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https://www.mdpi.com/1467-3045/44/3/73

Intracellular Reverse Transcription of Pfizer BioNTech COVID-19 mRNA Vaccine BNT162b2 In Vitro in Human Liver Cell Line

Open AccessArticle

¹

Department of Clinical Sciences, Lund University, 20502 Malmö, Sweden

²

Infection Medicine, Department of Clinical Sciences, Lund University, 22362 Lund, Sweden

^*

Author to whom correspondence should be addressed.

Curr. Issues Mol. Biol. 2022, 44(3), 1115-1126; https://doi.org/10.3390/cimb44030073

Received: 18 January 2022 / Revised: 19 February 2022 / Accepted: 23 February 2022 / Published: 25 February 2022

(This article belongs to the Topic Clinical, Translational and Basic Research on Liver Diseases)

Abstract

Preclinical studies of COVID-19 mRNA vaccine BNT162b2, developed by Pfizer and BioNTech, showed reversible hepatic effects in animals that received the BNT162b2 injection. Furthermore, a recent study showed that SARS-CoV-2 RNA can be reverse-transcribed and integrated into the genome of human cells. In this study, we investigated the effect of BNT162b2 on the human liver cell line Huh7 in vitro. Huh7 cells were exposed to BNT162b2, and quantitative PCR was performed on RNA extracted from the cells. We detected high levels of BNT162b2 in Huh7 cells and changes in gene expression of long interspersed nuclear element-1 (LINE-1), which is an endogenous reverse transcriptase. Immunohistochemistry using antibody binding to LINE-1 open reading frame-1 RNA-binding protein (ORFp1) on Huh7 cells treated with BNT162b2 indicated increased nucleus distribution of LINE-1. PCR on genomic DNA of Huh7 cells exposed to BNT162b2 amplified the DNA sequence unique to BNT162b2. Our results indicate a fast up-take of BNT162b2 into human liver cell line Huh7, leading to changes in LINE-1 expression and distribution. We also show that BNT162b2 mRNA is reverse transcribed intracellularly into DNA in as fast as 6 h upon BNT162b2 exposure.

Keywords:

COVID-19 mRNA vaccine; BNT162b2; liver; reverse transcription; LINE-1; Huh7

1. Introduction

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was announced by the World Health Organization (WHO) as a global pandemic on 11 March 2020, and it emerged as a devasting health crisis. As of February 2022, COVID-19 has led to over 430 million reported infection cases and 5.9 million deaths worldwide [1]. Effective and safe vaccines are urgently needed to reduce the morbidity and mortality rates associated with COVID-19.

Several vaccines for COVID-19 have been developed, with particular focus on mRNA vaccines (by Pfizer-BioNTech and Moderna), replication-defective recombinant adenoviral vector vaccines (by Janssen-Johnson and Johnson, Astra-Zeneca, Sputnik-V, and CanSino), and inactivated vaccines (by Sinopharm, Bharat Biotech and Sinovac). The mRNA vaccine has the advantages of being flexible and efficient in immunogen design and manufacturing, and currently, numerous vaccine candidates are in various stages of development and application. Specifically, COVID-19 mRNA vaccine BNT162b2 developed by Pfizer and BioNTech has been evaluated in successful clinical trials [2,3,4] and administered in national COVID-19 vaccination campaigns in different regions around the world [5,6,7,8].

BNT162b2 is a lipid nanoparticle (LNP)–encapsulated, nucleoside-modified RNA vaccine (modRNA) and encodes the full-length of SARS-CoV-2 spike (S) protein, modified by two proline mutations to ensure antigenically optimal pre-fusion conformation, which mimics the intact virus to elicit virus-neutralizing antibodies [3]. Consistent with randomized clinical trials, BNT162b2 showed high efficiency in a wide range of COVID-19-related outcomes in a real-world setting [5]. Nevertheless, many challenges remain, including monitoring for long-term safety and efficacy of the vaccine. This warrants further evaluation and investigations. The safety profile of BNT162b2 is currently only available from short-term clinical studies. Less common adverse effects of BNT162b2 have been reported, including pericarditis, arrhythmia, deep-vein thrombosis, pulmonary embolism, myocardial infarction, intracranial hemorrhage, and thrombocytopenia [4,9,10,11,12,13,14,15,16,17,18,19,20]. There are also studies that report adverse effects observed in other types of vaccines [21,22,23,24]. To better understand mechanisms underlying vaccine-related adverse effects, clinical investigations as well as cellular and molecular analyses are needed.

A recent study showed that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the genome of human cells [25]. This gives rise to the question of if this may also occur with BNT162b2, which encodes partial SARS-CoV-2 RNA. In pharmacokinetics data provided by Pfizer to European Medicines Agency (EMA), BNT162b2 biodistribution was studied in mice and rats by intra-muscular injection with radiolabeled LNP and luciferase modRNA. Radioactivity was detected in most tissues from the first time point (0.25 h), and results showed that the injection site and the liver were the major sites of distribution, with maximum concentrations observed at 8–48 h post-dose [26]. Furthermore, in animals that received the BNT162b2 injection, reversible hepatic effects were observed, including enlarged liver, vacuolation, increased gamma glutamyl transferase (γGT) levels, and increased levels of aspartate transaminase (AST) and alkaline phosphatase (ALP) [26]. Transient hepatic effects induced by LNP delivery systems have been reported previously [27,28,29,30], nevertheless, it has also been shown that the empty LNP without modRNA alone does not introduce any significant liver injury [27]. Therefore, in this study, we aim to examine the effect of BNT162b2 on a human liver cell line in vitro and investigate if BNT162b2 can be reverse transcribed into DNA through endogenous mechanisms.

