Flow Cytometry, Control, Fluorescence, Gene Expression, Standard Deviation, Comparison

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: BET inhibitor nanotherapy halts kidney damage and reduces chronic kidney disease progression after ischemia-reperfusion injury.

doi: 10.1016/j.biopha.2024.116492

Figure Lengend Snippet: Fig. 4. Mice treatment with JQ1-NPs following bilateral ischemia-reperfusion injury results in decreased kidney damage. (A) Schematic representation of experi mental design. A 45 min renal bilateral ischaemic injury was performed followed by administration of NPs-JQ1 at dosages of 10, 20, 30, and 40 mg/kg at 1 h after reperfusion. Mice were sacrificed at 24 h. (B) Lcn2 (NGAL) gene expression levels were analysed by qPCR and normalised to the empty nanoparticles (NPs) ischaemic group. Sham mice were used as the control group. (C) Levels of renal NGAL protein were assessed by western blotting. Images show the expression levels of 2 (sham) or 4 representative animals per group (left). Normalised quantification to the ischaemic group without treatment (empty NPs) is shown (right). β-actin was used as the loading control protein. (D) Blood urea nitrogen (BUN; mg/dl) and (E) serum creatinine (mg/dl) levels assessed in mice from the different groups at 24 h post- ischaemic damage in the 20, 30, and 40 mg/kg NPs-JQ1 administered mice. Results are mean ± standard deviation of 3–4 (Sham) to 5–6 (I/R) animals per group. Data are representative of three distinct experiments with comparable results. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparisons tests. # p<0.05 vs. Sham; and *p<0.05, **p<0.01 vs. empty NPs damage group.

Article Snippet: The inhibitor JQ1 (+) (supplied by James Bradner’s laboratory from Dana-Farber Cancer Institute, Boston, MA, USA) or its inactive enantiomer JQ1 (-) (Selleckchem, Houston, TX, USA) were used for 24 h at concentrations raging 50–500 nM.

Techniques: Gene Expression, Control, Western Blot, Expressing, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: BET inhibitor nanotherapy halts kidney damage and reduces chronic kidney disease progression after ischemia-reperfusion injury.

doi: 10.1016/j.biopha.2024.116492

Figure Lengend Snippet: Fig. 5. Equivalent treatment with free JQ1 does not improve renal damage. (A) Schematic representation of experimental design. A 45 min renal bilateral ischaemic injury was performed followed by the administration of the vehicle (DMSO/10% β-cyclodextrin 1:10) or non-encapsulated (free) JQ1 at a unique dose of 40 mg/kg 1 hour after reperfusion. Mice were sacrificed at 24 h. Expression levels of Lcn2 (NGAL) were quantified by qPCR (B) and protein levels were assayed by western blotting (C) in the vehicle (purple squares), free JQ1 (green triangles) and control groups. Gapdh and β-actin were used as loading controls. Absolute quantification of NGAL from the blots is shown (below). (D) Blood urea nitrogen (BUN; mg/dl) and (E) serum creatinine (mg/dl) levels are shown for each analysed group. For all experiments, error bars represent the mean ± standard deviation of n = 4 mice per group and data are representative of three separate experiments. (F) Repre sentative PAS-stained sections for sham and ischaemic animals (I/R) treated with empty nanoparticles (NPs), NPs-JQ1, and unencapsulated (free) JQ1 at a dose of 40 mg/kg. Asterisks (*) indicate presence of tubular casts and arrows highlight necrotic areas. Scale bar=100 µm.

Article Snippet: The inhibitor JQ1 (+) (supplied by James Bradner’s laboratory from Dana-Farber Cancer Institute, Boston, MA, USA) or its inactive enantiomer JQ1 (-) (Selleckchem, Houston, TX, USA) were used for 24 h at concentrations raging 50–500 nM.

