Journal: Journal of Chemical Information and Modeling

Article Title: SILVR: Guided Diffusion for Molecule Generation

doi: 10.1021/acs.jcim.3c00667

Figure Lengend Snippet: Schematic of the equivariant diffusion model with selective iterative latent variable refinement (SILVR) indicated for every denoising step. Here, the reference in blue on the left shows 3 small fragments. They evolve over time t in the diffusion process to resemble a Gaussian distribution at t = T , see eq . The β represents the noise added at each step, and the dots show the steps omitted from time t = 3 to t = T . As atoms effectively “diffuse”, they can be perceived as changing position. To generate a new sample, a sample is generated from p θ ( x ) according to eq , this distribution is from the learned EDM. At each denoising step, a set of reference fragments ( y t ) at that same level of noise t is used, which is indicated by the SILVR arrows to condition the EDM. This is controlled through SILVR at a given rate r S , until a new sample that resembles the reference is generated (following the bottom row along the yellow boxes and EDM arrows).

Article Snippet: The agreement in the shape of the samples and the binding site of MPro were determined using the OpenEye toolkit version 2022.2.2 Gaussian scoring function Shapegauss., This scoring function measures the shape complementarity between the ligand and receptor by considering each heavy atom as a Gaussian function.

Techniques: Diffusion-based Assay, Generated

Journal: Journal of Chemical Information and Modeling

Article Title: SILVR: Guided Diffusion for Molecule Generation

doi: 10.1021/acs.jcim.3c00667

Figure Lengend Snippet: Validation measures of the SILVR model using fragments x0072 and x0354 as reference coordinates. (A) Ratio of stable atoms—an atom is determined as stable if the valence matches the expected valence for the element. (B) RMSD from reference—the calculated RMSD between the reference and sample, using an absolute one-to-one mapping ignores atom identity with low RMSD meaning molecules are similar to the reference and high RMSD they are not. (C) OpenEye measure Shapegauss—a Gaussian scoring function describing the shape fit between Mpro and samples, ignoring chemical interactions. A lower score means a better shape fit of the molecule. (D) Geometry stability—AIMNet geometry optimization was completed with Auto3D using the SMILES string of each sample. RMSD was calculated between the predicted geometry and the sampled geometry using RDKit. Horizontal lines indicate the sample median and circles indicate the sample mean.

Article Snippet: The agreement in the shape of the samples and the binding site of MPro were determined using the OpenEye toolkit version 2022.2.2 Gaussian scoring function Shapegauss., This scoring function measures the shape complementarity between the ligand and receptor by considering each heavy atom as a Gaussian function.

Techniques: Biomarker Discovery

Journal: Cellular and molecular bioengineering

Article Title: Elucidation of Exosome Migration across the Blood-Brain Barrier Model In Vitro

doi: 10.1007/s12195-016-0458-3

Figure Lengend Snippet: (a) Schematic depiction of the isolation protocol for exosomes. (b) Size distribution of native and hGluc-Lact exosomes measured by nanoparticle tracking analysis (NTA). (c) Flow cytometry detection of exosome characterization. Exosomes were incubated with anti-CD63 Dynabeads and immunostained with exosomal surface markers (CD9, CD63, and CD81). Green histogram is the isotype control. Data were quantified and expressed as MFI (mean fluorescence intensity). (d) Western blot analysis of the marker proteins (i) CD63, (ii) CD81, and (iii) CD9 and (iv) Gluc on exosomes, respectively. Exosomes were purified from conditioned medium and characterized using western blot for the presence of typical exosomal markers CD63, CD81, and CD9. Additionally, Gluc detection by immunoblot of purified exosomes showed that hGluc-Lact fusion protein bound to the exosomal membranes.

Article Snippet: 25 μM (final concentration) of Gaussia luciferase substrate coelenterazine (CTZ, Nanolight) was added and bioluminescence activity was measured immediately using an IVIS Lumina (Caliper LifeSciences).

Techniques: Isolation, Flow Cytometry, Incubation, Control, Fluorescence, Western Blot, Marker, Purification

Journal: Cellular and molecular bioengineering

Article Title: Elucidation of Exosome Migration across the Blood-Brain Barrier Model In Vitro

doi: 10.1007/s12195-016-0458-3

Figure Lengend Snippet: (a) In vitro bioluminescence assay of conditioned medium, ultra-centrifugation supernatant and exosomes. (i) Conditioned medium collected from 293T cells, (ii) supernatant collected after serial steps of ultra-centrifugation and (iii) exosomes purified from 293T cells through ultra-centrifugation were diluted in PBS. CTZ was then added at a final concentration of 25 µM. Gaussia luciferase (hGluc) activity was measured using IVIS Lumina (exposure time 0.5s). (b) hGluc activity was significantly higher in conditioned medium. Error bar: mean ± SEM. **** P < 0.0001. (c) After ultra-centrifugation, hGluc activity was mainly detected in hGluc-Lact exosomes, indicating hGluc was enriched on exosomes. Error bar: mean ± SEM. **** P < 0.0001.

