Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 1. Rapid testing microfluidic platform for early thrombosis recapitulates SARS-CoV-2 and Spike-mediated technologies thrombotic effect. a) Schematic of SARS-CoV-2 infection-mediated thrombus formation in the microcirculation. In response to viral infection, inflammatory cells are recruited to activate the extrinsic and intrinsic coagulation pathways, leading to thrombin production. Thrombin cleaves fibrinogen to fibrin, which in turn, pro- motes platelet aggregation and fibrin deposition to form blood clots in the microcirculation. b) Similarly, SARS-CoV-2 vaccination therapies Spike-based technologies have the potential role to increment blood coagulation. c) SARS-CoV-2, Spike protein, and Spike variant for mimicking microcirculation environment were assessed for their thrombotic phenotypes in multiple endothelialized microfluidic channels (2 cm x 400 mm x 100 mm). Antibody anti-IL6 and decoy nanoliposome-hACE2 were also tested together with the aforementioned conditions. SARS-CoV-2, Spike protein, and Spike variant expressed using viral vectors were incubated in the PDMS-based microfluidic channels for 12 h at 37 °C, followed by a thrombosis assay in the presence of human subject-specific whole blood at wall shear stress of 25 dyne cm−2. Thrombus formation was quantified in terms of fibrin and platelet deposition.

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Infection, Coagulation, Variant Assay, Incubation, Shear

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 3. SARS-CoV-2 and Spike-mediated inflammatory cytokines regulates coagulation cascade. a) Tissue factor (TF) binds to coagulation factor VII (FVII) to initiate the thrombosis. SARS-CoV-2 infection also induces endothelial release of cytokines (such as IL-1, IL-6, and TNF-𝛼) that mediate platelet activation and coagulation cascades. SARS-COV-2 treatment for 48 h significantly upregulated HAEC mRNA expression of TNF-𝛼, IL-1, IL-6, and IL-15 as demonstrated by the heatmap (*p < 0.05 CTRL vs SARS-COV-2, n = 4 by qRT-PCR). The heat map was constructed by using Euclidean distance with average linkage. The Z-score centered log2-transformed gene in each sample is presented by a color scale, and gene upregulation is denoted in blue, and downregulation in red. b) Spike mutation D614G inflammatory effect was tested HAECs. A microarray heatmap represents 22 genes and selected control genes in HAECs in response to Spike D614G. Hierarchical clustering heatmap reveal differentially expressed genes in response to Lenti-S mutation (in the presence or absence of Lipo-hACE2), normalized to the lenti-CTRL respectively. The heatmap was constructed as previously described, and the Z-score centered log2-transformed gene in each sample was presented as a color scale. Each condition was performed in triplicate (n = 3). In addition to increased cytokines and chemokines mRNA expression level, higher mRNA expression level of endothelial marker of thrombosis, such as vWF and PAI-1, was also observed. Furthermore, the activation of the Toll-like receptor signaling pathway suggests its crucial role in enhancing downstream inflammation and thrombosis (see Figure S5, Supporting Information) (*p < 0.05: n = 3). c) Immunocytochemical analysis showed Lenti-S D614G increasing protein level of IL-6 (in red). Nuclei were stained with DAPI (scale bar: 50 μm).

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Coagulation, Infection, Activation Assay, Expressing, Quantitative RT-PCR, Construct, Transformation Assay, Mutagenesis, Microarray, Control, Marker, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 4. Lipo-hACE2 and anti-IL-6 attenuate SARS-CoV-2-mediated inflammation and thrombosis. a) In the endothelialized microfluidic platform, HAECs were exposed to SARS-CoV-2, Lenti-S D614G, or Lipo-S in the presence or absence of Lipo-hACE2 or anti-IL-6 (scale bar = 100 μm). Lipo-hACE2 and anti-IL-6 attenuated SARS-CoV-2, Lenti-S, or Lipo-S-mediated fibrin deposition. b) Both Lipo-hACE2 and anti-IL-6 reduced SARS-CoV-2, Lipo-S, and Lenti-S mediated increases in surface coverage of fibrin and platelet deposition (*p < 0.05 vs Lipo-S or Lenti-S mutation; ***p < 0.001 vs SARS-CoV-2, n = 4).

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Mutagenesis

Journal: Nature Communications

Article Title: Designer diatom episomes delivered by bacterial conjugation

doi: 10.1038/ncomms7925

Figure Lengend Snippet: ( a ) Conjugation from E. coli to T. pseudonana results in increased conjugation efficiency when the yeast CEN6-ARSH4-HIS3 region is included on the plasmid (pTpPuc3) compared with control plasmid lacking this region (pTpPuc4). Error bars denote one s.d. of the mean from at least three biological replicates. ( b , c ) Images of T. pseudonana wild type ( b ) and exconjugants expressing YFP translationally fused to PEPCK (Protein ID_5186) encoded on a p0521s-derived episome ( c ). Scale bar, 2.5 μm.