Techniques: Expressing, Western Blot, Control, Quantitative Proteomics, Standard Deviation, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: BET inhibitor nanotherapy halts kidney damage and reduces chronic kidney disease progression after ischemia-reperfusion injury.

doi: 10.1016/j.biopha.2024.116492

Figure Lengend Snippet: Fig. 6. Suppression of inflammation and inflammatory cell infiltration following JQ1-NP treatment. (A) Expression of proinflammatory cytokines genes were measured by qPCR in kidney samples obtained from the sham (n=3), ischemia/reperfusion (I/R) + empty nanoparticles (NPs), and the I/R + NPs-JQ1 mouse groups (n=5) at 24 h post-reperfusion. Gapdh was used as a control housekeeping gene. Data are summarised as the mean ± standard deviation. Statistical analyses involved use of the two-tailed Student’s unpaired t-test or Mann-Whitney U test. *p<0.05. Representative dot plots from flow cytometry analysis of neutrophil (B) and monocyte (D) cell infiltration within the kidneys of the aforementioned mice in panel (A). Representative percentages from parents are shown. Dead cells were excluded, as well as doublets before CD45+ cell gating. Complete gating strategy is shown in Supplementary Figure 6. Total cell number quantification per kidney of neutrophils (C) and monocytes (E) in the CD45+-gated population was determined by TruCount Tubes. The results are means ± standard deviation of 3 (sham) to 5 (I/R) animals per group, and statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparisons tests. *p<0.05, **p<0.01 compared to I/R + empty NPs group. All data are representative of three experiments.

Article Snippet: The inhibitor JQ1 (+) (supplied by James Bradner’s laboratory from Dana-Farber Cancer Institute, Boston, MA, USA) or its inactive enantiomer JQ1 (-) (Selleckchem, Houston, TX, USA) were used for 24 h at concentrations raging 50–500 nM.

Techniques: Expressing, Control, Standard Deviation, Two Tailed Test, MANN-WHITNEY, Flow Cytometry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: BET inhibitor nanotherapy halts kidney damage and reduces chronic kidney disease progression after ischemia-reperfusion injury.

doi: 10.1016/j.biopha.2024.116492

Figure Lengend Snippet: Fig. 7. Administration of JQ1-NPs following bilateral ischemia-reperfusion injury results in prevention of renal fibrosis. (A) Graphical depiction of experimental layout. 30 min ischaemic mice were administered empty nanoparticles (NPs) or JQ1-NPs at 40 mg/kg for a single boost 1 h after reperfusion and mice were sacrificed at day 21. Gapdh was used as a housekeeping gene. (B) Quantitative PCR analyses of fibrotic-related gene expression. (C) Western blots of α-SMA and fibronectin were performed. β-actin was used as a loading control protein. (D) Representative images of Masson’s trichrome staining at day 21 in ischaemic kidneys treated as indicated with stained area quantification (%). (E) Expression level of the damage marker NGAL (Lcn2 gene) was determined by qPCR in the kidneys and their function was estimated by the measure of blood urea nitrogen (BUN; mg/dl) serum levels (F). Values are expressed as the mean ± standard deviation of 3 (Sham) to 4–6 (I/R) animals per group. Statistical analysis was conducted with one-way ANOVA and Tukey’s post-hoc tests. *p < 0.05, ** p < 0.01, *** p < 0.001 compared to ischemia/reperfusion (I/R) + empty NPs group. Data are representative of three independent experiments with similar findings.

Article Snippet: The inhibitor JQ1 (+) (supplied by James Bradner’s laboratory from Dana-Farber Cancer Institute, Boston, MA, USA) or its inactive enantiomer JQ1 (-) (Selleckchem, Houston, TX, USA) were used for 24 h at concentrations raging 50–500 nM.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Control, Staining, Expressing, Marker, Standard Deviation

Journal: Biology Direct

Article Title: Dual-labeled nanoparticles based on small extracellular vesicles for tumor detection

doi: 10.1186/s13062-022-00345-7

Figure Lengend Snippet: In vitro assessment of radioactive and optical uptake of dual-sEVs by gamma counter and flow cytometry. A Radioactive uptake for RAW 264.7, HeLa, and U87 cells after 1 h, 4 h, and 24 h of dose addition. 99m Tc was used as a control. Data are represented as mean ± standard deviation (SD). B Median fluorescence intensity (MFI) inside the cells was evaluated at 24 h. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are represented as mean ± SD. C Flow cytometry diagrams of control cells (RAW 264.7, HeLa, and U87 cells, in blue) and treated cells (RAW 264.7, HeLa, and U87 cells, in purple)

Article Snippet: The human HeLa (ATCC® CCL-2TM) cell line was used as a model of cervical carcinoma and human U87 (ATCC® HTB-14TM) cell line as a model of glioblastoma.

