COVID-19 Research

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Coronavirus Causes Weaponized 'Tentacles' To Sprout From Infected Cells, Directly Inject Virus Into New Ones


Fri, 06/26/2020 - 19:25

The virus behind COVID-19 causes infected cells to sprout 'tentacles' which allow the virus to attack several nearby cells at once - poking holes which allow the disease to easily transfer inside.

This nightmare fuel was discovered by researchers led by the University of California, San Francisco.

"There are long strings that poke holes in other cells and the virus passes through the tube from cell to cell," said UCSF's Director of the Quantitative Biosciences Institute, Professor Nevan Krogan. "Our hypothesis is that these speed up infection."

The images taken by scientists at the National Institutes of Health (NIH) laboratory in the US and University of Freiburg in Germany will be published in the medical journal Cell on Saturday.

Most viruses do not cause infected cells to grow these tentacles. Even those that do, such as smallpox, do not have as many or the same type of branching as Sars-Cov-2, the virus behind Covid-19. -FT

According to the report, the silver lining is that the tentacle discovery may pave the way for a number of drugs to work against the disease - most of which were previously being used to treat cancer.

"It totally makes sense there's an overlap in anticancer drugs and an antiviral effect," said Prof. Krogan, who added that cancers, HIV and SARS-CoV-2 are all searching for the "Achilles heel of the cell."

Potential drugs include silmitasertib, made by Taiwan-based Senhwa Biosciences - which is working with the NIH on trials in the US. The drug works by inhibiting the CK2 enzyme which is used to build the tubes.

Harmine's inhibition-regulation of CK2:

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Exploiting the repertoire of CK2 inhibitors to target DYRK and PIM kinases☆
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GiorgioCozzaaLorenzo A.Pinnaa
https://doi.org/10.1016/j.bbapap.2013.01.018
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Abstract

Advantage has been taken of the relative promiscuity of commonly used inhibitors of protein kinase CK2 to develop compounds that can be exploited for the selective inhibition of druggable kinases other than CK2 itself. Here we summarize data obtained by altering the scaffold of CK2 inhibitors to give rise to novel selective inhibitors of DYRK1A and to a powerful cell permeable dual inhibitor of PIM1 and CK2. In the former case one of the new compounds, C624 (naphto [1,2-b]benzofuran-5,9-diol) displays a potency comparable to that of the first-in-class DYRK1A inhibitor, harmine, lacking however the drawback of drastically inhibiting monoamine oxidase-A (MAO-A) as harmine does. On the other hand the promiscuous CK2 inhibitor 4,5,6,7-tetrabromo-1H-benzimidazole (TBI,TBBz) has been derivatized with a sugar moiety to generate a 1-(β-D-2′-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (TDB) compound which inhibits PIM1 and CK2 with comparably high efficacy (IC50 values < 100 nM) and remarkable selectivity. TDB, unlike other dual PIM1/CK2 inhibitors described in the literature is readily cell permeable and displays a cytotoxic effect on cancer cells consistent with concomitant inhibition of both its onco-kinase targets. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Highlights
► We have discovered and characterized novel inhibitors of protein kinases. ► C624 was demonstrated to have good efficacy and selectivity as inhibitor of DYRK1A. ► TDB was demonstrated to be a specific dual inhibitor of CK2 and PIM1.

https://www.sciencedirect.com/science/article/abs/pii/S1570963913000290

FAST TRACK — RESEARCH LETTERS| VOLUME 361, ISSUE 9374, P2045-2046, JUNE 14, 2003

Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus

Prof J Cinatl, PhD
B Morgenstern, PhD
G Bauer
Prof P Chandra, PhD
Prof H Rabenau, PhD
Prof HW Doerr, PhD Published:June 14, 2003

DOI:https://doi.org/10.1016/S0140-6736(03)13615-X

Summary

The outbreak of SARS warrants the search for antiviral compounds to treat the disease. At present, no specific treatment has been identified for SARS-associated coronavirus infection. We assessed the antiviral potential of ribavirin, 6-azauridine, pyrazofurin, mycophenolic acid, and glycyrrhizin against two clinical isolates of coronavirus (FFM-1 and FFM-2) from patients with SARS admitted to the clinical centre of Frankfurt University, Germany. Of all the compounds, glycyrrhizin was the most active in inhibiting replication of the SARS-associated virus. Our findings suggest that glycyrrhizin should be assessed for treatment of SARS.