2. Materials and Methods

2.1. Cell Culture

Huh7 cells (JCRB Cell Bank, Osaka, Japan) were cultured in 37 °C at 5% CO₂ with DMEM medium (HyClone, HYCLSH30243.01) supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich, F7524-500ML, Burlington, MA, USA) and 1% (v/v) Penicillin-Streptomycin (HyClone, SV30010, Logan, UT, USA). For BNT162b2 treatment, Huh7 cells were seeded with a density of 200,000 cells/well in 24-well plates. BNT162b2 mRNA vaccine (Pfizer BioNTech, New York, NY, USA) was diluted with sterile 0.9% sodium chloride injection, USP into a final concentration of 100 μg/mL as described in the manufacturer's guideline [31]. BNT162b2 suspension was then added in cell culture media to reach final concentrations of 0.5, 1.0, or 2.0 μg/mL. Huh7 cells were incubated with or without BNT162b2 for 6, 24, and 48 h. Cells were washed thoroughly with PBS and harvested by trypsinization and stored in −80 °C until further use.

2.2. REAL-TIME RT-QPCR

RNA from the cells was extracted with RNeasy Plus Mini Kit (Qiagen, 74134, Hilden, Germany) following the manufacturer's protocol. RT-PCR was performed using RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, K1622, Waltham, MA, USA) following the manufacturers protocol. Real-time qPCR was performed using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, K0222, Waltham, MA, USA) with primers for BNT162b2, LINE-1 and housekeeping genes ACTB and GAPDH (Table 1).

Table 1. Primer sequences of RT-qPCR and PCR.

2.3. Immunofluorescence Staining and Confocal Imaging

Huh7 cells were cultured in eight-chamber slides (LAB-TEK, 154534, Santa Cruz, CA, USA) with a density of 40,000 cells/well, with or without BNT162b2 (0.5, 1 or 2 µg/mL) for 6 h. Immunohistochemistry was performed using primary antibody anti-LINE-1 ORF1p mouse monoclonal antibody (Merck, 3574308, Kenilworth, NJ, USA), secondary antibody Cy3 Donkey anti-mouse (Jackson ImmunoResearch, West Grove, PA, USA), and Hoechst (Life technologies, 34850, Carlsbad, CA, USA), following the protocol from Thermo Fisher (Waltham, MA, USA). Two images per condition were taken using a Zeiss LSM 800 and a 63X oil immersion objective, and the staining intensity was quantified on the individual whole cell area and the nucleus area on 15 cells per image by ImageJ 1.53c. LINE-1 staining intensity for the cytosol was calculated by subtracting the intensity of the nucleus from that of the whole cell. All images of the cells were assigned a random number to prevent bias. To mark the nuclei (determined by the Hoechst staining) and the whole cells (determined by the borders of the LINE-1 fluorescence), the Freehand selection tool was used. These areas were then measured, and the mean intensity was used to compare the groups.

2.4. Genomic DNA Purification, PCR Amplification, Agarose Gel Purification, and Sanger Sequencing

Genomic DNA was extracted from cell pellets with PBND buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.45% NP-40, 0.45% Tween-20) according to protocol described previously [32]. To remove residual RNA from the DNA preparation, RNase (100 µg/mL, Qiagen, Hilden, Germany) was added to the DNA preparation and incubated at 37 °C for 3 h, followed by 5 min at 95 °C. PCR was then performed using primers targeting BNT162b2 (sequences are shown in Table 1), with the following program: 5 min at 95 °C, 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min; finally, 72 °C for 5 min and 12 °C for 5 min. PCR products were run on 1.4% (w/v) agarose gel. Bands corresponding to the amplicons of the expected size (444 bps) were cut out and DNA was extracted using QIAquick PCR Purification Kit (Qiagen, 28104, Hilden, Germany), following the manufacturer's instructions. The sequence of the DNA amplicon was verified by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany).

Statistics

Statistical comparisons were performed using two-tailed Student's t-test and ANOVA. Data are expressed as the mean ± SEM or ± SD. Differences with p < 0.05 are considered significant.

2.5. Ethical Statements

The Huh7 cell line was obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank.

3. Results

3.1. BNT162b2 Enters Human Liver Cell Line Huh7 Cells at High Efficiency

To determine if BNT162b2 enters human liver cells, we exposed human liver cell line Huh7 to BNT162b2. In a previous study on the uptake kinetics of LNP delivery in Huh7 cells, the maximum biological efficacy of LNP was observed between 4–7 h [33]. Therefore, in our study, Huh7 cells were cultured with or without increasing concentrations of BNT162b2 (0.5, 1.0 and 2.0 µg/mL) for 6, 24, and 48 h. RNA was extracted from cells and a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed using primers targeting the BNT162b2 sequence, as illustrated in Figure 1. The full sequence of BNT162b2 is publicly available [34] and contains a two-nucleotides cap; 5′- untranslated region (UTR) that incorporates the 5′ -UTR of a human α-globin gene; the full-length of SARS-CoV-2 S protein with two proline mutations; 3′-UTR that incorporates the human mitochondrial 12S rRNA (mtRNR1) segment and human AES/TLE5 gene segment with two C→U mutations; poly(A) tail. Detailed analysis of the S protein sequence in BNT162b2 revealed 124 sequences that are 100% identical to human genomic sequences and three sequences with only one nucleotide (nt) mismatch in 19–26 nts (Table S1, see Supplementary Materials). To detect BNT162b2 RNA level, we designed primers with forward primer located in SARS-CoV-2 S protein regions and reverse primer in 3′-UTR, which allows detection of PCR amplicon unique to BNT162b2 without unspecific binding of the primers to human genomic regions.

