Defending Against the Spike Protein

 

Many people have been exposed to the spike protein by either Covid-19 and or the shot and many have been damaged and or damage is ongoing. 

You may have probably already heard, but in case you have not, a report just came out that indicates that a combination of Bromelain and NAC (N-Acetyl L-Cysteine) creates a synergistic effect that destroys the spike protein. 

I know many were coerced into introducing spike proteins into their body. God is a very loving and kind and merciful God and can provide a way of escape for those who ask forgiveness. And probably many of us have gotten COVID from exposure to others. Maybe this synergy, through God’s grace, can be a way of restoration for those who have been damaged.

 

The Combination of Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2

by  

¹Department of Surgery, St. George Hospital, Sydney, NSW 2217, Australia

²Mucpharm Pty Ltd., Sydney, NSW 2217, Australia

³CIRI, Centre International de Recherche en Infectiologie, Team VirPatH, Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007 Lyon, France

⁴Hospices Civils de Lyon, EMR 3738 (CICLY), Lyon 1 Université, F-69921 Lyon, France

⁵St. George & Sutherland Clinical School, University of New South Wales, Sydney, NSW 2217, Australia

⁶Laboratoire de Virologie, Institut des Agents Infectieux (IAI), Hospices Civils de Lyon, Groupement Hospitalier Nord, F-69004 Lyon, France

^*Author to whom correspondence should be addressed.

^†These authors contributed equally to this work.

^‡These authors contributed equally to this work.

Viruses 2021, 13(3), 425; https://doi.org/10.3390/v13030425

Received: 31 January 2021 / Revised: 25 February 2021 / Accepted: 1 March 2021 / Published: 6 March 2021

(This article belongs to the Special Issue Vaccines and Therapeutics against Coronaviruses)

 

1.   Introduction

The recently emergent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which can range from asymptomatic to severe and lethal forms with a systemic inflammatory response syndrome. As of 21 February 2021, over 111 million confirmed cases have been reported, with an estimated overall mortality of 2.2% [1]. There are currently few therapeutic agents proven to be beneficial in reducing early- and late-stage disease progression [2]. While there are fortunately many vaccine candidates, their widespread availability for vaccination may not be immediate, the length of immune protection may be limited [3,4], and the efficacy of the vaccines may be reduced by novel SARS-CoV-2 variants. The continued exploration of effective treatments is therefore still needed.

Structurally, SARS-CoV-2 contains surface spike proteins, membrane proteins, and envelope proteins, as well as internal nucleoproteins that package the RNA. The spike protein is a homotrimer glycoprotein complex with different roles accomplished through dynamic conformational modifications, based in part on disulfide bonds [5]. It allows the infection of target cells by binding to the human angiotensin-converting enzyme (ACE2) receptors, among others, which triggers proteolysis by transmembrane protease serine 2 (TMPRSS2), furin, and perhaps other proteases, leading to virion and host cell membrane fusion [6,7].

The entry of viruses into mammalian cells, or “virus internalization”, is a key mechanism of enveloped virus infection and is based on dynamic conformational changes of their surface glycoproteins, namely, as mediated by disulfide bond reduction and regulated by cell surface oxydoreductases and proteases [5,8,9,10,11]. SARS-CoV-2 entry into host cells has been shown to start with destabilization of the spike protein through allosteric mechanical transition, which induces a conformational change from the closed “down” state to open “up” state of the receptor binding domain (RBD) of the spike protein [12,13]. The conformational changes of RBD and virus binding are induced by TMPRSS2 or Cathepsin L, which trigger the transition from the pre-fusion to post-fusion state [5,12,13]. The energy liberated by disulfide bond reduction increases protein flexibility, which is maximal when the reduced state is complete [8], thus allowing the fusion of host–virus membranes, which is otherwise impossible due to the repulsive hydration forces present before reduction [5].

Bromelain is extracted mainly from the stem of the pineapple plant (Ananas comosus) and contains a number of enzymes that give it the ability to hydrolyze glycosidic bonds in complex carbohydrates [14]. Previous studies have indicated that Bromelain removes the spike and hemagglutinin proteins of Semliki Forest virus, Sindbis virus, mouse gastrointestinal coronavirus, hemagglutinating encephalomyelitis virus, and H1N1 influenza viruses [15,16]. As a therapeutic molecule, it is used for debriding burns. Acetylcysteine is a powerful antioxidant that is commonly nebulized into the airways for mucus accumulation and is also used as a hepatoprotective agent in paracetamol overdose. Most importantly in the present context, Acetylcysteine reduces disulfide bonds [17]. Moreover, the association of the spike and envelope proteins by their respective triple cysteine motifs warrants the hypothesis of impacting virion stability following disulfide bridge disruption by the action of Acetylcysteine [18]. The combination of Bromelain and Acetylcysteine (BromAc) exhibits a synergistic mucolytic effect that is used in the treatment of mucinous tumors [19,20] and as a chemosensitizer of several anticancer drugs [21]. These different actions are due to the ability of BromAc to unfold the molecular structures of complex glycoproteins, thus allowing binding to occur because of the high affinity between RBD and ACE2.

Therefore, in the current study we set out to determine whether BromAc can disrupt the integrity of SARS-CoV-2 spike and envelope proteins and subsequently examine its inactivation potential against in vitro replication of two viral strains, including one with a spike mutant alteration of the novel S1/S2 cleavage site.

2.   Materials and Methods

2.1. Materials

Bromelain API was manufactured by Mucpharm Pty Ltd (Kogarah, Australia) as a sterile powder. Acetylcysteine was purchased from Link Pharma (Cat# AUST R 170803; Warriewood, Australia). The recombinant SARS-COV-2 spike protein was obtained from SinoBiological (Cat# 40589-V08B1; Beijing, China). The recombinant envelope protein was obtained from MyBioSource (Cat# MBS8309649; San Diego, CA, USA). All other reagents were from Sigma Aldrich (St. Louis, MO, USA).

2.2. Recombinant Spike and Envelope Gel Electrophoresis

The spike or envelope proteins were reconstituted in sterile distilled water according to the manufacturer’s instructions, and aliquots were frozen at −20 °C. Two and a half micrograms of spike or envelope protein were incubated with 50 or 100 µg/mL Bromelain, 20 mg/mL Acetylcysteine, or a combination of both in Milli-Q water. The control contained no drugs. The total reaction volume was 15 µL each. After 30 min incubation at 37 °C, 5 µL of sample buffer was added into each reaction. A total of 20 µL of each reaction was electrophoresed on an SDS-PAGE (Cat# 456-1095; Bio-Rad Hercules, CA, USA). The gels were stained using Coomassie blue.