A new coronavirus has been identified in patients with severe acute respiratory syndrome (SARS).1 SARS is an infectious disease with a high potential for transmission to close contacts. The outbreak of SARS in several countries has led to the search for active antiviral compounds to treat this disease.
Here, we assessed the antiviral activities of ribavirin, 6-azauridine, pyrazofurin, mycophenolic acid, and glycyrrhizin against two clinical isolates of coronavirus (FFM-1 and FFM-2) from patients with SARS admitted to the clinical centre of Frankfurt University, Germany. All the compounds are available commercially and have been used in patients for their antiviral, antitumour, and immunosuppressive activity. We visually scored cytopathogenicity induced by the virus 72–96 h after infection in 96-well microplates on confluent layers of Vero cells. The selectivity index was determined as the ratio of the concentration of the compound that reduced cell viability to 50% (CC50) to the concentration of the compound needed to inhibit the cytopathic effect to 50% of the control value (EC50). We determined the cytotoxicity of the drugs with an MMT cell-proliferative Kit I (Roche, Mannheim, Germany).

Ribavirin and mycophenolic acid, inhibitors of inosine monophosphate dehydrogenase, did not affect replication of the SARS-associated coronaviruses (SARS-CV) (table). The inhibitors of orotidine monophosphate decarboxylase, 6-azauridine and pyrazofurin, inhibited replication of SARS-CV at non-toxic doses with selectivity indices of 5 and 12, respectively. The most potent inhibitor of SARS-CV replication in Vero cells was glycyrrhizin, which had a selectivity index of 67.

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(03)13615-X/fulltext

In vitro antiviral effects of Peganum harmala seed extract and its total alkaloids against Influenza virus

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Mohammad-TaghiMoradiaFatemehFotouhic

https://doi.org/10.1016/j.micpath.2017.06.014
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Highlights
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Peganum harmala L. extract and its total alkaloids showed the inhibitory effect against influenza A virus replication.

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The extract could inhibit viral RNA replication and viral polymerase activity.

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Antiviral activity of the extract against influenza virus is most probably associated with inhibiting viral RNA transcription.

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The crud extract and its total alkaloid should be further characterized to be developed as anti-influenza A virus agent.


Abstract

This research was aimed to evaluate the in vitro antiviral effect and the mechanism of the effect of Peganum. harmala seeds extract against influenza A virus infection using Madin-Darby canine kidney (MDCK) cells. In this research, ethyl alcohol extract of P. harmala seeds and its total alkaloids was prepared. The potential antiviral activity of the extract and its total alkaloids against influenza A/Puerto Rico/8/34 (H1N1; PR8) virus was assessed. The mode of action of the extract to inhibit influenza replication was investigated using virucidal activity, hemagglutination inhibition assay, time of addition assays, RNA replication, western blot analysis and RNA polymerase blocking assay. The crud extract of P. harmala seed and its total alkaloids showed the best inhibitory effect against influenza A virus replication in MDCK cells using MTT assay, TCID50 method and hemagglutination assay. Our results indicated that the extract inhibits viral RNA replication and viral polymerase activity but did not effect on hemagglutination inhibition and virucidal activity. This study showed that, in vitro antiviral activity of P. harmala seed extract against influenza virus is most probably associated with inhibiting viral RNA transcription. Therefore, this extract and its total alkaloid should be further characterized to be developed as anti-influenza A virus agent.

https://www.sciencedirect.com/science/article/abs/pii/S0882401017305843

DYRK1A Is a Novel Negative Regulator of Cardiomyocyte Hypertrophy

Key quote:

Similarly, treatment of cardiomyocytes with harmine, a specific inhibitor of DYRK1A, revealed cardiomyocyte hypertrophy on morphological and molecular level.

2009
Christian Kuhn, Derk Frank, [...], and Norbert Frey

Additional article information

Associated Data
Supplementary Materials

Abstract

Activation of the phosphatase calcineurin and its downstream targets, transcription factors of the NFAT family, results in cardiomyocyte hypertrophy. Recently, it has been shown that the dual specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) is able to antagonize calcineurin signaling by directly phosphorylating NFATs. We thus hypothesized that DYRK1A might modulate the hypertrophic response of cardiomyocytes. In a model of phenylephrine-induced hypertrophy, adenovirus-mediated overexpression of DYKR1A completely abrogated the hypertrophic response and significantly reduced the expression of the natriuretic peptides ANF and BNP. Furthermore, DYRK1A blunted cardiomyocyte hypertrophy induced by overexpression of constitutively active calcineurin and attenuated the induction of the hypertrophic gene program. Conversely, knockdown of DYRK1A, utilizing adenoviruses encoding for a specific synthetic miRNA, resulted in an increase in cell surface area accompanied by up-regulation of ANF- mRNA. Similarly, treatment of cardiomyocytes with harmine, a specific inhibitor of DYRK1A, revealed cardiomyocyte hypertrophy on morphological and molecular level. Moreover, constitutively active calcineurin led to robust induction of an NFAT-dependent luciferase reporter, whereas DYRK1A attenuated calcineurin-induced reporter activation in cardiomyocytes. Conversely, both knockdown and pharmacological inhibition of DYRK1A significantly augmented the effect of calcineurin in this assay. In summary, we identified DYRK1A as a novel negative regulator of cardiomyocyte hypertrophy.