Techniques: In Vitro, Flow Cytometry, Control, Standard Deviation, Fluorescence

Journal: Biology Direct

Article Title: Dual-labeled nanoparticles based on small extracellular vesicles for tumor detection

doi: 10.1186/s13062-022-00345-7

Figure Lengend Snippet: Optical uptake of dual-sEVs by confocal microscopy in RAW 264.7, HeLa, and U87 cells at 5 min, 1 h, 4 h, and 24 h after the administration of 5 μg/mL of dual-sEVs. Blue, DAPI; red, phalloidin; and white, dual-sEVs

Article Snippet: The human HeLa (ATCC® CCL-2TM) cell line was used as a model of cervical carcinoma and human U87 (ATCC® HTB-14TM) cell line as a model of glioblastoma.

Techniques: Confocal Microscopy

Journal: Biology Direct

Article Title: Dual-labeled nanoparticles based on small extracellular vesicles for tumor detection

doi: 10.1186/s13062-022-00345-7

Figure Lengend Snippet: In vivo and ex vivo assessment of dual-sEVs by nuclear techniques. A Blood half-life. B Ex vivo biodistribution of dual-sEVs in a U87 xenograft mouse model 24 h after tracer injection. Detailed radioactivity in the brain (control organ) compared to U87 tumor tissue. * p < 0.05. C Ratio of dual-SEV uptake in U87 tumor tissue to non-target tissues at 24 h. D Excretion profile of dual-sEVs in urine and feces collected from the animal 24 h post-injection. Radioactivity in tissues is expressed as % ID/g. Data are represented as mean ± SD

Article Snippet: The human HeLa (ATCC® CCL-2TM) cell line was used as a model of cervical carcinoma and human U87 (ATCC® HTB-14TM) cell line as a model of glioblastoma.

Techniques: In Vivo, Ex Vivo, Injection, Radioactivity, Control

Journal: Biology Direct

Article Title: Dual-labeled nanoparticles based on small extracellular vesicles for tumor detection

doi: 10.1186/s13062-022-00345-7

Figure Lengend Snippet: In vivo optical imaging of dual-sEVs. A In vivo optical imaging of tumor-bearing mice 24 h after i.v. injection in the lateral (left) and prone positions (right). B Ex vivo biodistribution of the excised organs (brain, spleen, kidneys, liver, tumor, heart, lungs; n = 11). Detailed quantification of the brain (control organ) compared to U87 tumor tissue. * p < 0.05. Data is represented as mean ± SD

Article Snippet: The human HeLa (ATCC® CCL-2TM) cell line was used as a model of cervical carcinoma and human U87 (ATCC® HTB-14TM) cell line as a model of glioblastoma.

Techniques: In Vivo, Optical Imaging, Injection, Ex Vivo, Control

Journal: Biomaterials Research

Article Title: Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate

doi: 10.1186/s40824-022-00328-9

Figure Lengend Snippet: Schematic diagram and application of PTK7-GEMs. A Schematic diagram showing GEM is transformed into a modular drug-type nucleotide and then combined with PTK7-Aptamer to produce PTK7-GEMs. B Application and anti-tumor effect of PTK7-GEMs in three different tumor models

Article Snippet: Thereafter, the membranes were reacted with rabbit polyclonal antibody recognizing PTK7 (Abcam; 1:500) or ACTB (quality control, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:800) overnight at 4 °C.