Article Snippet: E. coli strains containing plasmids p0521s (GenBank accession KP745602), pPtPuc3 (GenBank accession KP745601) and pTpPuc3 (GenBank accession KP745603) described in this manuscript have been deposited at Addgene.com and are available for request.

Techniques: Conjugation Assay, Plasmid Preparation, Control, Expressing, Derivative Assay

Journal: Nature Communications

Article Title: iFISH is a publically available resource enabling versatile DNA FISH to study genome architecture

doi: 10.1038/s41467-019-09616-w

Figure Lengend Snippet: iFISH implementation. a Scheme of iFISH4U. Pre-designed genome-wide databases of oligos (left) are used as input by the iFISH4U web interface (center) to select oligos within one or more user-specified genomic regions, based on the indicated features. Features 1–3 are used while designing single probes, whereas all the four features are used to design multiple probes on the same chromosome. The black dashed boxes indicate examples of probes within the same region of interest, with the same number of oligos (vertical bars), but suboptimal size (1), homogeneity (2), or centrality (3), whereas the orange box represents the probe of choice having optimal size, homogeneity and centrality. b Cumulative distribution of the distances between consecutive oligos in the human 40-mers database. c Median standard deviation (s.d.) of the distance between consecutive oligos, inside non-overlapping genomic windows of the indicated size, in the 40-mers database and OligoMiner (OM) hg19 databases. OMB, OM ‘Balance’. OMC, OM ‘Coverage’. OMS, OM ‘Stringent’. d Percentage of non-overlapping genomic windows of the indicated size, containing at least 96 oligos, in the 40-mers database and OM hg19 databases. e Scheme of oligos in iFISH probes. Each probe consists of n oligos differing in the T sequence. f Location of the 330 iFISH probes targeting all the human autosomes and chrX. Red dots, individually tested probes (see Fig ). g Scheme of the pipeline used to produce iFISH probes. (1) Up to 12,000 oligos, corresponding to a maximum of 125 probes each containing 96 oligos, are synthesized on an array and then pooled together. (2) The oligo-pool is dispensed into n 96-well plates, depending on the total number of probes ( p ) and colors per probe ( c ). (3) In each well, the oligos corresponding to the same probe are selectively amplified using a probe-specific PCR primer pair that incorporates the T7 promoter sequence (T7) and color adapter sequence (C), and (4) successfully amplified probes are purified and linearly amplified by in vitro transcription (IVT). (5) Purified IVT products are reverse transcribed (RT), (6) RNA is hydrolyzed, and finally (7) single-stranded DNA (ssDNA) is purified to obtain ready-to-use probes

Article Snippet: We make all the 380 probes described in this study—as well as additional probes which we are continuously adding to our database—available to the community as a non-profit DNA FISH probe repository ( http://ifish4u.org/browse ), following the model of Addgene for plasmids.

Techniques: Genome Wide, Standard Deviation, Sequencing, Synthesized, Amplification, Purification, In Vitro, Reverse Transcription

Journal: Nature Communications

Article Title: iFISH is a publically available resource enabling versatile DNA FISH to study genome architecture

doi: 10.1038/s41467-019-09616-w

Figure Lengend Snippet: Visualization of chromosomal territories by iFISH chromosome-spotting probes. a Representative images of chr1, 6, and 17 territories in IMR90 cells. Blue, DNA. Scale bar: 10 μm. b Frequency distributions of dot counts per nucleus in the images of which a is a representative example. Dashed lines, expected dot counts per nucleus. n , number of G1-phase cells analyzed. c Scheme of ‘dense’ chr17-spotting probe consisting of 63 probes spaced every ~1.25 Mb, in four alternating colors. AT542, ATTO 542 dye. AF594, Alexa Fluor 594 dye. AT647N, ATTO 647N dye. IR800, IRDye 800 dye. d Examples of chr17 territories visualized using the probes depicted in c in HAP1 cells. e Chr17 territory volume estimated based on FISH dots in one, two, three, or four colors, using the probes shown in c . P values (Wilcoxon test, two-tailed) are shown for each of the indicated pair-wise comparisons. In all the box plots, the central line represents the median, the bottom and upper bounds of the box represent the 25th and 75th percentile respectively, and the whiskers extend from −1.5 × IQR to + 1.5 × IQR from the closest quartile, where IQR is the inter-quartile range. f Simultaneous visualization of six different chromosomes in mitotic hESCs using chromosome-spotting probes. Scale bar: 10 μm. All the microscopy images in this figure are the maximum intensity z-projection of each channel

Article Snippet: We make all the 380 probes described in this study—as well as additional probes which we are continuously adding to our database—available to the community as a non-profit DNA FISH probe repository ( http://ifish4u.org/browse ), following the model of Addgene for plasmids.