2.3. UV Spectral Detection of Disulfide Bonds in Spike and Envelope Proteins

The method of Iyer and Klee for the measurement of the rate of reduction of disulfide bonds has been used to detect disulfide bonds in spike and envelope proteins [22]. The recombinant SARS-CoV-2 spike protein at a concentration of 3.0 µg/mL in phosphate-buffered saline (PBS) (pH 7.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA) was incubated with 0, 10, 20, 40, and 50 µL of Acetylcysteine (0.5 M), agitated at 37 °C for 30 min followed by equivalent addition of Dithiothreitol (DTT) (0.5 M), and agitated for a further 30 min at 37 °C. The spike protein was incubated in parallel only with DTT (0.5 M) as before without any Acetylcysteine and agitated at 37 °C for 30 min. The absorbance was then read at 310 nm. UV spectral detection of disulfide bonds in the envelope protein was performed in a similar manner.

2.4. SARS-CoV-2 Whole Virus Inactivation with BromAc

Fully respecting the World Health Organization (WHO) interim biosafety guidance related to the coronavirus disease, the SARS-CoV-2 whole virus inactivation tests were carried out with a wild-type (WT) strain representative of early circulating European viruses (GISAID accession number EPI_ISL_578176). A second SARS-CoV-2 strain (denoted as ∆S), reported through routine genomic surveillance in the Auvergne-Rhône-Alpes region of France, was added to the inactivation tests due to a rare mutation in the spike S1/S2 cleavage site and its culture availability in the laboratory (GISAID accession number EPI_ISL_578177).

These tests were conducted with incremental concentrations of Bromelain alone (0, 25, 50, 100, and 250 µg/mL), Acetylcysteine alone (20 mg/mL), and the cross-reaction of the different Bromelain concentrations combined with a constant 20 mg/mL Acetylcysteine formulation, against two virus culture dilutions at 10^5.5 and 10^4.5 TCID50/mL. Following 1 h of drug exposure at 37 °C, all conditions, including the control, were diluted 100-fold to avoid cytotoxicity, inoculated in quadruplicate on confluent Vero cells (CCL-81; ATCC©, Manassas, VA, USA), and incubated for 5 days at 36 °C with 5% CO₂. Cells were maintained in Eagle’s minimal essential medium (EMEM) with 2% Penicillin-Streptomycin, 1% L-glutamine, and 2% inactivated fetal bovine serum. Results were obtained by daily optical microscopy observations, an end-point cell lysis staining assay, and reverse-transcriptase polymerase chain reaction (RT-PCR) of supernatant RNA extracts. Briefly, the end-point cell lysis staining assay consisted of adding Neutral Red dye (Merck KGaA, Darmstadt, Germany) to cell monolayers, incubating at 37 °C for 45 min, washing with PBS, and adding citrate ethanol before optical density (OD) was measured at 540 nm (Labsystems Multiskan Ascent Reader, Thermo Fisher Scientific, Waltham, MA, USA). OD was directly proportional to viable cells, so a low OD would signify important cell lysis due to virus replication. In addition, RNA from well supernatants was extracted by the semi-automated eMAG^® workstation (bioMérieux, Lyon, FR), and SARS-CoV-2 RdRp IP2-targeted RdRp Institute Pasteur RT-PCR was performed on a QuantStudio™ 5 System (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA). Log₁₀ reduction values (LRV) of viral replication were calculated by the difference between treatment and control wells per condition divided by 3.3 (as 1 log₁₀ ≈ 3.3 PCR Cycle thresholds (Ct)).

2.5. Replication Kinetics by Real-Time Cell Analysis

To compare the in vitro replication capacity of both WT and ∆S SARS-CoV-2 strains, replication kinetics were determined by measuring the electrode impedance of microelectronic cell sensors on the xCELLigence Real-Time Cell Analyzer (RTCA) DP Instrument (ACEA Biosciences, Inc., San Diego, CA, USA). Vero cells were seeded at 20,000 cells per well on an E-Plate 16 (ACEA Biosciences, Inc., San Diego, CA, USA) and incubated with the same media conditions as described previously at 36 °C with 5% CO2. After 24 h, SARS-CoV-2 culture isolates were inoculated in triplicate at a multiplicity of infection of 10−2. Mock infections were performed in quadruplicate. Electronic impedance data (cell index) were continuously collected at 15-min intervals for 6 days. Area under the curve analysis of normalized cell index, established at time of inoculation, was then calculated at 12-h intervals. At each interval, cell viability was determined by normalizing against the corresponding cell control. Tukey multiple comparison tests were used to compare each condition on GraphPad Prism (software version 9.0; San Diego, CA, USA).

3.   Results

3.1. Alteration of SARS-CoV-2 Spike and Envelope Proteins

Treatment of the spike protein with Acetylcysteine alone did not show any alteration of the protein, whereas concentrations of Bromelain at 50 and 100 µg/mL and BromAc at 50 and 100 µg/20 mg/mL resulted in protein alteration (Figure 1A). Treatment with Acetylcysteine on the envelope protein did not alter the protein, whereas treatment with Bromelain at 50 and 100 µg/mL and BromAc at 50 and 100 µg/20 mg/mL also resulted in near complete and complete fragmentation, respectively (Figure 1A).

 

Figure 1. (A) Bromelain and Acetylcysteine present a synergistic effect on severe acute respiratory syndrome coronavirus (SARS-CoV-2) spike and envelope protein destabilization. SDS-PAGE of the recombinant SARS-CoV-2 spike protein S1 + S2 subunits (150 kDa) and envelope protein (25 kDa). Proteins were treated with 20 mg/mL Acetylcysteine alone, 100 and 50 µg/mL Bromelain alone, and a combination of 100 and 50 µg/20 mg/mL BromAc. (B) Disulfide reduction of recombinant SARS-CoV-2 spike protein by Acetylcysteine. The differential assay between Acetylcysteine (Ac) and Dithiothreitol (DTT) for the reduction of disulfide bonds found on the spike protein indicates that Acetylcysteine reduces 42% of the disulfide bonds before the addition of DTT. The remaining bonds are reduced by DTT to produce the chromogen detected at 310 nm. (C) Disulfide reduction of recombinant SARS-CoV-2 envelope protein by Acetylcysteine. The differential assay between Acetylcysteine (Ac) and Dithiothreitol (DTT) for the reduction of disulfide bonds found on the envelope protein indicates that Acetylcysteine reduces 40% of the bonds before the addition of DTT.