Mechanistically, this effect appears to be mediated via inhibition of NFAT transcription factors.

Cardiac hypertrophy accompanies a variety of heart diseases and is an independent predictor of cardiovascular morbidity and mortality (1). Hypertrophy may develop in response to a variety of pathological stimuli, e.g. arterial hypertension or heart valve disease (2–4). While numerous molecular pathways have been implicated in the development of cardiomyocyte hypertrophy, the phosphatase calcineurin and its downstream targets, transcription factors of the nuclear factor of activated T-cells (NFAT)2 family, appear to play a central role. Activation of calcineurin leads to dephosphorylation of NFATs, subsequent nuclear translocation, and initiation of the hypertrophic gene program as well as marked cardiomyocyte hypertrophy (5). In contrast, genetic ablation of calcineurin Aβ renders the heart unresponsive to hypertrophic stimuli (6). In addition, inhibitors of calcineurin signaling have been shown to attenuate cardiac hypertrophy (7–9).

Recently, the dual specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) has been identified as a novel modifier of NFAT transcription factors in Drosophila (10) and vertebrate neurons (11). DYRK1A directly phosphorylates NFATs, thereby promoting their nuclear export followed by a reduction of transcriptional activity. DYRK1A, which belongs to a family of dual specificity kinases, is ubiquitously expressed with high levels in the developing nervous system and the heart (12). All isoforms share the ability to autocatalyze their own phosphorylation at an YXY motif (13), while all other substrates are phosphorylated at Ser/Thr residues. DYRK1A, DYRK1B, and the Drosophila homolog, minibrain, are predominantly localized to the nucleus. In addition to NFAT, DYRK1A is capable of phosphorylating other transcription factors, including FKHR/Foxo1 (14), STAT3 (15), and Gli1 (16). Furthermore DYRK1A stimulates the phosphorylation of CREB (17). Because the DYRK1A gene is localized to the Down syndrome critical region and DYRK1A is overexpressed in Down syndrome fetal brain, it was proposed to contribute to the phenotype of trisomy 21. Consistent with this notion, transgenic mouse models with overexpression of DYRK1A reveal learning impairment and hyperactivity (18, 19).

In contrast, little is known about the function of DYRK1A in the heart. Because DYRKs were recently recognized as kinases that phosphorylate NFATs, we hypothesized that the cardiac-enriched isoform DYRK1A might modulate cardiac hypertrophy. Here we show for the first time that DYRK1A overexpression potently inhibits cardiomyocyte hypertrophy. Conversely, miRNA-mediated knockdown or pharmacological inhibition of DYRK1A causes a hypertrophic phenotype accompanied by induction of the hypertrophic gene program and increased NFAT activity.

More at link.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719367/#__ffn_sectitle

https://gladstone.org/news/new-insights-how-covid-19-causes-heart-damage

More at link. Looks like Harmine again protects cells against damage from SARS.

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New Insights into How COVID-19 Causes Heart Damage

ARTICLE BY JULIE LANGELIER
August 25, 2020
5 MIN READ

Bruce Conklin, Melanie Ott, and Todd Mcdevitt standing outside the Gladstone building wearing masks

New study from Bruce Conklin (left), Melanie Ott (center), and Todd McDevitt (right) may shed light on the long-term consequences for COVID-19 patients.


COVID-19 was initially identified as a respiratory disease, but scientists now appreciate that it also affects several other organs in the body, including the heart. Heart damage is a major determinant of COVID-19 related deaths, and even patients who experience only mild COVID-19 symptoms exhibit signs of cardiac dysfunction several months after recovery.

A new study by scientists at Gladstone Institutes helps explain how SARS-CoV-2, the virus that causes COVID-19, inflicts damage on heart cells. The team’s findings, shared publicly on bioRxiv, show the virus’s unexpected effects on the structure of heart cells in the lab, as well as in heart tissue from COVID-19 patients.

The team, led by Gladstone Senior Investigators Todd C. McDevitt, PhD, and Bruce R. Conklin, MD, was uniquely positioned to tackle this work, due to their experience in deriving various types of cardiac cells in the lab from induced pluripotent stem cells.

In collaboration with Director of the Gladstone Institute of Virology Melanie Ott, MD, PhD, they exposed the cells to varying doses of SARS-CoV-2. The virus only productively infected the cardiomyocytes, or heart muscle cells, meaning that the virus could enter those cells and make new copies of itself.

“Early on in our experiments, we noticed that many of the cardiomyocytes were exhibiting some very strange features,” says McDevitt, who is also a professor of bioengineering and therapeutic sciences at UC San Francisco (UCSF). “What we were seeing was completely abnormal; in my years of looking at cardiomyocytes, I had never seen anything like it before.”

