Endotoxin Mouse Killing understood by Australian application of PCR, Synchrotron Crystallography and Mathematical Models

Prompted by subscriber questions and links to papers, I look at one paper funded by Pfizer that made a major contribution published in 2013.

A paid subscriber kindly sent me a link to a website including a Wellcome Trust Grant to a multinational team researching a topic I will discuss later.

I saw a scientist at Monash University in Australia was included, so I had a quick look and found Professor Jamie Rossjohn was actually born and educated in Wales, so we can call him a migrant.

A search of PubMed finds he has published 538 papers, and refining the PubMed search yields 4 papers covering Endotoxin. His field of study is Immunity.

Deeper dives into his papers would find many more that deal with Jab contaminant Endotoxin, because he is an expert in TLRs.

That’s a challenge where I have been offered some help by skilled data miners.¹²

But now I concentrate on the paper in Nature that I find so useful, starting with their Figure 3 that shows what they found going on at molecular level.³

Figure 3 Contacts between IFNAR1 and IFN-β.

(a) Contacts between IFNAR1 SD1 and SD2 around the IFN-β helix CD and N terminus of helix D.

(b) Contacts between IFNAR1 SD1 and SD2 with Trp122 of IFN-β.

(c) Contacts between IFNAR1 SD2 and SD3 with the IFN-β helices B and D.

(d) Contacts between IFNAR1 SD3 and IFN-β helix C.

(e) Contacts between the β2–β3 loop of IFNAR1 SD2 that bind over the point of structural and sequence conservation between helices B of the IFN-β and IFN-ω structures.

IFNAR1 SD1 is in yellow, SD2 in green, SD3 in blue and SD4 in magenta; IFN-β helices A–E are in orange.

SD = Subdomain; IFNAR = The interferon-α/β receptor; IFN-β = Interferon beta

A few readers will be excited by this, but let’s wind back to their Figure 2 that shows Interferon-β (IFN-β) snuggling up to its receptor IFNAR1.

Readers will recall that IFN-β is the only type I Interferon induced by Endotoxin (Lipopolysaccharide, LPS) of Gram-negative Bacteria, activating Interferon Regulatory Factor 3 (IRF3) and Nuclear Factor kappa B (NF-κB) via the TLR4 pathway leading to Death among countless other Jab Harms.

Figure 2

Structure of the IFNAR1–IFN-β complex.

(a) Orthogonal views of the IFNAR1–IFN-β complex with IFNAR1 SD1 in yellow, SD2 in green, SD3 in blue and SD4 in magenta. Helices of IFN-β (orange; labeled A–E) are also indicated.

(b,c) Footprints mapped to surfaces of IFNAR1 (b) and IFN-β (c), with residues colored in each case according to the interacting IFNAR1 domain, as in a.

Killing the Mice via IFNAR1

Now let’s remember this paper is all about how Endotoxin Kills Mice, as shown in their Figure 5, part of which I show here

Figure 5

Pathophysiological relevance of the IFNAR1–IFN-β signaling axis.

(a) Kaplan-Meier plot showing the percentage survival over time of age-matched wild-type (WT), Ifnar1−/− and Ifnar2−/− mice (8 mice in each group) after in vivo intraperitoneal stimulation with Endotoxin (LPS) (4 mg/ mouse).

**P < 0.01 (log-rank (Mantel-Cox) test).

In simple terms, when they used GMO Mice with Interferon A Receptor 1 knocked out (Ifnar1−/−), survival time of the Mice after the normally Lethal Dose of Endotoxin improved dramatically. They used Endotoxin from K-235 E. coli purchased from Sigma-Aldrich and repurified using a method developed by Australian researchers in the group in 2011 to avoid contaminating proteins that would stimulate TLR pathways other than TLR4.⁴

There was no statistical difference for knockout of IFNAR2 in Mice.

Peter Hotez, pioneer in Killing Mice with “intraperitoneal stimulation” with Endotoxin would have been proud.⁵

Crystal Growth

One of my favourite pastimes since childhood and a major part of my career was crystal growth. For the Monash University study,

Crystals of the IFNAR1–IFN-β complex were grown by the hanging-drop vapor diffusion method at 24 °C of IFNAR1–IFN-β (1 μl at 12 mg/ml), in 10 mM Tris-HCl, pH 8.0, and 150 mM NaCl was mixed with 1 μl of reservoir solution comprising 12% (v/v) PEG 3350, 8% (v/v) tacsimate pH 6.0

I would have liked to have been a fly on the wall.