3.2. UV Spectral Detection Demonstrates the Alteration of Disulfide Bonds in Spike and Envelope Proteins

The comparative reduction of disulfide bonds on the spike protein between DTT alone and DTT with Acetylcysteine demonstrated a 42% difference (Figure 1B), based on the slope of the graphs [0.002599/0.006171 (100) = 42 %]. Acetylcysteine was thus able to reduce 58% of the disulfide linkages in the sample, after which the remaining disulfide bonds were reduced by DTT to produce the chromogen that was monitored in the spectra. Similarly, the differential assay between Acetylcysteine and DTT for the reduction of disulfide bonds found in the envelope protein [0.007866/0.01293 (100) = 60%] indicates that Acetylcysteine reduces 40% of the disulfide bonds before the addition of DTT (Figure 1C).

3.3. In Vitro SARS-CoV-2 Inactivating Potential of Bromelain, Acetylcysteine, and BromAc

For both SARS-CoV-2 strains tested, the untreated virus controls at 10^5.5 and 10^4.5 TCID₅₀/mL yielded typical cytopathic effects (CPE), and no cytotoxicity was observed for any of the drug combinations on Vero cells. Optical CPE results were invariably confirmed by end-point Neutral Red cell staining. Overall, Bromelain and Acetylcysteine treatment alone showed no viral inhibition, all with CPE comparable to virus control wells, whereas BromAc combinations displayed virus inactivation in a concentration-dependent manner (Figure 2). Treatment on 10^4.5 TCID₅₀/mL virus titers (Figure 2B,D) yielded more consistent inhibition of CPE for quadruplicates than on 10^5.5 TCID₅₀/mL virus titers (Figure 2A,C).

Figure 2. Cell lysis assays demonstrated in vitro inactivation potential of Acetylcysteine and Bromelain combined (BromAc) against SARS-CoV-2. Cell viability was measured by cell staining with Neutral Red, where optical density (OD) is directly proportional to viable cells. Low OD would signify important cell lysis due to virus replication. The wild-type (WT) SARS-CoV-2 strain at 5.5 and 4.5 log₁₀TCID₅₀/mL titers (A and B, respectively) showed no inhibition of cytopathic effect (CPE) for single agent treatment, compared to the mock treatment virus control condition. BromAc combinations were able to inhibit CPE, compared to the mock infection cell controls. Treatment of a SARS-CoV-2 spike protein variant (∆S) with a mutation at the S1/S2 junction at 5.5 and 4.5 log₁₀TCID₅₀/mL titers (C and D, respectively) showed similar results. Bars represent the average of each quadruplicate per condition, illustrated by white circles. Ordinary one-way ANOVA was performed, using the mock treatment virus control as the control condition (**** p < 0.0001, *** p < 0.0005, ** p < 0.003, and * p < 0.05).

Based on the virus inactivation guidelines established by the WHO, a robust and reliable process of inactivation will be able to reduce replication by at least 4 logs [Log₁₀ reduction value (LRV) = (RT-PCR Ct treatment – RT-PCR Ct control)/3.3; as 1 log₁₀ ≈ 3.3 Ct]. As such, RT-PCR was performed on the RNA extracts to directly measure virus replication. For the wild-type (WT) strain at 10^4.5 TCID₅₀/mL, successful LRV > 4 were observed with 1 out of 4 wells, 2 out of 4 wells, 3 out of 4 wells, and 4 out of 4 wells for 25, 50, 100 and 250 µg/20 mg/mL BromAc, respectively (Figure 3). It is worth noting that at 10^5.5 TCID₅₀/mL, LRV were slightly below the threshold at, on average, 3.3, with 3 out of 4 wells and 2 out of 4 wells for 100 and 250 µg/20 mg/mL BromAc, respectively (Table 1). For the spike protein mutant (∆S) at 10^4.5 TCID₅₀/mL, no successful LRV > 4 was observed for 25 µg/20 mg/mL BromAc, but it was observed in 4 out of 4 wells for 50, 100, and 250 µg/20 mg/mL BromAc (Figure 3). Of note, at 10^5.5 TCID₅₀/mL, LRV were slightly below the threshold at, on average, 3.2, with 1 out of 4 wells, 2 out of 4 wells, and 4 out of 4 wells for 50, 100, and 250 µg/20 mg/mL BromAc, respectively (Table 1). Overall, in vitro inactivation of both SARS-CoV-2 strains’ replication capacity was observed in a dose-dependent manner, most strongly demonstrated at 100 and 250 µg/20 mg/mL BromAc against 10^4.5 TCID₅₀/mL of virus.

Figure 3. Threshold matrix of log₁₀ reduction values (LRV) of in vitro virus replication 96 h after BromAc treatment on WT and ∆S SARS-CoV-2 strains at 5.5 and 4.5 log₁₀TCID₅₀/mL titers. LRV were calculated with the following formula: LRV = (RT-PCR Ct of treatment—RT-PCR Ct virus control)/3.3; as 1 log10 ≈ 3.3 Ct. The color gradient matrix displays the number of quadruplicates per condition yielding an LRV > 4, corresponding to a robust inactivation according to the WHO. WT = wild-type; ∆S = S1/S2 spike mutant.

Table 1. Log₁₀ reduction values (LRV) of in vitro virus replication 96 h after BromAc treatment on WT and ∆S SARS-CoV-2 strains at 5.5 and 4.5 log₁₀TCID₅₀/mL titers. LRV were calculated with the following formula: LRV = (RT-PCR Ct of treatment – RT-PCR Ct virus control)/3.3; as 1 log₁₀ ≈ 3.3 Ct. Each replicate is described. TCID₅₀/mL = Median Tissue Culture Infectious Dose; WT = wild-type; ∆S = S1/S2 spike mutant.