Techniques: Transformation Assay

Journal: Biomaterials Research

Article Title: Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate

doi: 10.1186/s40824-022-00328-9

Figure Lengend Snippet: Serum stability and cell binding ability of PTK7-GEMs. Serum stability analysis of PTK7-GEMs using 12% polyacrylamide gel electrophoresis (PAGE). A PTK7-GEMs were incubated for 0, 1, 2, 4, 8, 12, 16, and 24 h 10% FBS-supplemented or 10% NMS-supplemented RPMI-1640 medium, respectively. B The abscissa indicates that the same sample of PTK7-GEMs was incubated in 10% NMS (red) and 10% FBS (blue) for 0, 1, 2, 4, 8, 12, 16, and 24 h respectively, and the ordinate indicates the respective degradation percentage of the samples. The binding ability of PTK7-GEMs to PTK7 overexpressing cells at 4 °C, as determined using flow cytometry. C , D The binding ability of various FITC-labeled samples to 5637 cells and SV-HUC-1 cells

Article Snippet: Thereafter, the membranes were reacted with rabbit polyclonal antibody recognizing PTK7 (Abcam; 1:500) or ACTB (quality control, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:800) overnight at 4 °C.

Techniques: Binding Assay, Polyacrylamide Gel Electrophoresis, Incubation, Flow Cytometry, Labeling

Journal: Biomaterials Research

Article Title: Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate

doi: 10.1186/s40824-022-00328-9

Figure Lengend Snippet: CCK-8 assays of PTK7-GEMs cytotoxicity. A , B Viability of 5637 cells and SV-HUC-1 cells respectively treated with LIB-GEMs, GEM or PTK7-GEMs evaluated by CCK8 assay. C IC 50 of PTK7-GEMs and GEM against 5637 cells (NS, not significant). Data represents the mean ± SEM, n = 3. D , E Cell cycle distributions of 5637 and SV-HUC-1 cells treated with LIB-GEMs, GEM or PTK7-GEMs respectively for 8 h and then incubated for 72 h with complete medium before being tested using flow cytometry

Article Snippet: Thereafter, the membranes were reacted with rabbit polyclonal antibody recognizing PTK7 (Abcam; 1:500) or ACTB (quality control, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:800) overnight at 4 °C.

Techniques: CCK-8 Assay, Incubation, Flow Cytometry

Journal: Biomaterials Research

Article Title: Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate

doi: 10.1186/s40824-022-00328-9

Figure Lengend Snippet: Investigation of the GEM release mechanisms from PTK7-GEMs. The profile of PTK7-GEMs release from A Crude cell lysate (untreated by inhibitors) or B Lysate of cells treated with phosphatase inhibitors, The DNA maximum absorption peak was measured at 260 nm. C Cumulative release of GEM when PTK7-GEMs were incubated with crude cell lysate, phosphatase inhibitor-treated cell lysate, buffer at pH 5.5, 10% FBS and 10 ⋅ 10 − 3 м GSH respectively

Article Snippet: Thereafter, the membranes were reacted with rabbit polyclonal antibody recognizing PTK7 (Abcam; 1:500) or ACTB (quality control, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:800) overnight at 4 °C.

Techniques: Incubation

Journal: Biomaterials Research

Article Title: Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate

doi: 10.1186/s40824-022-00328-9

Figure Lengend Snippet: PTK7-GEMs internalization and trafficking in cells. Images generated using confocal microscopy revealing the co-localization of PTK7-GEMs-cy5 (red) with markers of endocytosis dextran ( A ), choleratoxin ( B ), transferrin ( C ) labeled with Alexa Fluor 488 respectively. D Pearson’s correlation coefficient analysis of PTK7-GEMs-cy5 and endocytosis marker co-localization in 5637 cells using Image J. Confocal microscopy images revealing the co-localization of PTK7-GEMs-cy5 (red) with EIPA ( E ) (inhibitor of macropinocytosis), Filipin ( F ) (inhibitor of the caveolae pathway), and Chlorpromazine ( G ) (inhibitor of the clathrin pathway) (green). DAPI was used to counterstain the nuclei (blue). Scale bar, 10 μm. The means ± standard deviation ( n = 3 per group) is indicated using error bars. Each replicate was from a single biological experiment and 10 independent fields of view were chosen from quantification. * P < 0.05; NS = not significant

Article Snippet: Thereafter, the membranes were reacted with rabbit polyclonal antibody recognizing PTK7 (Abcam; 1:500) or ACTB (quality control, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:800) overnight at 4 °C.

