Journal: Nature

Article Title: Latent human herpesvirus 6 is reactivated in CAR T cells

doi: 10.1038/s41586-023-06704-2

Figure Lengend Snippet: a, Quantification of HHV-6B by qPCR at day 19 for four donors normalized to the cell count. Bars are shown in order of increasing abundance. b, Longitudinal qPCR surveillance of HHV-6B U31 gene copies in CAR T cell culture from two donors. c, Schematic of the single-cell sequencing workflow to detect HHV-6+ cells from the CAR T culture. Two models are presented that would explain HHV-6 reactivation: model 1 (top), in which all cells express HHV-6 transcripts; and model 2 (bottom), in which only a subset of cells express HHV-6B. Both the host and HHV-6B viral RNA can be directly quantified using the 10x Genomics scRNA-seq workflow. d, Summary of HHV-6B expression from an individual donor (98). The top 0.2% of cells contain 99% of the HHV-6B transcript UMIs from this experiment. e, Tabulated summary of scRNA-seq profiling for four CAR T donors, including number of cells profiled, percentage expressing HHV-6, U31 qPCR value and number of shared TCR clones between the HHV-6B+ cells. N/A, not applicable. f, Extended longitudinal sampling of HHV-6B through U31 qPCR. Number at right end of each plot line indicates the fold (×) increase from the first qPCR measurement (day 21) to the final measurement (day 27; black outline) for each donor. g, Schematic and summary of HHV-6B expression in donor 34 after 19 and 25 days, showing evidence of HHV-6B spreading in the culture as depicted in the schematic. h, Correlation analyses of host factor gene expression with HHV-6B RNA abundance in individual cells for donor 34 on day 25. The per-gene correlation statistics are shown in black against a permutation of the HHV-6B expression in grey. Select genes are indicated. i, Pathway enrichment analysis of Molecular Signatures Database Hallmark gene sets for gene set enrichment analysis. A positive normalized enrichment score corresponds to genes that are overexpressed in cells with large amounts of HHV-6B transcript.

Article Snippet: Both the host and HHV-6B viral RNA can be directly quantified using the 10x Genomics scRNA-seq workflow. d , Summary of HHV-6B expression from an individual donor (98).

Techniques: Cell Counting, Cell Culture, Sequencing, Expressing, Clone Assay, Sampling, Gene Expression

Journal: Nature

Article Title: Latent human herpesvirus 6 is reactivated in CAR T cells

doi: 10.1038/s41586-023-06704-2

Figure Lengend Snippet: (a) Schematic of the experiment where CAR T cells from D98 were profiled using the 10x Genomics Multiome workflow to detect both viral DNA and RNA. (b) Scatter plot of the abundance of viral DNA and RNA at single-cell resolution. Pearson correlation between the log10 abundances is shown. (c) Per-cell viral gene expression signatures. The proportion of viral gene expression belonging to each class (late, early, immediate early) per cell is shown. (d) Same plot as in (c) but colored by the log2 number of viral DNA fragments. The population of cells highly expressing early HHV-6 transcripts show a corresponding high HHV-6 DNA copy number is highlighted by the arrow. (e) Pearson correlation of HHV-6 transcript signatures with their log abundance of DNA fragments per cell. The two-sided p-value for the Pearson correlation test is noted by each bar. (f) Bulk-level RNA and DNA correspondence in the four donors studied in the day 19 allogeneic CAR products.

Article Snippet: Both the host and HHV-6B viral RNA can be directly quantified using the 10x Genomics scRNA-seq workflow. d , Summary of HHV-6B expression from an individual donor (98).

Techniques: Gene Expression, Expressing

Journal: Nature

Article Title: Latent human herpesvirus 6 is reactivated in CAR T cells

doi: 10.1038/s41586-023-06704-2

Figure Lengend Snippet: (a) Schematic of CAR T product reculture experiment. Donor D97, which at day 19 showed a low but detectable level of HHV-6, was selected for reculture for five days. (b) Summary of RT-qPCR at the control and two treatment levels of Foscarnet. Each dot represents a technical replicate over one biological replicate per condition (validated in panel d). HHV-6 was not detected (n.d.) at the 1 mM concentration. Error bars represent the standard error of the mean. Comparison of foscarnet treated to untreated resulted in significantly lower abundance of HHV-6 RNA (p = 0.00026; two-sided ordinary least squares linear model). (c) Schematic of D34 reculture +/− foscarnet at 1 mM. (d) Difference between untreated and treated in the abundance of HHV-6+ cells. Comparing the two 10x Genomics scRNA-seq data channels, foscarnet-treated cells had a lower incidence of HHV-6 positive cells (OR = 6.25; p = 8.3e-122; Fisher’s exact test, two-sided). (e) Reduced dimensionality analysis of treated and untreated D34 cells profiled with scRNA-seq. Host gene expression was used for the analysis, showing overlapping clustering of populations irrespective of treatment status. (f) Differential gene expression analysis comparing foscarnet treated and control CAR T cells. The three most significant differential genes are noted. 0 genes were differentially expressed with a minimum log2 fold-change exceeding 1 (noted by the red).

Article Snippet: Both the host and HHV-6B viral RNA can be directly quantified using the 10x Genomics scRNA-seq workflow. d , Summary of HHV-6B expression from an individual donor (98).

Techniques: In Vitro, Quantitative RT-PCR, Control, Concentration Assay, Comparison, Gene Expression

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: Time Savings Using a Digital Workflow versus a Conventional for Intraocular Lens Implantation in a Corporate Chain Hospital Setting

doi: 10.2147/OPTH.S439930

Figure Lengend Snippet: Time assessments for digital cataract workflow and existing conventional workflow. *Time 1 was calculated as the sum of the mean times recorded for each step: (IOL Master) + (Pentacam) + (OCT) + (endothelial cell count). **Time 3: Digital transfer step not applicable in digital cataract workflow as it is automated. # Times 4 and 5: IOL axis marking is not applicable in the digital cataract workflow as it is done via the FORUM ® platform/CALLISTO eye.