Real-time cell analysis demonstrated comparable replication kinetics for both WT and ∆S SARS-CoV-2 strains (Figure 4). No significant difference in cell viability was observed between WT and ∆S at any time point. From 48 h post-infection, WT and ∆S cell viability were significantly different compared to the mock infection (p < 0.05).

Figure 4. SARS-CoV-2 replication capacity of WT and ∆S SARS-CoV-2 measured by Real-Time Cell Analysis. Data points correspond to area under the curve analysis of normalized cell index (electronic impedance of RTCA established at time of inoculation) at 12-h intervals. Cell viability was then determined by normalizing against the corresponding cell control. WT = wild-type; ∆S = S1/S2 spike mutant.

4.   Discussion

The combination of Bromelain and Acetylcysteine, BromAc, synergistically inhibited the infectivity of two SARS-CoV-2 strains cultured on Vero cells. Protein confirmation and its molecular properties are dependent on its structural and geometric integrity, which are dependent on both the peptide linkages and disulfide bridges. Acetylcysteine, as a good reducing agent, tends to reduce the disulfide bridges and hence alter the molecular properties of most proteins. This property has been widely exploited in the development of several therapies (chronic obstructive pulmonary disease, allergic airways diseases, cystic fibrosis, pseudomyxoma peritonei, etc.) [20,23,24,25,26,27]. More recently, Acetylcysteine has been used in the development of therapies for respiratory infections such as influenza and COVID-19 [28,29,30], where the integrity of the spike protein is vital for infection [12,13]. A hypothesized mechanism of action could be the unfolding of the spike glycoprotein and the reduction of its disulfide bonds.

The SARS-CoV-2 spike protein is the cornerstone of virion binding to host cells and hence represents an ideal therapeutic target. A direct mechanical action against this spike protein is a different treatment strategy in comparison to most of the existing antiviral drugs, which prevents viral entry in host cells rather than targeting the replication machinery. BromAc acts as a biochemical agent to destroy complex glycoproteins. Bromelain’s multipotent enzymatic competencies, dominated by the ability to disrupt glycosidic linkages, usefully complement Acetylcysteine’s strong power to reduce disulfide bonds [17]. Amino acid sequence analysis of the SARS-CoV-2 spike glycoprotein identified several predetermined sites where BromAc could preferentially act, such as the S2’ site rich in disulfide bonds [31], together with three other disulfide bonds in RBD [32]. In parallel, the role of the glycosidic shield in covering the spike, which is prone to being removed by BromAc, has been highlighted as a stabilization element of RBD conformation transitions as well as a resistance mechanism to specific immune response [5,33,34].

Mammalian cells exhibit reductive functions at their surface that are capable of cleaving disulfide bonds, and the regulation of this thiol-disulfide balance has been proven to impact the internalization of different types of viruses, including SARS-CoV-2 [8,35,36,37,38]. Both ACE2 and spike proteins possess disulfide bonds. When all the spike protein RBD disulfide bonds were reduced to thiols, ACE2 receptor binding to spike protein became less favorable [8]. Interestingly, the reduction of ACE2 disulfide bonds also induced a decrease in binding [8]. Moreover, other reports suggested that Bromelain alone could inhibit SARS-CoV-2 infection in VeroE6 cells through an action on disulfide links [39,40]. As such, the loss of SARS-CoV-2 infectivity observed after pre-treatment with BromAc could be correlated to the cumulative unfolding of the spike and envelope proteins, with a significant reduction of their disulfide bonds by Acetylcysteine, demonstrated in vitro.

Interestingly, a similar effect of BromAc was observed against both WT and ∆S SARS-CoV-2. The main difference in amino acid sequences between SARS-CoV-2 and previous SARS-CoV is the inclusion of a furin cleavage site between S1 and S2 domains [41]. This distinct site of the spike protein and its role in host spill-over and virus fitness is a topic of much debate [41,42,43,44]. Of note, ∆S, which harbors a mutation in this novel S1/S2 cleavage site and alters the cleavage motif, exhibits no apparent difference in replication capacity compared to the WT strain. The slightly increased sensitivity of ∆S to BromAc treatment is therefore not due to a basal replication bias, but the mutation could perhaps be involved in enhancing the mechanism of action of BromAc. These results would nevertheless suggest that, from a threshold dose, BromAc could potentially be effective on spike mutant strains. This may be a clear advantage for BromAc over specific immunologic mechanisms of a spike-specific vaccination [3,4].

To date, different treatment strategies have been tested, but no molecules have demonstrated a clear antiviral effect. In addition, given the heterogeneous disease outcome of COVID-19 patients, the treatment strategy should combine several mechanisms of action and be adapted to the stage of the disease. Thus, treatment repurposing remains an ideal strategy against COVID-19, whilst waiting for sufficient vaccination coverage worldwide [45,46]. In particular, the development of early nasal-directed treatment prone to decreasing a patient’s infectivity and preventing the progression towards severe pulmonary forms is supported by a strong rationale. Hou et al. demonstrated that the first site of infection is the nasopharyngeal mucosa, with secondary movement to the lungs by aspiration [47]. Indeed, the pattern of infectivity of respiratory tract cells followed ACE2 receptor expression, decreasing from the upper respiratory tract to the alveolar tissue. The ratio for ACE2 was five-fold greater in the nose than in the distal respiratory tract [40]. Other repurposing treatments as a nasal antiseptic have been tested in vitro, such as Povidone-Iodine, which has shown activity against SARS-CoV-2 [48]. In the present study, we showed the in vitro therapeutic potential of BromAc against SARS-CoV-2 with a threshold efficient dose at 100 µg/20 mg/mL. As animal airway safety models in two species to date have exhibited no toxicity (unpublished data), the aim is to test nasal administration of the drug in a phase I clinical trial (ACTRN12620000788976). Such treatment could help mitigate mild infections and prevent infection of persons regularly in contact with the virus, such as health-care workers.