Techniques: Two Tailed Test, Microscopy

Journal: Nature Communications

Article Title: Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox

doi: 10.1038/s41467-019-09678-w

Figure Lengend Snippet: Design of SpyDock. a All small residues except D blocked SpyCatcher reaction. E77 was mutated to the indicated residue and incubated with SpyTag-MBP for 24 h, before boiling in SDS and analysis by SDS-PAGE with Coomassie staining. b Additional mutations in SpyCatcher enhanced Spy&Go purification. The original SpyCatcher, the evolved SpyCatcher002 or SpyCatcher2.1 bearing the E77A mutation were compared for capture and elution of SpyTag-MBP (SDS-PAGE with Coomassie staining). Further E77X mutations were similarly explored on SpyCatcher2.1. c Positions of mutations screened for SpyDock acceleration, based on Protein Data Bank 4MLI; CnaB2 triad represented in spheres, anchoring sites in yellow, and SpyCatcher2.1 accelerating mutations in green. d S49C resin anchoring allowed efficient SpyDock purification. SpyCatcher2.1 E77A S49C was coupled to SulfoLink resin and tested for SpyTag-MBP capture and release (SDS-PAGE with Coomassie staining)

Article Snippet: Inserts were verified by Sanger sequencing. pDEST14-SpyCatcher2.1 was derived from pDEST14-SpyCatcher002 (GenBank MF974388 and Addgene plasmid ID 102827) with additional A89P, Q97D and K108E mutations (see below). pDEST14-SpyCatcher2.1 S49C E77A (SpyDock) (Supplementary Fig. , GenBank MK637462, Addgene plasmid ID 124618) has the organization: His 6 , SpyCatcher2.1 with E77A S49C mutations, GSSGS. pDEST14-SpyCatcher2.1 E77X S49C has the same organization as SpyDock except with E77X mutations instead, with X being D, G, N, Q, S, T, or V. pDEST14-SpyCatcher2.1 E77A N-term Cys has the same organization as SpyDock except with a cysteine-anchoring mutation at the 6 th amino acid residue preceding SpyCatcher2.1 E77A instead of S49C. pDEST14-SpyCatcher2.1 S55C E77A has the same organization as SpyDock except with a S55C mutation instead of S49C mutation. pET28a-SpyTag-MBP (Addgene plasmid ID 35050) and pET28a-SpyTag002-MBP (GenBank MF974389 and Addgene plasmid ID 102831) were as described. pENTR4-EpCAM-SpyTag (GenBank MK637463) was derived from pENTR4-EpCAM-SnoopTagJr (GenBank MH511516) with the organization: tissue plasminogen activator (tPA) secretion leader sequence, extracellular domain of human EpCAM protein (EpCAM; residue 24–265), (GSG) , SpyTag, GEGS, His 6 . pENTR4-LPTOS-CyRPA-SpyTag (GenBank MH425516) was previously published. pET28a-αDR5-SpyTag (GenBank MK637464) was derived from pET28a-SnoopTag-αDR5-SpyTag (GenBank KU500643) with the organization: αDR5 (4E6 nanobody ), (GGGGS) , SpyTag. pET28a-SpyTag-mClover3 has the organization: SpyTag, SGGGSG, mClover3 , GSGSGS, His 6 . pET28a-scPvuII-SpyTag (GenBank MK637465) has the organization: His 6 , SSG, PvuII from Proteus vulgaris , GSG, TEV cleavage site, GGSG, SpyTag, GSGG, PvuII.

Techniques: Residue, Incubation, SDS Page, Staining, Purification, Mutagenesis

Journal: STAR Protocols

Article Title: Cloning BRD4 long isoform into overexpression vectors for stable overexpression of BRD4-L in mammalian cells

doi: 10.1016/j.xpro.2022.101785

Figure Lengend Snippet:

Article Snippet: Download the appropriate sequences. a. Download the Reference Sequence for Homo sapiens bromodomain containing 4 (BRD4), transcript variant long, mRNA (GenBank: NM_058243.3) from the NCBI’s website, using the GenBank data base ( BRD4-L RefSeq ). b. Download the sequence for LentiV_Blast (Addgene, cat# 111887) from Addgene’s website ( LentiV_Blast ).

Techniques: Virus, Recombinant, Cloning, Gel Extraction, Plasmid Preparation, Sequencing, Over Expression, Expressing, Software, Imaging