Article Snippet: In conclusion, significant time savings at each step of cataract surgery planning were observed with the Zeiss digital cataract workflow compared with the existing conventional workflow.

Techniques: Cell Counting

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: Time Savings Using a Digital Workflow versus a Conventional for Intraocular Lens Implantation in a Corporate Chain Hospital Setting

doi: 10.2147/OPTH.S439930

Figure Lengend Snippet: Time Measurements at Various Steps in Site’s Existing Conventional and Digital Cataract Workflow

Article Snippet: In conclusion, significant time savings at each step of cataract surgery planning were observed with the Zeiss digital cataract workflow compared with the existing conventional workflow.

Techniques: Cell Counting

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: Time Savings Using a Digital Workflow versus a Conventional for Intraocular Lens Implantation in a Corporate Chain Hospital Setting

doi: 10.2147/OPTH.S439930

Figure Lengend Snippet: Inter observer variability in the digital cataract workflow versus existing conventional workflow for preoperative assessments.

Article Snippet: In conclusion, significant time savings at each step of cataract surgery planning were observed with the Zeiss digital cataract workflow compared with the existing conventional workflow.

Techniques:

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: Time Savings Using a Digital Workflow versus a Conventional for Intraocular Lens Implantation in a Corporate Chain Hospital Setting

doi: 10.2147/OPTH.S439930

Figure Lengend Snippet: Overall time savings in digital cataract workflow versus existing conventional workflow.

Article Snippet: In conclusion, significant time savings at each step of cataract surgery planning were observed with the Zeiss digital cataract workflow compared with the existing conventional workflow.

Techniques:

Journal: bioRxiv

Article Title: A Cryo-/Liquid Phase Correlative Light Electron Microscopy Workflow to Visualize Crystallization Processes in Graphene Liquid Cells

doi: 10.1101/2023.05.08.539575

Figure Lengend Snippet: Schematic overview of the cryo-/LP-CLEM workflow. Graphene liquid cell preparation is completely automated. The machine etches away the copper (7°C), transfers the graphene to a TEM grid via loop-assisted transfer and seals the GLCs by blotting away excess liquid. The reaction starts once the liquid pockets are sealed. Live fluorescence microscopy can be used to determine not only the location, but the specific time to image at high resolution, at which point the process is arrested by rapid vitrification. ROIs are located using cryo-fluorescence microscopy and hereafter they are imaged at nanometer resolution (cryo-TEM). The process is reinitiated inside the microscope by heating-up the grid using a cryo-holder, and the reaction dynamics are directly recorded (LP-TEM). To confirm that GLCs remain intact and retain liquid after TEM observation, fluorescence microscopy is used.

Article Snippet: After vitrification, the TEM grids were loaded into a universal TEM cryo-holder (349559-8100-010) using the ZEISS Correlative Cryo Workflow solution, which fit into the PrepDek® (PP3010Z, Quorum technologies, Laughton, UK).

Techniques: Fluorescence, Microscopy

Journal: bioRxiv

Article Title: A Cryo-/Liquid Phase Correlative Light Electron Microscopy Workflow to Visualize Crystallization Processes in Graphene Liquid Cells

doi: 10.1101/2023.05.08.539575

Figure Lengend Snippet: Cryo-/LP-CLEM workflow to visualize crystallization processes inside a GLC. a) TEM overview imaged two days after thawing, overlaid with live-FM (green) to indicate the GLCs. Insert shows a high magnification LP-TEM image of the GLC in the blue box. b) SAED pattern taken at the position indicated by the black dashed circle in . Inner ring in the DP shows the contribution of the [100] plane on NaCl and outer ring the contribution of the graphene. c) Enlargement of the area marked by the orange box in were multiple GLCs are present (green, yellow and purple boxes) and a crystal that is not encapsulated by graphene (red box). D-f) Large crystal inside a GLC imaged at multiple time points after thawing (3d: 2 days; 3e: 5 days; 3f: 7 days) shows morphological changes. Graphene wrinkles (close yellow arrow), outline of the GLC (dotted yellow line) and the intensity gradient of the liquid surrounding the crystal (open yellow arrow) are visible at all time points. g) Two different but interconnected GLCs imaged two days after thawing (open and closed purple arrows). h) Condensation (solid arrow), and phase transformation (open purple arrow) observed within the pockets five days after thawing. I) Cubic NaCl crystal resulting from an Ostwald ripening process observed seven days after thawing j) Liquid pockets (green dashed circles) containing an amorphous-like phase. k) Nucleation within the amorphous phase contained in the GLCs (green striped circles). l) Dissolution of small crystals (top circle) and further crystallization (bottom circle) inside the GLCs. m) Crystal not encapsulated by graphene outlined by the red dotted line shows only minimum morphological changes five (3n) and seven (3o) days after thawing. Accumulative dose: a) 0.75e - /Å ; c, d, g, j, m) 0.15 e - /Å ; e, h, k, n) 0.30 e - /Å ; f, i, l, o) 0.45 e - /Å

Article Snippet: After vitrification, the TEM grids were loaded into a universal TEM cryo-holder (349559-8100-010) using the ZEISS Correlative Cryo Workflow solution, which fit into the PrepDek® (PP3010Z, Quorum technologies, Laughton, UK).

Techniques: Crystallization Assay, Transformation Assay