Figure 1. PCR primer set used to detect mRNA level and reverse-transcription of BNT162b2. Illustration of BNT162b2 was adapted from previously described literature [34].

RT-qPCR results showed that Huh7 cells treated with BNT162b2 had high levels of BNT162b2 mRNA relative to housekeeping genes at 6, 24, and 48 h (Figure 2, presented in logged 2^−ΔΔCT due to exceptionally high levels). The three BNT162b2 concentrations led to similar intracellular BNT162b2 mRNA levels at the different time points, except that the significant difference between 1.0 and 2.0 µg/mL was observed at 48 h. BNT162b2 mRNA levels were significantly decreased at 24 h compared to 6 h, but increased again at 48 h.

Figure 2. BNT162b2 mRNA levels in Huh7 cells treated with BNT162b2. Huh7 cells were treated without (Ctrl) or with 0.5 (V1), 1 (V2), and 2 µg/mL (V3) of BNT162b2 for 6 (green dots), 24 (orange dots), and 48 h (blue dots). RNA was purified and qPCR was performed using primers targeting BNT162b2. RNA levels of BNT162b2 are presented as logged 2^−ΔΔCT values relative to house-keeping genes GAPDH and ACTB. Results are from five independent experiments (n = 5). Differences between respective groups were analyzed using two-tailed Student's t-test. Data are expressed as the mean ± SEM. (* p < 0.05; ** p < 0.01; *** p < 0.001 vs. respective control at each time point, or as indicated).

3.2. Effect of BNT162b2 on Human Endogenous Reverse Transcriptase Long Interspersed Nuclear Element-1 (LINE-1)

Here we examined the effect of BNT162b2 on LINE-1 gene expression. RT-qPCR was performed on RNA purified from Huh7 cells treated with BNT162b2 (0, 0.5, 1.0, and 2.0 µg/mL) for 6, 24, and 48 h, using primers targeting LINE-1. Significantly increased LINE-1 expression compared to control was observed at 6 h by 2.0 µg/mL BNT162b2, while lower BNT162b2 concentrations decreased LINE-1 expression at all time points (Figure 3).

Figure 3. LINE-1 mRNA levels in Huh7 cells treated with BNT162b2. Huh7 cells were treated without (Ctrl) or with 0.5 (V1), 1 (V2), and 2 µg/mL (V3) of BNT162b2 for 6 (green dots), 24 (red dots), and 48 h (blue dots). RNA was purified and qPCR was performed using primers targeting LINE-1. RNA levels of LINE-1 are presented as 2^−ΔΔCT values relative to house-keeping genes GAPDH and ACTB. Results are from five independent experiments (n = 5). Differences between respective groups were analyzed using two-tailed Student's t-test. Data are expressed as the mean ± SEM. (* p < 0.05; ** p < 0.01; *** p < 0.001 vs. respective control at each time point, or as indicated; † p < 0.05 vs. 6 h-Ctrl).

Next, we studied the effect of BNT162b2 on LINE-1 protein level. The full-length LINE-1 consists of a 5′ untranslated region (UTR), two open reading frames (ORFs), ORF1 and ORF2, and a 3′UTR, of which ORF1 is an RNA binding protein with chaperone activity. The retrotransposition activity of LINE-1 has been demonstrated to involve ORF1 translocation to the nucleus [35]. Huh7 cells treated with or without BNT162b2 (0.5, 1.0 and 2.0 µg/mL) for 6 h were fixed and stained with antibodies binding to LINE-1 ORF1p, and DNA-specific probe Hoechst for visualization of cell nucleus (Figure 4a). Quantification of immunofluorescence staining intensity showed that BNT162b2 increased LINE-1 ORF1p protein levels in both the whole cell area and nucleus at all concentrations tested (Figure 4b–d).

Figure 4. Immunohistochemistry of Huh7 cells treated with BNT162b2 on LINE-1 protein distribution. Huh7 cells were treated without (Ctrl) or with 0.5, 1, and 2 µg/mL of BNT162b2 for 6 h. Cells were fixed and stained with antibodies binding to LINE-1 ORF1p (red) and DNA-specific probe Hoechst for visualization of cell nucleus (blue). (a) Representative images of LINE-1 expression in Huh7 cells treated with or without BNT162b2. (b–d) Quantification of LINE-1 protein in whole cell area (b), cytosol (c), and nucleus (d). All data were analyzed using One-Way ANOVA, and graphs were created using GraphPad Prism V 9.2. All data is presented as mean ± SD (** p < 0.01; *** p < 0.001; **** p < 0.0001 as indicated).

3.3. Detection of Reverse Transcribed BNT162b2 DNA in Huh7 Cells

A previous study has shown that entry of LINE-1 protein into the nucleus is associated with retrotransposition [35]. In the immunofluorescence staining experiment described above, increased levels of LINE-1 in the nucleus were observed already at the lowest concentration of BNT162b2 (0.5 µg/mL). To examine if BNT162b2 is reversely transcribed into DNA when LINE-1 is elevated, we purified genomic DNA from Huh7 cells treated with 0.5 µg/mL of BNT162b2 for 6, 24, and 48 h. Purified DNA was treated with RNase to remove RNA and subjected to PCR using primers targeting BNT162b2, as illustrated in Figure 1. Amplified DNA fragments were then visualized by electrophoresis and gel-purified (Figure 5). BNT162b2 DNA amplicons were detected in all three time points (6, 24, and 48 h). Sanger sequencing confirmed that the DNA amplicons were identical to the BNT162b2 sequence flanked by the primers (Table 2). To ensure that the DNA amplicons were derived from DNA but not BNT162b2 RNA, we also performed PCR on RNA purified from Huh7 cells treated with 0.5 µg/mL BNT162b2 for 6 h, with or without RNase treatment (Ctrl 5 and 6 in Figure 5), and no amplicon was detected in the RNA samples subjected to PCR.