Although our results are encouraging, there are a number of points to consider regarding this demonstration. Namely, the in vitro conditions are fixed and could be different from in vivo. Any enzymatic reaction is influenced by the pH of the environment, and even more so when it concerns redox reactions such as disulfide bond reduction [9]. The nasal mucosal pH is, in physiological terms, between 5.5 and 6.5 and increases in rhinitis to 7.2–8.3 [49]. Advanced age, often encountered in SARS-CoV-2 symptomatic infections, also induces a nasal mucosa pH increase [49]. Such a range of variation, depending on modifications typically induced by a viral infection, may challenge the efficacy of our treatment strategy. Further in vitro experiments to test various conditions of pH are ongoing, but ultimately, only clinical studies will be able to assess this point. Our experiments were led on a monkey kidney cell line known to be highly permissive to SARS-CoV-2 infectivity. With the above hypothesis of S protein lysis thiol-disulfide balance disruption, BromAc efficacy on SARS-CoV-2 should not be influenced by the membrane protease pattern. Reproducing this experimental protocol with the human pulmonary epithelial Calu-3 cell line (ATCC^® HTB-55™) would allow these points to be addressed, as virus entry is TMPRSS2-dependent and pH-independent, as in airway epithelium, while virus entry in Vero cells is Cathepsin L-dependent, and thus pH-dependent [50].

Overall, results obtained from the present study in conjunction with complementary studies on BromAc properties and SARS-CoV-2 characterization reveal a strong indication that BromAc can be developed into an effective therapeutic agent against SARS-CoV-2.

5.   Conclusions

There is currently no suitable therapeutic treatment for early SARS-CoV-2 aimed at preventing disease progression. BromAc is under clinical development by the authors for mucinous cancers due to its ability to alter complex glycoprotein structures. The potential of BromAc on SARS-CoV-2 spike and envelope proteins stabilized by disulfide bonds was examined and found to induce the unfolding of recombinant spike and envelope proteins by reducing disulfide stabilizer bridges. BromAc also showed an inhibitory effect on wild-type and spike mutant SARS-CoV-2 by inactivation of its replication capacity in vitro. Hence, BromAc may be an effective therapeutic agent for early SARS-CoV-2 infection, despite mutations, and even have potential as a prophylactic in people at high risk of infection.

Author Contributions

Conceptualization, J.A., K.P., S.J.V., and D.L.M.; methodology, J.A., G.Q., K.P., S.B., and A.H.M.; validation, J.A., G.Q., K.P., V.K., S.B., and A.H.M.; investigation, J.A., G.Q., K.P., V.K., S.B., and A.H.M.; writing—original draft preparation, G.Q., K.P., V.K, A.H.M., E.F., and S.J.V.; supervision, D.L.M. and E.F.; project administration, S.J.V.; funding acquisition, S.J.V. and D.L.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research is partly funded by Mucpharm Pty Ltd., Australia.

Data Availability Statement

A preprint of this manuscript was archived on www.biorxiv.org (accessed on 31 January 2021) due to the emergency of COVID-19.

Conflicts of Interest

David L. Morris is the co-inventor and assignee of the Licence for this study and director of the spin-off sponsor company, Mucpharm Pty Ltd. Javed Akhter, Krishna Pillai, and Ahmed Mekkawy are employees of Mucpharm Pty Ltd. Sarah Valle is partly employed by Mucpharm for its cancer development and is supported by an Australian Government Research Training Program Scholarship. Vahan Kepenekian thanks the Foundation Nuovo Soldati for its fellowship and was partly sponsored for stipend by Mucpharm Pty Ltd.

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MDPI and ACS Style

Akhter, J.; Quéromès, G.; Pillai, K.; Kepenekian, V.; Badar, S.; Mekkawy, A.H.; Frobert, E.; Valle, S.J.; Morris, D.L. The Combination of Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2. Viruses 2021, 13, 425. https://doi.org/10.3390/v13030425

AMA Style

Akhter J, Quéromès G, Pillai K, Kepenekian V, Badar S, Mekkawy AH, Frobert E, Valle SJ, Morris DL. The Combination of Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2. Viruses. 2021; 13(3):425. https://doi.org/10.3390/v13030425

Chicago/Turabian Style

Akhter, Javed, Grégory Quéromès, Krishna Pillai, Vahan Kepenekian, Samina Badar, Ahmed H. Mekkawy, Emilie Frobert, Sarah J. Valle, and David L. Morris. 2021. "The Combination of Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2" Viruses 13, no. 3: 425. https://doi.org/10.3390/v13030425

Do you want to understand what will happen in the end times and when, so you can be prepared? Read The Coming Epiphany;—Your Guide to Understanding End Times Bible Prophecy. 

Not sure that you will go to heaven? The end times are knocking on the door and so is Jesus. He wants to save you; will you let him in? Find out what you must do to be saved, click here. 

Watch ye therefore, and pray always, that ye may be accounted worthy to escape all these things that shall come to pass, and to stand before the Son of man. Luke 21:36 

About the Author: My name is William Frederick and my book entitled “The Coming Epiphany” is available on Amazon.com.  

When you purchase this book you help to support the work that I am doing, and one way that you can really help is by sending digital copies as gifts through Amazon to family and friends.  

Time is short, and I need help getting these warnings into the hands of as many people as possible.  I have published thousands of articles on The End Times Forecaster Blog which are read all over the world. 

 I always freely and happily allow others to republish my articles on their own websites, but I also ask that they include this “About the Author” section with each article. 

The material contained in this article is for general information purposes only, and readers should consult licensed professionals before making any legal, business, financial, or health decisions. 

The California Mega Earthquake: Is Joe Brandt's Dream About to be Fulfilled?

 

The California mega earthquake; you have probably all heard that it is coming. Some are saying it is time for it to occur. Let’s take a look at some data and see what it reveals.

 

John Paul Jackson

 

 

“There is a an earthquake that has been predicted to devastate California. Meaning skyscrapers are going to fall that the shape of the United States will change after that earthquake. That won’t happen until after there’s a storm – a major storm is going to come to California. It’s either a hurricane of incredible force, or it is a storm of incredible force. But a great, great hurricane or incredible force is going to come to California, and the earthquake that destroys it will not happen until after that takes place. So there’s a way of saying, okay, I have time, but that doesn’t mean it won’t be an earthquake tomorrow 7.5 you know, or next week or two weeks from now of some some magnitude. I’m talking about the one that changes the shape of California where you don’t want to live in California, anywhere in California, when that happens or perhaps even most of the west coast. Where an inland ocean is formed and Baja becomes an island and the mouth of the inland ocean forms between between San Diego and Los Angeles. That is not going happen before that that storm comes. That is a sign that God is giving to the people- don’t worry about that big one. Because this sign will happen first.” 