Synchrotron Data Collection

As mentioned before I have a number of brilliant crystallographer friends working at the Australian Synchrotron adjacent to the Moderna factory in Clayton.⁶

The Pfizer funded researchers in the present paper state:

crystals were equilibrated in a solution consisting of the reservoir solution enriched with 30% (v/v) PEG 3350 and then flash-cooled in a liquid nitrogen stream at –173 °C. Native data to 2.9 Å resolution were collected on the Macromolecular Crystallography (MX2) beamline of the Australian Synchrotron.

Structural determination was achieved by a combination of molecular replacement and single-wavelength anomalous diffraction (SAD) phasing as implemented in PHENIX. The position of the IFN-β cytokine was found by molecular replacement using PHASER with the crystal structure of IFN-β (Protein Data Bank (PDB): 1WU3; ref. 18) used as the search model. The position of SD2 and SD3 of IFNAR1 were similarly placed using PHASER with SD2 and SD3 of human IFNAR1 used as the search model (PDB: 3SE4; ref. 9). SD1 and SD4 of IFNAR1 were visualized in density modified electron density maps resulting from MR-SAD phasing using the partially determined molecular replacement model and SAD phases from a Ta6Br12 soaked crystal. Models of SD1 and SD4 of IFNAR1 were generated from the structure of SD2 of IFNAR1 using ROSETTA43 and then placed by a computational search of the electron density map as implemented in MOLREP. The structure was refined with iterative rounds of manual building in COOT and refinement with BUSTER

Fascinating details there including the use of the Tantalum cluster for Single-wavelength Anomalous Diffraction (SAD)!

Quite different to when I determined a crystal structure in 1979 using paper tape punched data collection under instruction by the marvelous Maureen Mackay.

PCR helped identify dysregulated Genes

The authors used quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR) to measure mRNA upregulated or downregulated for 235 genes after treatment of Ifnar2−/− Mice with purified IFN-β.

The sequences of primers used for qRT-PCR are shown in their Supplementary Table 4 for readers interested in such things.

Here are the Genes that showed at least a 3.3 fold change in expression.

I have briefly covered the Epigenetics of Endotoxin poisoning and observant readers will see some very interesting genes related to Jab Harms.⁷

Patent Application Abandoned

The paper states that the work was the subject of a patent application by Monash University, with No Inventors Mentioned which I found was abandoned.⁸

Perhaps Monash University and Pfizer could not agree on sharing costs of pursuing the patent, or it might have been deemed already covered by prior art?

If you search your collection of pdf downloads, expect to see IFNAR1.

1.

2. One generous paid subcriber suggested I look at data “pythons” to help, but as I mentioned, the process used by Humans involves looking at the superscripts of each author at the beginning and analyzing the linked affiliations, then recognizing the country. So a computer program would have to “think” like a human and also sometimes go to the end of the paper and match initials of each author to get the country.

3. Nicole A de Weerd, Julian P Vivian, Thao K Nguyen, Niamh E Mangan, Jodee A Gould, Susie-Jane Braniff, Leyla Zaker-Tabrizi, Ka Yee Fung, Samuel C Forster, Travis Beddoe, Hugh H Reid, Jamie Rossjohn and Paul J Hertzog. 2013. Structural basis of a unique interferon-b signaling axis mediated via the receptor IFNAR1. https://www.nature.com/articles/ni.2667

4. Claire J Greenhill , Stefan Rose-John, Rami Lissilaa, Walter Ferlin, Matthias Ernst, Paul J Hertzog, Ashley Mansell, Brendan J Jenkins. 2011. IL-6 trans-signaling modulates TLR4-dependent inflammatory responses via STAT3. https://journals.aai.org/jimmunol/article/186/2/1199/84608/IL-6-Trans-Signaling-Modulates-TLR4-Dependent

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8. No Inventors Mentioned. Method for identifying an interferon receptor (IFNAR) modulator, and uses thereof. 10 July 2013. https://patents.google.com/patent/AU2013902549A0/en