Techniques: Generated, Confocal Microscopy, Labeling, Marker, Standard Deviation

Journal: Biomaterials Research

Article Title: Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate

doi: 10.1186/s40824-022-00328-9

Figure Lengend Snippet: Optical imaging and Anti-tumor activity of PTK7-GEMs in xenotransplanted subcutaneous tumors. A Time-lapse fluorescence imaging at 0.5, 1, 2, 3, and 4 h after tail vein injection of Cy5-labeled PTK7-GEMs (right) or Lib-GEMs (left). The tumor site is indicated using a red circle. B Biodistribution of PTK7-GEMs-cy5 or Lib-GEMs-cy5 in the major viscera and tumors at 4 h after injection. C Growth curve of the tumors during therapy. In vivo evaluation of antitumor effects in the 5637 xenografted bladder cancer mouse model ( n = 6 mice per group, n = 6 groups). D The tumor weights from the different groups are shown as a scattergram. E Images of tumor tissues from mice sacrificed at 32 days post inoculation. F Body weight changes during therapy. Data represents the mean ± SEM, n = 6; NS = not significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Thereafter, the membranes were reacted with rabbit polyclonal antibody recognizing PTK7 (Abcam; 1:500) or ACTB (quality control, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:800) overnight at 4 °C.

Techniques: Optical Imaging, Activity Assay, Fluorescence, Imaging, Injection, Labeling, In Vivo

Journal: Biomaterials Research

Article Title: Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate

doi: 10.1186/s40824-022-00328-9

Figure Lengend Snippet: The antitumor activity of PTK7-GEMs in a mouse model of lung metastasis bladder cancer induced by tail vein injection. A Photographs of lungs excised from mice bearing bladder cancer cell tumors treated as indicated. B Photographs of lung tissue sections from mice bearing bladder cancer cell tumors under different treatments stained with H&E. C Comparison of pulmonary metastatic nodules of bladder cancer cells tumor-bearing mice treated as indicated. D Changes in the body weights of bladder cancer cells bearing mice under various treatments. All data are presented as the mean ± SD ( n = 5). ** P < 0.01, *** P < 0.001

Article Snippet: Thereafter, the membranes were reacted with rabbit polyclonal antibody recognizing PTK7 (Abcam; 1:500) or ACTB (quality control, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:800) overnight at 4 °C.

Techniques: Activity Assay, Injection, Staining, Comparison

Journal: Biomaterials Research

Article Title: Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate

doi: 10.1186/s40824-022-00328-9

Figure Lengend Snippet: The antitumor activity of PTK7-GEMs in a model of in-situ bladder cancer model. A Photographs of H&E-stained tissue sections (muscle invasive bladder cancer at stage pT2 or higher and excised bladders after treatment with PBS. B Photographs of H&E-stained tissue sections (muscle invasive bladder cancer at stage pT2 or higher and excised bladders after treatment with PTK7. ( C ) Photographs of H&E-stained tissue sections (muscle invasive bladder cancer at stage pT2 or higher and excised bladders after treatment with LIB. D Photographs of H&E-stained tissue sections (noninvasive papillary carcinoma: stage pT1) and excised bladders after treatment with GEM. E Photographs of H&E-stained tissue sections (noninvasive papillary carcinoma: stage pT1) and excised bladders after treatment with LIB-GEMs. F Photographs of H&E-stained tissue sections (noninvasive papillary carcinoma: at stage pTa or lower) and excided bladders after treatment with PTK7-GEMs.

Article Snippet: Thereafter, the membranes were reacted with rabbit polyclonal antibody recognizing PTK7 (Abcam; 1:500) or ACTB (quality control, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:800) overnight at 4 °C.

Techniques: Activity Assay, In Situ, Staining