 

Joe Brandt’s Dream 

I remember, vaguely, the fall from my horse—Blackie. As I lay there, pictures began to form in my mind—pictures that stood still. I seemed to be in another world…I thought about Hollywood Boulevard, and I found myself there. Whether this is true, I do not know, but there were a lot of guys my age with beards and wearing, some of them, earrings. All the girls, some of them keen-o, wore real short skirts… 

I noticed there was a quietness about the air, a kind of stillness. Something else was missing, something that should be there. At first, I couldn't figure it out, I didn't know what it was—then I did. There were no birds…I saw a newspaper on the corner with a picture of the President. It surely wasn't Mr. Roosevelt. He was bigger, heavier, big ears… I wondered if I went into a movie (since nobody could see me) if I'd like it. Some cardboard blonde was draped over the marquee with her leg six feet long...

It was ten minutes to four. Something big was going to happen…More like early spring…There was a funny smell. I don't know where it came from. I didn't like it. A smell like sulphur, sulfuric acid, a smell like death. For a minute I thought I was back in chem…It was five minutes to four on a sunny afternoon… The ground shook, just an instant. People looked at each other, surprised. Then they laughed. I laughed, too. So this was what I had been waiting for. This funny little shake. It meant nothing… 

There was that smell again, coming up from the ocean. I was getting to the 5 and 10 store and I saw the look on the kids' faces. Two of them were right in front of me, coming my way. "Let's get out of this place. Let's go back East." He seemed scared.… One young lady just sat down on the sidewalk all doubled up. She kept saying, "earthquake, its the earthquake," over and over…. 

Then, when it came, how it came. Like nothing in God's world. Like nothing. It was like the scream of a siren, long and low, or the scream of a woman I heard having a baby when I was a kid. It was awful. It was as if something—some monster—was pushing up the sidewalks… But then I saw the streets of Los Angeles—and everything between the San Bernardino mountains and Los Angeles. It was still tilting towards the ocean, houses, everything that was left…. I knew it was going to happen to San Francisco—it was going to turn over—it would turn upside down. It went quickly, because of the twisting, I guess. It seemed much faster than Hollywood… 

When I looked at Grand Canyon, that great big gap was closing in, and Boulder Dam was being pushed, from underneath. And then, Nevada, and on up to Reno. Way down south, way down. Baja, California. Mexico too. It looked like some volcano down there was erupting, along with everything else. I saw the map of South America, especially Colombia. Another volcano—eruption— shaking violently. I seemed to be seeing a movie of three months before—before the Hollywood earthquake. Venezuela seemed to be having some kind of volcanic activity. Away off in the distance, I could see Japan, on a fault, too. It was so far off—not easy to see because I was still on Big Bear Mountain, but it started to go into the sea… 

Then, I saw again. Boulder Dam (Hoover Dam), going down—pushing together, pushing together breaking apart—no, Grand Canyon was pushing together, and Boulder Dam was breaking apart. It was still daylight. All these radio stations went off at the same time—Boulder Dam had broken… 

 

Canadian Earthquake Researcher

 

A super mega earthquake is on the way!!! That is the claim being made by a man that goes by the name of Canadian Earthquake Researcher, which has been featured before on this site. He has come out with his latest forecast for the Super Mega earthquake. As pictured above he believes it will happen between now and June of 2023.  

His research has led him to believe that the plates are locking up and have extreme pressure against them and will one day result in a super mega earthquake event(s). 

It is interesting to note that his theory coincides nicely with the researcher who is monitoring the magnetic pole shift and believes earth effecting events, including earthquakes will greatly increase after the pole reaches the 40 degree mark, which he believes will occur in about 4 months. 

 

Here are some of his latest forecasts.

 

IPG2 

In the evil infamous IPG2 an earthquake is shown in this scene.

 

So according to that scene it looks like the earthquake precedes the arrival of the ac. 

 

Analysis 

Let’s analyze the data. According to John Paul Jackson’s vision, if the catastrophic atmospheric rivers that are impacting the West Coast are the storm(s) he foresaw, then that precursor event is fulfilled and the mega earthquake will most likely follow sometime in the near future. 

The Canadian Earthquake Researcher believes the super mega earthquake will occur between now and June of 2023. Whether or not he believes it will involve California, I do not know. He also believes there will be a devastating 10.4 in Japan in the near future. 

Joe Brandt’s dream listed several precursor events such as a devastating earthquake in Japan, Boulder dam/Hoover dam being destroyed, and big earthquakes and volcanoes south of the border. 

So, if we put all the data together here are the major precursor events to look for.

 

California storm

President with big ears on the front page of the newspaper

Cardboard blonde with 6 ft. legs over the marquee in L.A.

Earthquakes and volcanoes going off down south

Devastating Japan earthquake

Hoover Dam destroyed

 

So, in light of all the data, I would say that possibly only one of the prerequisites have occurred, maybe 2—the storms and possibly a President with big ears. 

The cardboard blonde with 6 ft. legs on the marquee in L.A. may possibly be fulfilled this summer or just before with the scheduled release of the Barbie movie on 7/21/23. BTW the movie trailer featured Barbie, a blonde, with what looked like 6 ft. long legs. 

At present there are earthquakes and volcanoes going off down south, but I do not think to the extent that Joe Brandt saw them. The devastating Japan earthquake has not occurred either. 

The Boulder dam renamed Hoover dam has not been destroyed yet either. My research has led me to believe that the Hoover will be destroyed in a breaking of the waters antichrist birthing ritual. This ties in with the IPG2 scene where the earthquake precedes the arrival of the ac. 

So, are we there yet? Taking all into account, and if the data is valid, then I would say that possibly only one prerequisite has been fulfilled so far. But if Barbie is the blonde with 6ft long legs that Joe foresaw, then I would say that we are close, and the big California earthquake could be between now and the fall of 2023. 

But again, if the data is to be trusted, for this to happen we would have to see the prerequisites fulfilled including the devastating Japan earthquake and the Hoover dam destroyed. If we see those, then I believe the California mega earthquake will be imminent. 

And before we end this discussion let’s consider some things from the film 2012. First off, in case you did not know, the 2012 movie contained predictive programming for the 3/11/11 Japan earthquake and tsunami. They said, "So this time we hit Japan" and then a little tsunami comes rolling in.