Figure 5. Detection of DNA amplicons of BNT162b2 in Huh7 cells treated with BNT162b2. Huh7 cells were treated without (Ctrl) or with 0.5 µg/mL of BNT162b2 for 6, 24, and 48 h. Genomic DNA was purified and digested with 100 µg/mL RNase. PCR was run on all samples with primers targeting BNT162b2, as shown in Figure 1 and Table 1. DNA amplicons (444 bps) were visualized on agarose gel. BNT: BNT162b2; L: DNA ladder; Ctrl1: cultured Huh7 cells; Ctrl2: Huh7 cells without BNT162b2 treatment collected at 6 h; Ctrl3: Huh7 cells without BNT162b2 treatment collected at 24 h; Ctrl4: Huh7 cells without BNT162b2 treatment collected at 48 h; Ctrl5: RNA from Huh7 cells treated with 0.5 µg/mL of BNT162b2 for 6 h; Ctrl6: RNA from Huh7 cells treated with 0.5 µg/mL of BNT162b2 for 6 h, digested with RNase.

Table 2. Sanger sequencing result of the BNT162b2 amplicon.

4. Discussion

In this study we present evidence that COVID-19 mRNA vaccine BNT162b2 is able to enter the human liver cell line Huh7 in vitro. BNT162b2 mRNA is reverse transcribed intracellularly into DNA as fast as 6 h after BNT162b2 exposure. A possible mechanism for reverse transcription is through endogenous reverse transcriptase LINE-1, and the nucleus protein distribution of LINE-1 is elevated by BNT162b2.

Intracellular accumulation of LNP in hepatocytes has been demonstrated in vivo [36]. A preclinical study on BNT162b2 showed that BNT162b2 enters the human cell line HEK293T cells and leads to robust expression of BNT162b2 antigen [37]. Therefore, in this study, we first investigated the entry of BNT162b2 in the human liver cell line Huh7 cells. The choice of BNT162b2 concentrations used in this study warrants explanation. BNT162b2 is administered as a series of two doses three weeks apart, and each dose contains 30 µg of BNT162b2 in a volume of 0.3 mL, which makes the local concentration at the injection site at the highest 100 µg/mL [31]. A previous study on mRNA vaccines against H10N8 and H7N9 influenza viruses using a similar LNP delivery system showed that the mRNA vaccine can distribute rather nonspecifically to several organs such as liver, spleen, heart, kidney, lung, and brain, and the concentration in the liver is roughly 100 times lower than that of the intra-muscular injection site [38]. In the assessment report on BNT162b2 provided to EMA by Pfizer, the pharmacokinetic distribution studies in rats demonstrated that a relatively large proportion (up to 18%) of the total dose distributes to the liver [26]. We therefore chose to use 0.5, 1, and 2 μg/mL of vaccine in our experiments on the liver cells. However, the effect of a broader range of lower and higher concentrations of BNT162b2 should also be verified in future studies.

In the current study, we employed a human liver cell line for in vitro investigation. It is worth investigating if the liver cells also present the vaccine-derived SARS-CoV-2 spike protein, which could potentially make the liver cells targets for previously primed spike protein reactive cytotoxic T cells. There has been case reports on individuals who developed autoimmune hepatitis [39] after BNT162b2 vaccination. To obtain better understanding of the potential effects of BNT162b2 on liver function, in vivo models are desired for future studies.

In the BNT162b2 toxicity report, no genotoxicity nor carcinogenicity studies have been provided [26]. Our study shows that BNT162b2 can be reverse transcribed to DNA in liver cell line Huh7, and this may give rise to the concern if BNT162b2-derived DNA may be integrated into the host genome and affect the integrity of genomic DNA, which may potentially mediate genotoxic side effects. At this stage, we do not know if DNA reverse transcribed from BNT162b2 is integrated into the cell genome. Further studies are needed to demonstrate the effect of BNT162b2 on genomic integrity, including whole genome sequencing of cells exposed to BNT162b2, as well as tissues from human subjects who received BNT162b2 vaccination.

Human autonomous retrotransposon LINE-1 is a cellular endogenous reverse transcriptase and the only remaining active transposon in humans, able to retrotranspose itself and other nonautonomous elements [40,41], and ~17% of the human genome are comprised of LINE-1 sequences [42]. The nonautonomous Alu elements, short, interspersed nucleotide elements (SINEs), variable-number-of-tandem-repeats (VNTR), as well as cellular mRNA-processed pseudogenes, are retrotransposed by the LINE-1 retrotransposition proteins working in trans [43,44]. A recent study showed that endogenous LINE-1 mediates reverse transcription and integration of SARS-CoV-2 sequences in the genomes of infected human cells [25]. Furthermore, expression of endogenous LINE-1 is often increased upon viral infection, including SARS-CoV-2 infection [45,46,47]. Previous studies showed that LINE-1 retrotransposition activity is regulated by RNA metabolism [48,49], DNA damage response [50], and autophagy [51]. Efficient retrotransposition of LINE-1 is often associated with cell cycle and nuclear envelope breakdown during mitosis [52,53], as well as exogenous retroviruses [54,55], which promotes entrance of LINE-1 into the nucleus. In our study, we observed increased LINE-1 ORF1p distribution as determined by immunohistochemistry in the nucleus by BNT162b2 at all concentrations tested (0.5, 1, and 2 μg/mL), while elevated LINE-1 gene expression was detected at the highest BNT162b2 concentration (2 μg/mL). It is worth noting that gene transcription is regulated by chromatin modifications, transcription factor regulation, and the rate of RNA degradation, while translational regulation of protein involves ribosome recruitment on the initiation codon, modulation of peptide elongation, termination of protein synthesis, or ribosome biogenesis. These two processes are controlled by different mechanisms, and therefore they may not always show the same change patterns in response to external challenges. The exact regulation of LINE-1 activity in response to BNT162b2 merits further study.