Article Snippet: 25 μM (final concentration) of Gaussia luciferase substrate coelenterazine (CTZ, Nanolight) was added and bioluminescence activity was measured immediately using an IVIS Lumina (Caliper LifeSciences).

Techniques: In Vitro, ATP Bioluminescent Assay, Centrifugation, Purification, Concentration Assay, Luciferase, Activity Assay

Journal: Cellular and molecular bioengineering

Article Title: Elucidation of Exosome Migration across the Blood-Brain Barrier Model In Vitro

doi: 10.1007/s12195-016-0458-3

Figure Lengend Snippet: (a) Schematic representation of the in vitro model of the BBB. hGluc-Lact exosomes were added to the luminal chamber of the transwell and incubated with BMECs for various time points. Both luminal and abluminal chambers of conditioned medium were collected for bioluminescence assay. (b) and (c) Exosomes can cross BMECs in stroke-like conditions. (b) Representation of in vitro bioluminescence assay. Conditioned medium from both luminal and abluminal chambers were collected after exosome incubation and CTZ was added at a final concentration of 25 µM. Gaussia luciferase activity was measured immediately thereafter using IVIS Lumina (exposure time 0.5s). (c) Quantitative analysis of in vitro bioluminescence assay of hGluc-Lact exosomes crossing both live and fixed BMECs at different time points. Relative Gluc Activity = (abluminal chamber signal - native exo signal) / (luminal chamber signal -native exo signal) × 100%. Error bar: mean ± SEM. Native vs. TNF-α: n.s ., not significant, * P < 0.05 and ** P < 0.01. Live BMECs (TNF-α) vs. fixed BMECs (TNF-α) at 24 hours: # P < 0.05.

Article Snippet: 25 μM (final concentration) of Gaussia luciferase substrate coelenterazine (CTZ, Nanolight) was added and bioluminescence activity was measured immediately using an IVIS Lumina (Caliper LifeSciences).

Techniques: In Vitro, Incubation, ATP Bioluminescent Assay, Concentration Assay, Luciferase, Activity Assay

Journal: Cellular and molecular bioengineering

Article Title: Elucidation of Exosome Migration across the Blood-Brain Barrier Model In Vitro

doi: 10.1007/s12195-016-0458-3

Figure Lengend Snippet: (a) Exosomes can cross the BMECs carrying hGluc in vitro . hGluc-Lact exosomes were labeled with the lipophilic dye PKH67, and were added to the luminal chamber of the transwell. Conditioned medium from abluminal chambers were collected and then incubated with a monolayer of BMEC on coverglass to further confirm the hGluc activity observed from bioluminescence assay was directly from exosomes. (b) Exosomes uptake by BMECs. hGluc conditioned medium of abluminal chamber stained with PKH67 was used as a control. Scale bar: 20 µm. (c) The schematic representation of exosome migration from abluminal chamber to the luminal chamber under native and TNF-α-treated conditions. (d) Quantitative analysis of exosome migration from abluminal to luminal chamber at 6 hours and 18 hours. Relative bioluminescence activity suggested that there was no significant difference between native and TNF-α-treated conditions at 6 hours, whereas the relative bioluminescence activity is significant higher in BMECs treated with TNF-α at 18 hours. Relative Gluc Activity = (luminal chamber signal - native exo signal) / (abluminal chamber signal - native exo signal) × 100%. Error bar: mean ± SEM. n.s ., not significant and * P < 0.05.

Article Snippet: 25 μM (final concentration) of Gaussia luciferase substrate coelenterazine (CTZ, Nanolight) was added and bioluminescence activity was measured immediately using an IVIS Lumina (Caliper LifeSciences).

Techniques: In Vitro, Labeling, Incubation, Activity Assay, ATP Bioluminescent Assay, Staining, Control, Migration

Journal: Cellular and molecular bioengineering

Article Title: Elucidation of Exosome Migration across the Blood-Brain Barrier Model In Vitro

doi: 10.1007/s12195-016-0458-3

Figure Lengend Snippet: BMECs were pretreated with indicated inhibitors: amiloride (1mM), CPZ (15 µM), cytochalasin D (20 µM), filipin III (5 µM), MβCD (5 mM), nystatin (5 µM) for 30 minutes at 37°C, respectively. hGluc-Lact exosomes were subsequently added to the luminal chamber of each transwell and incubated with BMECs for various time points (6 and 18 hours). Cells treated with vehicles (no inhibitor) alone were used as a negative control. To study the temperature effect on endocytosis, BMECs containing exosomes were incubated at either 37°C or 4°C for (a) 6 and (b) 18 hours, and then conditioned medium from both luminal and abluminal chambers were collected and Gaussia luciferase activity was measured immediately after addition of its substrate CTZ (IVIS Lumina, exposure time: 0.5s). Relative Gluc Activity = (abluminal chamber signal -native exo signal) / (luminal chamber signal - native exo signal) × 100%. Error bar: mean ± SEM. Native vs. TNF-α: n.s ., not significant, * P < 0.05 and ** P < 0.01. # P < 0.05 and ## P < 0.01, compared to native no inhibitor or TNF-α no inhibitor conditions, respectively.