So, realizing the movie that featured the big CA earthquake contained predictive programming, is it possible that it also contains predictive programming for the big CA earthquake itself? 

Well, let’s look at some numbers from the film. One of the numbers that stood out to me was the number 4420, which was associated with a precursor earthquake to the big California earthquake in the movie.

 

Let’s calculate; starting with 3/11/11 and adding 4420 days we come to 4/17/2023. 

Is this predictive programming, could this be a signal to the day of a precursor earthquake to the big one, or even the big one itself? 

Now the number 4420 in the 2012 film might just be a random number, right? Now look at the time on the watch given to the airplane pilot to rent the escape plane before the big earthquake hit—7:22—it’s a match.

 

The time of 7:22 equals 442 minutes. (7 hours x 60 minutes = 420 minutes + 22 minutes = 442 minutes.)

So there is a match with the numbers on the apartment building of 4420 after you drop the zero as they do, and the time on the watch of 442 minutes. BTW: I calculate the odds of those numbers matching at 7,200,000 to one. 

And let me remind you that the Barbie movie is scheduled to be released on 7/21/23, which is the day before 7/22 so the watch time of 7:22 could possibly be a signal to 4420 and also a signal to 7/22. 

So, is 442 and 4420 a signal to 4/17/2023 and or 7/22/23? Will we see a California earthquake on one of those days? 

The movie was released on 11/13/2009, and from that date to 7/22/23 is exactly 5000 days.

Here is another sync; from 7/22/23 to 11/3/23—our highly watched date of interest— is 2520 hours. As most of you are aware 2520 is the number of days in the last 7 years. And from 4/17/23 to 11/3/23 is 4800 hours, and at some point in that day it will be 288,888 minutes.

 

Conclusion 

Is the data to be trusted? Are the storms John Paul Jackson saw as a precursor to the earthquake occurring now? Is Joe Brandt’s dream valid, is Barbie the 6 ft. blonde he foresaw on the marquee? Is the Canadian Earthquake Researcher’s forecast accurate? 

If the answers to all those questions are yes, then we will see a devastating earthquake in Japan, the Hoover dam destroyed, and the super mega earthquake in California before the fall of 2023. And if the earthquake occurs, then look for the antichrist to come onto “the scene” shortly thereafter. But if the answers to any of those questions is no, then we have more time. 

Watch ye therefore, and pray always, that ye may be accounted worthy to escape all these things that shall come to pass, and to stand before the Son of man. Luke 21:36 

Have you had your epiphany? You need to be prepared for the end times! Read The Coming Epiphany;—Your Guide to Understanding End Times Bible Prophecy. 

Not sure that you will go to heaven? The end times are knocking on the door and so is Jesus. He wants to save you; will you let him in? Find out what you must do to be saved, click here. 

About the Author: My name is William Frederick and my book entitled “The Coming Epiphany” is available on Amazon.com.  

When you purchase this book you help to support the work that I am doing, and one way that you can really help is by sending digital copies as gifts through Amazon to family and friends.  

Time is short, and I need help getting these warnings into the hands of as many people as possible.  I have published thousands of articles on The End Times Forecaster Blog which are read all over the world. 

I always freely and happily allow others to republish my articles on their own websites, but I also ask that they include this “About the Author” section with each article. 

The material contained in this article is for general information purposes only, and readers should consult licensed professionals before making any legal, business, financial, or health decisions.

Will a Loaf of Bread Cost $18 in 2023!?

  

Will the average price of a loaf of white bread reach $18 in 2023? In light of Bible prophecy that is definitely a possibility, here is what I base that assertion on. 

5 And when he had opened the third seal, I heard the third beast say, Come and see. And I beheld, and lo a black horse; and he that sat on him had a pair of balances in his hand. 

6 And I heard a voice in the midst of the four beasts say, A measure of wheat for a penny, and three measures of barley for a penny; and see thou hurt not the oil and the wine. Revelation 6:5,6. 

As I have mentioned in my writings on this blog many times, I believe it is possible that Seals 1 and 2 have been opened, and if so, Seal 3 is next. And as you can see above Seal 3 involves inflation to the point that a measure of wheat costs a penny and three measures of barley cost the same. 

Now what do those Biblical amounts and prices mean to us today? Consider what these commentators say. 

A quart of wheat for a denarius, and three quarts of barley for a denarius: These prices are about twelve times higher than normal. It means that it would cost a day’s wage to buy the ingredients for a loaf of bread. This describes “a time of famine when life will be reduced to the barest necessities.” (Walvoord) 

6:6 a denarius. A Roman silver coin that had a nominal purchasing power of 10 qt. of wheat or 30 qt. of barley. One qt. of wheat was the daily ration for a soldier. (Ryrie) 

Thus, if a denarius could buy 10 qts., and after Seal 3 is opened, now can only buy one qt., the price increased ten times. 

So according to those analyses during Seal 3 the price of wheat would go up ten to twelve times and thus the price of bread would go up at least that much. 

According to the official stats the average price of a loaf of white bread in the USA in 2020 was about $1.45. Ten times that would put us at about $15 for a loaf of white bread, twelve times would be about $18! 

Will Seal 3 be opened in 2023? Will the price of bread go up to $18 per loaf? Here is what a few financial types are saying about the economy in general. 

So, when the housing bubble burst, all of these assets had to be revalued at much lower values... resulting in the global banking system imploding during the Great Financial Crisis of 2008. 

What did the Fed do to address this situation? 

It attempted to corner/ create a bubble in U.S. sovereign bonds, also called Treasuries. 

These are the senior most asset class in the world. These bonds act as the bedrock of our current financial system, with their yields representing the “risk free” rate of return against which all assets (stocks, bonds, real estate, etc.) are valued. 

Put simply, when the Fed created a bubble in these bonds it was actually creating a bubble in EVERYTHING, because ALL asset classes would eventually be repriced based on Treasuries were doing. 

This is why I coined the term “the Everything Bubble” in 2014. 

And that bubble has now burst. 

The yield on the all-important 10-year U.S. Treasury has broken its 35 year down trend. The era of Serial Bubbles is over. And there is nothing the Fed can do to fix this situation. 