The cell model that we used in this study is a carcinoma cell line, with active DNA replication which differs from non-dividing somatic cells. It has also been shown that Huh7 cells display significant different gene and protein expression including upregulated proteins involved in RNA metabolism [56]. However, cell proliferation is also active in several human tissues such as the bone marrow or basal layers of epithelia as well as during embryogenesis, and it is therefore necessary to examine the effect of BNT162b2 on genomic integrity under such conditions. Furthermore, effective retrotransposition of LINE-1 has also been reported in non-dividing and terminally differentiated cells, such as human neurons [57,58].

The Pfizer EMA assessment report also showed that BNT162b2 distributes in the spleen (<1.1%), adrenal glands (<0.1%), as well as low and measurable radioactivity in the ovaries and testes (<0.1%) [26]. Furthermore, no data on placental transfer of BNT162b2 is available from Pfizer EMA assessment report. Our results showed that BNT162b2 mRNA readily enters Huh7 cells at a concentration (0.5 µg/mL) corresponding to 0.5% of the local injection site concentration, induce changes in LINE-1 gene and protein expression, and within 6 h, reverse transcription of BNT162b2 can be detected. It is therefore important to investigate further the effect of BNT162b2 on other cell types and tissues both in vitro and in vivo.

5. Conclusions

Our study is the first in vitro study on the effect of COVID-19 mRNA vaccine BNT162b2 on human liver cell line. We present evidence on fast entry of BNT162b2 into the cells and subsequent intracellular reverse transcription of BNT162b2 mRNA into DNA.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cimb44030073/s1.

Author Contributions

M.A., F.O.F., D.Y., M.B. and C.L. performed in vitro experiments. M.A. and F.O.F. performed data analysis. M.R. and Y.D.M. contributed to the implementation of the research, designed, and supervised the study. Y.D.M. wrote the paper with input from all authors. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Swedish Research Council, Strategic Research Area Exodiab, Dnr 2009-1039, the Swedish Government Fund for Clinical Research (ALF) and the foundation of Skåne University Hospital.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data supporting the findings of this study are available within the article and supporting information.

Acknowledgments

The authors thank Sven Haidl, Maria Josephson, Enming Zhang, Jia-Yi Li, Caroline Haikal, and Pradeep Bompada for their support to this study.

Conflicts of Interest

The authors declare no conflict of interest.

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Aldén, M.; Olofsson Falla, F.; Yang, D.; Barghouth, M.; Luan, C.; Rasmussen, M.; De Marinis, Y. Intracellular Reverse Transcription of Pfizer BioNTech COVID-19 mRNA Vaccine BNT162b2 In Vitro in Human Liver Cell Line. Curr. Issues Mol. Biol. 2022, 44, 1115-1126. https://doi.org/10.3390/cimb44030073

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Aldén M, Olofsson Falla F, Yang D, Barghouth M, Luan C, Rasmussen M, De Marinis Y. Intracellular Reverse Transcription of Pfizer BioNTech COVID-19 mRNA Vaccine BNT162b2 In Vitro in Human Liver Cell Line. Current Issues in Molecular Biology. 2022; 44(3):1115-1126. https://doi.org/10.3390/cimb44030073

Chicago/Turabian Style

Aldén, Markus, Francisko Olofsson Falla, Daowei Yang, Mohammad Barghouth, Cheng Luan, Magnus Rasmussen, and Yang De Marinis. 2022. "Intracellular Reverse Transcription of Pfizer BioNTech COVID-19 mRNA Vaccine BNT162b2 In Vitro in Human Liver Cell Line" Current Issues in Molecular Biology 44, no. 3: 1115-1126. https://doi.org/10.3390/cimb44030073

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CPR? Rules and Regulations



https://www.verywellhealth.com/do-i-have-to-do-cpr-if-im-cpr-certified-1298422

When and How to Respond to an Emergency If You're CPR Certified

Don't skip the training for fear of being sued

Every year, at least 350,000 people go into cardiac arrest outside of a hospital setting. Of those, around 90% do not survive. Knowing how to do cardiopulmonary resuscitation (CPR) and having the confidence to administer it saves lives. In fact, an estimated 45% of people who go into cardiac arrest will survive because a bystander gave them CPR.1

Being trained and certified to perform CPR is one thing, but doing so without fear or hesitation is another. Addressing any reluctance you have before a situation arises may help you stay safe while saving someone's life.

SCIENCE PHOTO LIBRARY / Getty Images

What Does It Mean to Be Certified?

There are a variety of CPR certifications that you can earn online or in a classroom setting. Both the American Heart Association (AHA) and the Red Cross offer courses that are designed to help participants recognize and respond to cardiac emergencies. These certifications are generally valid in any state for two years.

The modules you take will depend on your certification. For example, if you are a first responder or a daycare provider, your training may be contextualized to fit your field. There are also courses for the general public and people whose employer requires them to get certified to meet Occupational Safety and Health Administration (OSHA) regulations.

Whichever course you choose, expect to learn more than the technical aspects of administering CPR.

Training will likely touch on:

• The ins and outs of Good Samaritan laws and how they can protect you from legal liability if you choose to provide care
• How to identify the difference between expressed consent and implied consent
• What you need to know to reduce the risk of disease transmission when administering CPR

Whether your course is self-directed or guided by an instructor, you will typically need to complete a sequence of training modules and hands-on simulations. You will then be tested with an in-person demonstration of your skills, and possibly a written exam.

