Genetically Engineered MRI-Trackable Extracellular Vesicles as SARS-CoV-2 Mimetics for Mapping ACE2 Binding In Vivo

SciScore for 10.1101/2022.03.27.485958: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IACUC: All animal studies were approved in accordance with the Weizmann Institute’s Animal Care and Use Committee (IACUC) guidelines and regulations (approval number 00580120-3). Euthanasia Agents: The mice were measured under general isoflurane inhalation anesthesia (5% induction, 1% maintenance) up to four hours after EVs injection using a gradient echo Fast Low Angle Shot (FLASH) with the following parameters: TR = 300 ms; TE = 2 ms; resolution of 0.23×0.23×0.7 mm3.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After overnight blocking in 5% …

SciScore for 10.1101/2022.03.27.485958: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IACUC: All animal studies were approved in accordance with the Weizmann Institute’s Animal Care and Use Committee (IACUC) guidelines and regulations (approval number 00580120-3). Euthanasia Agents: The mice were measured under general isoflurane inhalation anesthesia (5% induction, 1% maintenance) up to four hours after EVs injection using a gradient echo Fast Low Angle Shot (FLASH) with the following parameters: TR = 300 ms; TE = 2 ms; resolution of 0.23×0.23×0.7 mm3.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After overnight blocking in 5% milk in TBST, specific antibodies were applied to the membranes for 1 h to detect markers: CD81 (B-II, Santa Cruz, USA); c-myc (9E10, Santa Cruz, USA) (all 1:500); and beta-actin (C4, Santa Cruz, USA) (1:1000).	CD81 (B-II, Santa Cruz, USA) suggested: None c-myc suggested: (Covance Cat# MMS-150P-1000, RRID:AB_291322) 9E10 suggested: (Covance Cat# MMS-150R-500, RRID:AB_291327) beta-actin suggested: None C4 suggested: None
HRP-conjugated anti-mouse secondary HRP goat anti-mouse IgG antibody (#4053, Biolegend, USA) (1:5000 in TBST) was applied for 1 h before imaging using enhanced chemiluminescence substrate EZ-ECL Kit (Biological industries, catalog no. 20-500-120).	anti-mouse suggested: None anti-mouse IgG suggested: None
The expression of ACE2 protein in cells was also confirmed by Western blot analysis, as described above, using a rabbit monoclonal ACE2 antibody (ab239924; 1:1000; Abcam, USA).	ACE2 suggested: (Abcam Cat# ab239924, RRID:AB_2861381)
Then the cells were detached from wells by PBS and incubated with ACE2 primary antibody (ab239924; 1:1000; Abcam, USA) for 1 hour on ice, washed with PBS and then incubated with anti-rabbit fluorescently-conjugated secondary antibody (Alexa Fluor 647 nm) for 1 hour on ice.	anti-rabbit fluorescently-conjugated secondary suggested: None
Experimental Models: Cell Lines
Sentences	Resources
The stably expressing ACE2 HEK293T cell line was kindly obtained from the lab of Dr. Ron Diskin (Weizmann Institute of Science) and kept under puromycin antibiotics (0.5 μg/ml, Invitrogen, USA).	HEK293T suggested: RRID:CVCL_HA71)
HEK-pAGDisplay-RBD stable cell line generation: The pAGDisplay-based plasmids expressing RBD (1 ug of DNA) were transfected in a 60 mm culture dish with 80% confluent HEK293 cells by a JetPrime transfection reagent (Polyplus, France) according to the manufacturer’s protocol.	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
Binding assays and affinity curve determination using cell-display: The HEK-pAGDisplay-RBD stable cells grown to 80% confluency were gently detached by Accutase (1/2 solution in PBS, 3 min, Sigma Aldrich cat.	HEK-pAGDisplay-RBD suggested: None
Recombinant DNA
Sentences	Resources
Colony PCR and sequencing were used for analysis and verification. pAGDisplay vector construction: pDisplay Mammalian Expression vector was purchased from Invitrogen (V66020).	pAGDisplay suggested: None
The pAGDisplay vector backbone was assembled by combining a pET28b fragment bearing KanR and origin of replication, a pLVX vector fragment bearing WPRE, PuroR, and IRES sequences, and a pDisplay CMV promoter with a PDGFRβ expression cassette by a restriction-free three-component assembly 58.	pLVX suggested: RRID:Addgene_174088)
Protein production, purifications, and labeling procedures: The designed ALFA-tag binding nanobody (DnbALFA) and its mNeonGreen fusion were expressed by using expression plasmid pET28bdSUMO 62 and E.coli BL21(DE3) cells as described previously 60.	pET28bdSUMO suggested: None
Soluble his-tagged peptidase domain of ACE2 protein (Q18 – S740), inserted in pHLsec plasmid, was expressed in an Expi293F cell system with an ExpiFectamine 293 Transfection Kit (ThermoFisher, USA) according to the manufacturer’s protocol and purified as described previously 45.	pHLsec suggested: None
HEK-pAGDisplay-RBD stable cell line generation: The pAGDisplay-based plasmids expressing RBD (1 ug of DNA) were transfected in a 60 mm culture dish with 80% confluent HEK293 cells by a JetPrime transfection reagent (Polyplus, France) according to the manufacturer’s protocol.	pAGDisplay-based suggested: None
Software and Algorithms
Sentences	Resources
Mean FL-4 fluorescence signal values of RBD+ cells, subtracted by RFnano and the nonspecific signal of the RBD-population, were used to determine the KD of binding constants using a non-cooperative Hill equation and a nonlinear least-squares regression using Python 3.7 45.	Python suggested: (IPython, RRID:SCR_001658)
The fluorescence signal (APC filter) of cells was measured by a FACS machine (LRS-II) and analyzed by the FlowJo software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
MR images were analyzed by the ImageJ software or by a custom-made script written in MATLAB (MathWorks, USA).	MATLAB suggested: (MATLAB, RRID:SCR_001622)
After tuning and matching to the1H frequency, shimming of the magnetic field, and B0 correction, the tumor area was measured with the following parameters: both axial and coronal images were acquired; T2 and T2* maps were reconstructed in the Paravision software; and were followed by analysis in the ImageJ software.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software Inc., USA).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Genetically Engineered MRI-Trackable Extracellular Vesicles as SARS-CoV-2 Mimetics for Mapping ACE2 Binding In Vivo

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