After all, what can it do? There isn't a larger more systemically important asset class the Fed could use to create another bubble. And introducing more extraordinary monetary policy would make the situation worse… 

Source: https://gainspainscapital.com/

 

Lawyer John Titus is an ardent Fed watcher.  He has some of the most popular videos on the internet explaining complicated Federal Reserve actions and policies.  Titus says the Fed is in a dangerous situation where the slightest wrong move in any direction could cause a financial system meltdown worse than 2008.  Titus explains, “When you have a debt based monetary system and you take away the drugs, you risk a withdrawal process that can get very nasty, and that’s exactly what happened in 2008.  You start this downward deflationary spiral, and suddenly, people start calling in loans.  Oh my goodness, the collateral is not good, and we all know what happened in 2008.  That’s why the Fed has got to be careful now.  They are between a rock and a hard place.  They don’t want the money supply, the bank money supply, at the retail level to rocket up.  They’ve got to stop that.  They have stopped that on one hand, but on the other hand, they don’t want the deflationary spiral.  The last thing they want in the world is the twin nightmare.  You’ve got raging inflation, and by the way, no one has a job, and nobody has any money.  The money you do have doesn’t buy anything.  The Fed is staring right at it.” 

Titus contends the Fed has printed more money than ever before and at a much faster pace than ever before.  Titus warns, “The confetti party has a way of ending rather rudely and abruptly.” 

Source: https://usawatchdog.com/fed-confetti-party-will-end-rudely-abruptly-john-titus/ 

And another 

Greyerz Just Warned Investors To Prepare For A Hyperinflationary Collapse 

So, will 2023 be the year that we see the black horse ride?  Will the Jubilee year we are in now be the year that the markets crash, and we see hyperinflation as we forecasted? 

Forecast:

Financial 

If Seal 1 and 2 have already been opened, then 2023 could be the year that we see the Seal 3 hyperinflationary financial collapse and famine, which could follow a major earthquake--Joe Brandt's horse was named Blackie. 

Has Seal 1 and 2 Been Opened, Is Seal 3 Next? 

Remember that 2021/22 was a Shemitah year and 2022/23 is a Jubilee year and historically in those years stock markets do not do well. Thus, I issued a forecast for a financial downturn, and here is what happened. 

In 2022 the Dow lost about 9%, the S & P about 19% and the Nasdaq about 33%. 

I look for this financial downturn to continue and accelerate into 2023. Also look out for Elul 29, the last day of the Jubilee year, which is a date that has the potential for a big crash. In our calendar that would be Friday September 15, 2023. 

Will 2023 be the year that Seal 3 is opened or has it already been opened? Remember horses gallop slowly in comparison to other forms of transportation.

If the last 7 years began in 2020, then I say, yes 2023 will be the year of that we see the results of Seal 3 and loaf of white bread will go up to at least $15. If the last 7 years did not begin in 2020, then we have more time. Are you prepared for when it does happen? 

Watch ye therefore, and pray always, that ye may be accounted worthy to escape all these things that shall come to pass, and to stand before the Son of man. Luke 21:36 

Have you had your epiphany? You need to be prepared for the end times! Read The Coming Epiphany;—Your Guide to Understanding End Times Bible Prophecy. 

Not sure that you will go to heaven? The end times are knocking on the door and so is Jesus. He wants to save you; will you let him in? Find out what you must do to be saved, click here. 

About the Author: My name is William Frederick and my book entitled “The Coming Epiphany” is available on Amazon.com.  

When you purchase this book you help to support the work that I am doing, and one way that you can really help is by sending digital copies as gifts through Amazon to family and friends.  

Time is short, and I need help getting these warnings into the hands of as many people as possible.  I have published thousands of articles on The End Times Forecaster Blog which are read all over the world. 

 I always freely and happily allow others to republish my articles on their own websites, but I also ask that they include this “About the Author” section with each article. 

The material contained in this article is for general information purposes only, and readers should consult licensed professionals before making any legal, business, financial, or health decisions.

Catastrophic Flooding in California: Is Our Forecast of a January Water Event Being Fulfilled!?

 

Atmospheric rivers are flowing down into California. They are calling it the worst flooding in a long time, and it is not over yet. May our thoughts and prayers be with the people of California.

Is our forecast of a water event for January of 2023 being fulfilled? 

Forecast:

Water Events 

If 2023 is the year depicted in this screenshot, then we may say several water events (floods, tsunamis, hurricanes) in 2023. Notice the puddles are under January, February, July and or December, and November, that is if I have the months correct. 

 

Keep in mind that on 1/22/2023 the year of the water rabbit begins--note the rabbit in the background. The last year of the water rabbit was 1963 and that was the year of a mega tsunami. Also note that the April figure is shaded purple which may point to an ac event and or a purple rain--nuclear—event... 

Here is what AccuWeather is saying to look out for in the latest round of storms.

 

And here is what the long-range models are showing, storm after storm and more rain and snow.

 

So, with this catastrophic January water event occurring, we ask, is 2023 the year depicted in the IPG2 screenshot? I guess we will have to wait and see. But if it is, then we are in for a rough 2023 and if the last 7 years began in 2020 and Seal 1 and 2 are already open, then Seal 3 is next, and the ac will take over the world in the spring of 2024. If the last 7 did not begin in 2020, then we have more time.

Watch ye therefore, and pray always, that ye may be accounted worthy to escape all these things that shall come to pass, and to stand before the Son of man. Luke 21:36 

Have you had your epiphany? You need to be prepared for the end times! Read The Coming Epiphany;—Your Guide to Understanding End Times Bible Prophecy. 

Not sure that you will go to heaven? The end times are knocking on the door and so is Jesus. He wants to save you; will you let him in? Find out what you must do to be saved, click here. 

About the Author: My name is William Frederick and my book entitled “The Coming Epiphany” is available on Amazon.com.  

When you purchase this book you help to support the work that I am doing, and one way that you can really help is by sending digital copies as gifts through Amazon to family and friends.  

Time is short, and I need help getting these warnings into the hands of as many people as possible.  I have published thousands of articles on The End Times Forecaster Blog which are read all over the world. 

 I always freely and happily allow others to republish my articles on their own websites, but I also ask that they include this “About the Author” section with each article. 

The material contained in this article is for general information purposes only, and readers should consult licensed professionals before making any legal, business, financial, or health decisions.