Most CPR certifications are valid for two years, but according to the American Red Cross Scientific Advisory Council, only 50% of people can pass a CPR skills test one year after earning their certification. Keep your skills sharp by renewing your certification often.2

Do You Have to Perform CPR?

Rescuers who are part of an organized ambulance agency, fire department, law enforcement organization, or rescue squad have what is known as a duty to act and are usually bound to provide help.

Vermont's Good Samaritan law requires rescuers to help in the case of an emergency, and those who do not help face a fine of $100. Training of any sort is not mentioned in Vermont law. In other words: Being trained does not require that you help, but you might want to help so you can save a person's life.3

In all other 49 states, whether or not you choose to administer CPR is up to you. Every second counts in an emergency situation. But while it is critical that you act swiftly, it's also important that you provide reasonable care.

Reasonable Care

To help protect yourself from liability, be cautious and attentive of your surroundings and avoid anything that could endanger you or the person you are rescuing. Do not involve yourself in a situation if it is not safe.4

Good Samaritan Laws

Every state has Good Samaritan laws to protect people who provide voluntary medical care to a victim of a medical emergency. This protection is intended, in part, to encourage people to help others without fear of being sued—so long as they are not reckless or negligent with the care they provide.

To qualify for the protection of Good Samaritan laws, there are three things you must do before providing someone with emergency medical care:

• Ask for consent. The person must verbally express their consent or consent with body language. If they are unconscious or non-responsive, don't hesitate. Assume they want your help.
• Only do what you're trained to do. If you choose to give someone CPR and have been trained, give them CPR; but do not reset their shoulder or perform any other medical procedure that you are not trained for.
• Provide care voluntarily. Good Samaritan laws may not protect you if you accept a gift for your help. Only volunteer to help if you do not expect compensation.5

Attempting CPR Without a Certification

You do not need formal training or a certification to perform CPR on a person in cardiac arrest. If you see someone who needs help, you are encouraged to ask for consent to provide care, regardless of the status of your CPR credentials. Your ability to jump in and use your skills quickly matter more than the date of completion on your certification.

Common Concerns

Taking command of an emergency situation by offering to administer CPR is brave. If you have ever felt nervous or reluctant about having to use CPR, you're not alone.

Fear of Being Sued

If you are worried about injuring someone while performing CPR, keep in mind that your risk of being sued is significantly higher if you do not intervene.

To study the legal risk of bystanders who rescue, researchers reviewed 274 cases between 1989 and 2019 where the use or non-use of CPR led to a personal injury or wrongful death lawsuit. In 64% of the lawsuits, the bystander either provided inadequate CPR or did not perform CPR quickly enough. Additionally, more than $620 million had been issued in settlements where CPR was delayed—in contrast to the $120,000 that was issued in damages from performing CPR.6

The overall sentiment is that it's best to help, but it also doesn't hurt to be trained at what you're doing, either.

Fear of Disease

In 2020, the AHA issued guidelines for managing disease spread when administering CPR. The guidelines were written in light of COVID-19, but the principles are relevant to any other respiratory illness that may be present.

Emphasized are the following:

• Make sure you always have a mask that you can easily grab, and consider keeping a pair of glasses close that can shield your eyes.
• Know that you do not need to perform mouth-to-mouth. Cover the person's face with a cloth and use hands-only CPR to minimize your risk of coming into contact with a communicable disease.
• Any other bystanders who are willing to perform CPR should stand at a reasonable distance away until they are needed to take over.7

Fear of Making a Mistake

Any time you need to use CPR, you will be taking a chance; there's a chance you will succeed and there is a chance you will not. You can train and be certified as much as you want, but if you aren't willing to "fail," none of your training will matter. Remind yourself that being afraid does not mean you can't help, and accept that you may not be able to save everyone.

As with most things in life, the more prepared you are, the less afraid you will be. In addition to keeping your CPR certification current, it may ease your fears to occasionally rehearse scenarios in your mind where you safely perform CPR and save a life.

Being Prepared

Time has a way of slowing down when an emergency situation unfolds. Before you spring into action, take a deep breath.

Then cautiously move through the steps to perform rescue breathing:

• Be careful and reasonable when sizing up a situation. Make sure there are no hazards that could injure you, such as passing cars or falling objects.
• Ask for consent. If the person is unconscious or non-responsive, their consent is implied. Call 911 and begin resuscitation.
• Use your skills to the best of your ability and do not attempt anything that you are not trained for.
• Don't expect to be rewarded for your help. To qualify for protection under Good Samaritan laws, your care must be voluntary.
• Your safety comes first, every time. If you are reckless or negligent when providing care, your actions could have grave consequences for everyone involved.

Using Defibrillators

An automated external defibrillator (AED) is a device that can detect when someone is going into cardiac arrest and send an electric shock to restore their heart's rhythm. These devices are made to be easy for the general public to use and anyone can purchase a device for their home or organization.

Using an AED with CPR can greatly increase a person's chance of survival, but there are some precautions.8

For example:

• Never deliver an electric shock to someone who is wet or lying in water.
• To use an AED if the person has a pacemaker, the pads should be placed adjacent to the pacemaker and not directly on it.
• Remove any medication patches they may be wearing before attaching the AED pads.
• Always ensure that nobody is touching the person receiving an electric shock.

Hands-on experience is invaluable. If you are intimidated by the prospect of using an AED, many certification courses include AED training modules that you can take to feel more prepared.

Post-Certification Training

Administering CPR safely and effectively isn't quite like riding a bike. This is skill set that you will want to sharpen routinely, especially if you live or work with someone at risk of cardiac arrest.