Article Snippet: 25 μM (final concentration) of Gaussia luciferase substrate coelenterazine (CTZ, Nanolight) was added and bioluminescence activity was measured immediately using an IVIS Lumina (Caliper LifeSciences).

Techniques: Incubation, Negative Control, Luciferase, Activity Assay

Journal: The Analyst

Article Title: Modular calibrant sets for the structural analysis of nucleic acids by ion mobility spectrometry mass spectrometry

doi: 10.1039/c6an00453a

Figure Lengend Snippet: ATD's obtained from a) −8 charge state of 64b; b) −8 of 32a:b; and c) −9 of 64b. Red lines are Gaussian-fitted curves generated by Peakfit (see Experimental).

Article Snippet: In order to accurately identify the apex of each time-domain signal, the ATD traces were submitted to Gaussian fitting in PeakFit (v4.2 SeaSolve Software Inc., Framingham, MA) without any prior smoothing of the original raw data.

Techniques: Generated

Journal: Molecular Systems Biology

Article Title: A dynamic, spatially periodic, micro‐pattern of HES5 underlies neurogenesis in the mouse spinal cord

doi: 10.15252/msb.20209902

Figure Lengend Snippet: Schematic of extracting kymograph information from tissue data by averaging Venus::HES5 intensities observed in E10.5 heterozygous spinal cord slices to generate one intensity profile in the dorsal–ventral axis per timepoint (see Materials and Methods). Representative kymograph data showing spatiotemporal Venus::HES5 expression profile along ventral–dorsal direction in a 15 μm wide apical region and observed over 14 h; local bands of 20 μm width in D‐V; region of interest markers indicate: *low to high, **high to low and ***re‐occurring high/low activity in the same area. Hierarchical clustering of apical Venus::HE5 expression from one representative experiment showing behaviour in the same area over time; columns represent fluctuations in Venus::HES5 intensity in small local areas (bands) obtained by dividing the spatial signal into non‐overlapping 20 μm regions and normalising to the mean and standard deviation of each region over time (z‐scoring); data have been subject to a Gaussian blur pre‐processing step (see Appendix Fig S2B and Materials and Methods). Persistence of Venus::HES5 in 20 μm regions expressed as continuous time intervals when signal in the band is high or low compared with its mean (see Materials and Methods); individual datapoints (grey) indicate quantification of high and low persistence time obtained from over 300 thin bands collected from multiple tissues with 2 z‐stacks per tissue and two repeats (left and right of ventricle) per z‐stack; dots indicate paired medians of five independent experiments; statistical test is paired t ‐test of median per experiment with two‐tail significance and P = 0.7171. Persistence of Venus::HES5 levels in high and low states taken from 60 tracked single cells collected from three independent experiments; paired t ‐test not significant P = 0.0533. Relative distance between cell pairs computed from relative 3D Euclidean distance between nuclei over 12–15 h; dots indicate median distance over tracking period; horizontal lines show mean and SD of 14 cell pairs from three experiments. Spearman correlation coefficients computed in the same cell pairs from Venus::HES5 and H2B::mCherry (control) nuclear intensity timeseries; markers in each condition indicate pairs; black dots indicate median correlation coefficients per experiment (four pairs, three pairs and seven pairs); lines show median of 14 pairs from three experiments; paired t ‐test with significance P = 0.0058. Representative example timeseries of Venus::HES5 in cells pairs identified as remaining in close proximity; r ‐values indicate Spearman correlation coefficients between time traces over all co‐existing timepoints. Detrended Venus::HES5 fluorescent intensity timeseries (after z‐scoring) corresponding to examples in (H); red arrows indicate in‐phase peaks. Density phase plots from instantaneous Hilbert phase reconstruction at multiple timepoints over a 12–14 h period; dots indicate the phase angle in Cell 1 and Cell 2 from 14 pairs collected from three experiments; colormap indicates probability density showing accumulation of phase values predominantly along the (0,0) and (2π, 2π) diagonal; light colours indicate most frequent. Graphic representation of a neuroepithelial tissue with nuclei colour‐coded to indicate clusters of high or low HES5 expression. The tissue is illustrated at three different time points to depict how clusters of cells can dynamically switch from high to low or low to high while the periodic spatial pattern is maintained. In the example time traces (corresponding to the three grey and one red highlighted nuclei), synchronised ultradian oscillations are shown as being overlayed on the slow‐varying higher‐amplitude switching dynamics. Source data are available online for this figure.

Article Snippet: To account for any single‐cell movement in DV, we applied a 2 μm Gaussian blur filter onto the kymograph data using the MATLAB routine imgaussfilt.m prior to extracting timeseries per region.

Techniques: Expressing, Activity Assay, Standard Deviation, Control