The tetrazolium-based MTT assay was carried out as described in with minor modifications. Briefly, 2 × 104 cells/well were seeded into 96-well plates. Cells were treated with different concentrations of CFZ (0.5, 2, 8, and 32 nM) or BET inhibitors (I-BET762 and I-BET151) for 72 h in triplicates. For the assay, 10 µL of MTT (5 mg/mL) was added to each well and incubated for 1 h, followed by two washes with PBS. To dissolve the formazan crystals, 100 µl of DMSO was added to each well and absorbance was measured at 560 nm using a GloMax Explorer microplate reader.
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Next, to understand the impact of BET inhibitors in this context on a functional level, we turned to proteasome recovery assays that measure the ability of the cells to bounce back from proteasome inhibition . It is important to note that CFZ binds irreversibly to the proteasome β5 active site, whereas BTZ and IXA binding are reversible. We have previously demonstrated that in cells pulse treated with an irreversible proteasome inhibitor, the bounce-back response of proteasome activity almost exclusively relies on the Nrf1 pathway . Taking advantage of these facts, here, we treated three different cancer cells with CFZ for an hour such that the residual proteasome activity was ~10% as compared with the vehicle control. Then, we washed away the excess CFZ and followed the proteasome activity for 24 h, either in the absence or presence of BET inhibitors JQ1 and I-BET762 .
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Then, quantitative PCR was carried out with iTaq universal SYBR green supermix (Bio-Rad) using a C1000 Touch Thermal cycler (Bio-Rad). CFX manager 3.1 (Bio-Rad) was used for the analysis of data. 18S rRNA levels were used for normalization. Primers used for the assays are listed in Table S1.
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This highly questions the scientific validity of the test and makes it unsuitable as a specific diagnostic tool to identify the SARS-CoV-2 virus. The PCR products have not been validated at the molecular level. This fact makes the protocol useless as a specific diagnostic tool to identify the SARS-CoV-2 virus.
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Rapid tests have received a wave of negative attention following reports of inaccuracies in two such tests used in Nevada. The White House once relied on ID Now to screen visitors, a practice many scientific experts regarded as misguided. The task force worked with the researchers to design the study and make it financially possible for those who tested positive to isolate.
The pateint sample was cultured and the virus separated from the culture supernatant and then sequenced. I don’t know enough about sequencing to critique their mehtods but it would seem it has been isolated. I’d like to get a response about this paper. An exemplary scientific critique of the subject matter that deserves worldwide attention. It is abundantly clear to me that the so-called pandemic was driven by non-scientific agendas.
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Some remarks by Prof. Ulrike Kämmerer in an interview by the “Corona Ausschuss” meade it clear to me that there is another source of false positives that are not covered by the statistical definition. The dark agenda seems to be coordinated by the U.N. The virus outbreak in Wuhan was even negated by WHO for a while, allowing the virus to spread worldwide. That the more SAR-Cov2 virus-specific primers as negative/positive controls were not recommended tells a tale by itself. However, a review within 24 hours is very unusual at least in the field of my experience.
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The test has been validated on negative, positive and other viral controls (read the paper!). The RdRp-assay indeed has not the best design. However, this is a confirmatory assay and it has rather a lower sensitivity (see Voghels et al.). Yes, the E-gene assay might also detect Sars-CoV-1, but this mr bet bewertung virus is not really going around . There are extraneous factors that introduce contamination during the lab test procedure “on the bench” (or before!). Possibilities are the operator sneezing or positive control mnaterial finding its way into the test, or cross- contamination from other samples.
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- It is the general consensus to write genetic sequences always in the 5’-3’ direction, including the reverse primers.
- Thus, after that period of time, it cannot diagnose past Covid infection.
- Drosten is an author on this paper that declares the need to replace the primers that were reviewed in 24 hours.
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To address this question, we used a previously characterized NIH-3T3 cell line where Nrf1 has been knocked out using CRISPR/Cas9 . We treated both control wild-type and Nrf1KO cell lines for either 6 or 24 h with CFZ and analyzed the changes in gene expression using RNA-sequencing. Consistent with an established role for Nrf1 in inducing proteasome genes , we saw a robust increase in these genes in the WT, but not in the Nrf1KO cells in response to CFZ . We also observed a general increase in UPR-related genes in the WT cells in response to CFZ. Strikingly, under these conditions in the Nrf1KO cells treated with CFZ, we saw that a number of those UPR-related genes were hyperinduced .
Author: Bill Ordine