Online training certifications like those provided by the Red Cross are good for keeping you up-to-date on techniques and best practices. However, hands-on training is preferable; without passing an in-person skills test, you may not meet workplace safety requirements. Furthermore, gaining tactile experience will help you feel more prepared and deliberate if you eventually need to apply your skills.

The Red Cross and the American Heart Association aren't the only two places where you can receive great training. Your local YMCA or recreation center may offer classes in person or online, and if CPR training is required for your job, your employer should be able to point you in the right direction. Most fire departments offer regular CPR courses that you can attend, too, and getting to hear the real-world experience of first responders could be useful for you later on.

Frequently Asked Questions

Are there standard requirements for CPR certification courses?

No, CPR certification is not regulated by the state or federal government, so there are no overarching standards or national accreditation. That means that not all CPR courses are equal. OSHA does state that online-only CPR training is insufficient, and individual employers may require specific training courses for employees. Healthcare professionals such as EMTs and nurses are required to be licensed, which ensures that they receive a standard level of training in CPR and other skills required for their jobs.

How do I get recertified in CPR?

If your two-year CPR certification has expired, you're required to take another full training course. If your certification hasn't expired, you can take an abridged renewal course through the organization that first certified you, such as the American Red Cross or the American Heart Association. These organizations have different requirements for in-person and online training for renewal and recertification, so check their websites for details.

A Word From Verywell

You probably didn't earn your CPR certification because you wanted to be a bystander. If there's a reason you don't feel confident, look to see if there's a solution for your worries. After renewing your CPR credentials, acquaint yourself with the Good Samaritan laws in your location.

If you're still on the fence about renewing your CPR certification, remind yourself that being certified doesn't require you to go out and save lives. No matter what situation you are in, remember that you have options and your safety always comes first.

By Rod Brouhard, EMT-P
Rod Brouhard is an emergency medical technician paramedic (EMT-P), journalist, educator, and advocate for emergency medical service providers and patients.

Now, does THIS make sense???  (CPR for developmentally disabled persons….)   

P.s. Designers: wtf?

~A.

Fwd: The 3 Unexpected Secrets for Finding Common Ground Between Strangers | ideo.com

Begin forwarded message:

From: Adrienne den Tex <adentex@gmail.com>

Subject: The 3 Unexpected Secrets for Finding Common Ground Between Strangers | ideo.com

Date: August 16, 2022 at 4:54:42 PM EDT

https://www.ideo.com/blog/3-unexpected-secrets-for-finding-common-ground-between-strangers

~A.

Philosophy 2023

https://www.masterclass.com/classes/cornel-west-teaches-philosophy/chapters/honesty-trust-and-individuality-in-community?utm_campaign=Share&utm_medium=More&utm_source=lesson_page_logged_out


~A.

When?

JOE DOUCET x PARTNERS JDXP

Oooooooooo…….

https://joedoucet.com/index


~A.

Can Dragon Fruit Be Eaten At Night -Yes!

https://iupilon.com/can-dragon-fruit-be-eaten-at-night/

Can Dragon Fruit Be Eaten At Night

written by iupilonJanuary 1, 2023

Although most fruits are best when eaten first thing in the morning so that the body can swiftly break down the sugar and supply the body with all the nutrients, dragon fruit may be eaten at any time. Indeed, it promotes restful sleep when consumed in the evening.

Dragon pearl, cactus, and pitaya are a few other names for this exotic fruit. Recent research suggests it may also have health benefits, although most people appreciate it for its unusual appearance and flavor.

The tropical fruit, often known as "dragon fruit," also goes by the names "pitahaya" and "strawberry pear." its bright red skin conceals a sweet, seed-studded pulp. Its unusual appearance and widely-touted superfood capabilities have made it a hit with health-conscious consumers.

Simply put, free radicals are unbalanced chemicals that can damage cells and contribute to inflammation and illness. Consuming antioxidant-rich foods, including dragon fruit, can help with this.

Yet a pitahaya, when sliced open, reveals a deep magenta color within. White Asian cultivars are typically highly tasteless and nutrient-poor. On the other hand, Central American pitayas have a flavor reminiscent of an earthy strawberry or raspberry.

What Is The Best Time To Eat Dragon Fruit?

There's no optimal time to eat this fruit—you can eat it at any time and still gain benefits. Simply put, free radicals are unbalanced chemicals that can damage cells and contribute to inflammation and illness. Eating antioxidant-rich foods like dragon fruit can help with this.

Antioxidants protect cells from damage and inflammation by eliminating harmful free radicals. As a result, antioxidant-rich diets have shown promise in decreasing the risk of chronic diseases like cardiovascular illness, cancer, diabetes, and arthritis.

Problems include asthma and persistent cough. Some of the B vitamins found in dragon fruits are B1, which aids in the digestion of carbohydrates; B2, which aids in healing and improves appetite; and B3, which lowers harmful cholesterol levels and improves skin health.

The high fiber content in dragon fruit aids in maintaining healthy blood sugar levels. It also prevents the sugar surges that can occur after consuming foods high in glycemic index. So, it's safe for diabetics, though it's best to get the OK from your doctor beforehand.

Antioxidants abound in this fruit. It helps neutralize harmful free radicals in the body. When free radicals are diminished, it also aids in delaying the aging process. This results in skin that looks fresh, supple, and toned.

Can I Eat Dragon Fruit After Dinner?

Yes, dragon fruit is acceptable late-night fare. You can indulge in the exotic flavors of this fruit whenever you like, as was said earlier. You probably already know that eating dragon fruit regularly can have a favorable effect on your health and wellness because it is rich in vitamins and nutrients and contains potentially disease-fighting antioxidants.

The dietary fiber found in dragon fruit can aid in weight loss and maintenance for those with type 2 diabetes, which can help keep blood sugar levels stable.

The skin also improves from eating dragon fruit. Potential benefits include attenuating the appearance of blemishes and fine lines and moisturizing and hydrating dry skin. In addition, the fruit's vitamin C plus antioxidants may positively affect the skin. These nutrients may help repair damaged cells over time, giving you a more glowing complexion.

Eating dragon fruit may reduce inflammation and soreness if you endure chronic pain due to rheumatoid arthritis. Because of its anti-inflammatory characteristics, the fruit can alleviate these pains naturally.

No miracle diet will cause your body to lose weight and shape immediately. However, you can do beautiful things for your body with the appropriate diet. For example, snacking on dragon fruit between meals is a good idea because of its few calories.

Does Dragon Fruit Help With Sleep?

Insomnia can be caused by magnesium deficiency; therefore, including dragon fruit in one's diet may assist in enhancing one's ability to sleep. In addition, magnesium has a wide range of health advantages, involving lowering the risk of developing heart disease, reducing feelings of anxiety, and bringing blood pressure down.

Dragon fruit is a favorite among those interested in eating healthy foods because it has a peculiar appearance and is packed with minerals. However, no scientific research currently suggests that the dragon fruit has any exceptional powers above other fruits rich in nutrients.

You have almost certainly heard of probiotics, the beneficial microorganisms that aid in maintaining a healthy digestive system. Prebiotics are essential for forming beneficial gut bacteria, which foods like dragon fruit can feed.

The body weight may benefit from consuming dragon fruit due to its high fiber content. In addition, there is a correlation between eating a diet high in fiber and having a better body weight and a slimmer waistline.

This may help produce a feeling of fullness, which in turn may reduce the total number of calories taken during the day. In addition, eating meals that are high in fiber can assist in maintaining stable blood sugar levels, which can combat cravings.

What Are The Disadvantages Of Dragon Fruit?

Consuming an excessive amount of dragon fruit can adversely affect our bodies, even though dragon fruit is a source of fiber. However, they have many benefits, one of which is that they enhance the health of our digestive system.

However, consuming an abnormally high amount of fiber can negatively impact a person's health. In addition, you may encounter symptoms that make it difficult for you to digest regularly due to the accumulation of gas in your digestive tract.

Not only can eating an excessive amount of dragon fruit cause abdominal pain but doing so causes a significant amount of flatulence. But, of course, this is true of eating an excessive amount of any other fruit as well.

In the end, eating dragon fruit can be beneficial to one's health in several ways, including the prevention of a variety of illnesses. On the other hand, taking an excessive amount of dragon fruit can cause allergic reactions; however, this only occurs in a subset of the population.

Therefore, if you are having problems, you should take a modest amount of extract from dragon fruit and observe the results. Because there are no established protocols for the manufacture of fruit extracts, consuming the fruit in its natural form consistently and using dietary supplements both have the potential to provide varying outcomes over time.

Dragon Fruit

~A.

Alessi’s first foray into furniture collection with Philippe Starck is inspired by frying pans - Yanko Design

Throwing a 'failure party' YIPf-kingYIP!!!

Throwing a 'failure party'

By Riva Gold, Editor at LinkedIn News

Celebrating success can boost morale, but how about toasting failure? In this excerpt from his book Sparking Success, author Adam Kingl urges leaders to throw their teams a "failure party." Instead of glossing over what isn't working, we can learn from mistakes and clear the way for innovation, says Kingl. The book shows that creative leadership can have the biggest impact, especially when staff are under pressure to overachieve. Discussing failure without criticism also enhances the psychological safety of the team, while leaders can "share directly and obliquely what the boundaries are of acceptable risk," the author concludes.

Adam KinglAdam Kingl• 3rd+• 3rd+Author, Keynote Speaker, Educator, AdviserAuthor, Keynote Speaker, Educator, Adviser

4d • Edited • 

We know intuitively, however, that without failure, we're unlikely ever to experience dramatic success. And if we don't share failure, our colleagues are more likely to make the same mistakes. Read more on how to move a team's attitude toward failure in the #LinkedinBookClub excerpt from my new book, Sparking Success. 

To shift a team's attitude toward failure requires fostering an environment of vulnerability, where colleagues are comfortable, eager even, to share their learning from every experiment they attempt. One way to do this is for the leader to host a failure party. I hear you guffaw but hear me out. One of the many reasons that Pixar's mantra is always to 'Display' is to invite critique, sharing, building on ideas and socialising what isn't working. A failure party is simply to celebrate that spirit of Display. Colleagues become more comfortable as psychological safety grows to push the boundaries of what they think is possible, knowing that their personal reputation does not depend on inauthentically trying to communicate an aura of omniscience. 

In the failure party, each person in the team who has explored a new avenue, experimented with a hypothesis, tested a prototype or interviewed a stakeholder or customer, and it did not go to plan, would share their activity, the result and, most importantly, the learning. Colleagues may ask questions, can build on the ideas and even pursue avenues that the speaker had not. The one thing that no one must do is belittle or criticise the speaker. Each dialogue ends with the conversation 'what we learned here'. Some leaders of failure parties even toast every speaker when they finish with a ritual, be it ringing a bell or everyone shouting 'Huzzah!!' The speaker's candour, vulnerability and creativity are acknowledged. The 'party' element, which can simply involve refreshment and an informal environment, is proposed in order to put people at ease as quickly as possible. 

The failure party is also a tremendous opportunity for the leader to share directly and obliquely what the boundaries are of acceptable risk. Over time, the team understands explicitly and implicitly what they can try right away and what they need to share first before trying.  

For more on my book 'Sparking Success: Why every leaders needs to develop a creative mindset': https://lnkd.in/eKUyD4kF

from someecards...

https://youtu.be/gPZl4fXLFRA


~A.