The team observed that when they exposed cardiomyocytes to SARS-CoV-2, the sarcomeres in some of the cells appeared to be diced into small, regularly sized fragments. Typically, sarcomeres—units of the muscle fibers in heart cells—are organized into long filaments aligned in the same direction. These sarcomeres control the coordinated contraction of heart cells to produce the normal heartbeat.

Healthy heart cells compared to those infected with Sars-CoV-2

Healthy heart muscle (left) created from adult stem cells have long fibers which allow them to contract. SARS-CoV-2 infection causes these fibers to break apart into small pieces (right), which can cut off the cells ability to beat and may explain lasting cardiac defects in COVID-19 patients.

“The sarcomere disruptions we discovered would make it impossible for the heart muscle cells to beat properly,” explains Conklin, who is also a professor of medicine, cellular and molecular pharmacology, and ophthalmology at UCSF.

The scientists also noted that the nuclear DNA seemed to be missing from many of the heart cells. Without DNA, cells can no longer perform any normal functions.

“It’s the cell equivalent of being brain dead,” adds Conklin. “Even after scouring scientific literature and conferring with colleagues, we cannot find these abnormal cell features in any other cardiac disease model. We believe they are unique to SARS-CoV-2 and could explain the prolonged heart damage seen in many COVID-19 patients.”

Discoveries in a Dish Predict Changes in Human Tissue

To understand whether these changes to cells in culture were relevant to COVID-19 in humans, the researchers sought out heart tissue from COVID-19 patients. However, patient tissue was hard to find.

“Most people don’t appreciate how difficult it has been to access patient samples,” says McDevitt. “Since this virus is so contagious, many hospitals lack the special equipment they need to safely perform autopsies on COVID-19 patients.”

When the team was able to receive patient samples, what they saw corroborated the structural changes they saw in the lab. Remarkably, even in patients who had not been diagnosed with COVID-19 related heart disease, there was evidence of structural abnormalities in the heart muscle cells. Additional testing needs to be done to validate these findings further, but the immediate similarities are striking.

“These abnormalities haven’t been identified in patients before, so they may have been overlooked,” says McDevitt. “I hope our work motivates doctors to review their patients’ samples to start looking for these features at a higher magnification, which will be the true test of our hypotheses.”

More on Harmine and SARS..it stops the abnormal protein folding prevelant in most SARS cases...keep in mind it radiates UV light in the bloodstream and Brain:
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Harmine Acts as an Indirect Inhibitor of Intracellular Protein Aggregation

Swati Jain, Venkataharsha Panuganti, Sonali Jha, and Ipsita Roy*
Cite this: ACS Omega 2020, 5, 11, 5620–5628
Publication Date:March 11, 2020
https://doi.org/10.1021/acsomega.9b02375
Copyright 2020 American Chemical Society

SUBJECTS:Aggregation,Carbohydrates,Fungi,Oxidative stress,Peptides and proteins
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ACS Omega

Abstract

Protein aggregation and oxidative stress are two pathological hallmarks of a number of protein misfolding diseases, including Huntington’s disease (HD). Whether protein aggregation precedes elevation of oxidative stress or follows it remains ambiguous. We have investigated the role of harmine, a beta-carboline alkaloid, in aggregation of a mutant huntingtin fragment (103Q-htt) in a yeast model of HD. We observed that harmine was able to decrease intracellular aggregation of 103Q-htt, and this reduction was higher than that observed with trehalose, a conventional protein stabilizer. The presence of harmine also decreased prion formation. Decreased protein aggregation was accompanied by reduction in oxidative stress. However, harmine had no effect on aggregation of the mutant huntingtin fragment in vitro. Thus, based on experimental data, we conclude that the antioxidant harmine lowers aggregation-induced elevation in oxidative stress, which slows down intracellular protein aggregation.

Introduction

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder. Mutation in HTT (huntingtin) gene results in polyglutamine expansion at the N-terminus of huntingtin protein, leading to its misfolding and aggregation, and ultimately cell death. (1) The length of the CAG (coding for glutamine) repeat (at the 5′-end of huntingtin/IT-15 gene) varies from 6 to 36 in healthy individuals and between 38 and 182 in HD patients. The misfolded protein drives inappropriate interactions with transcription factors and proteins involved in cell signaling and maintenance of cell integrity. (2,3) Aggregation of mutant huntingtin generates oxidative stress within the cell, (4− that is, an imbalance in the amount of reactive oxygen species (ROS) and antioxidative action of the cell. ROS have the ability to damage all biomolecules, including lipid, protein, carbohydrates, and DNA, either directly or indirectly. (9) In neurological disorders such as multiple sclerosis, stroke, and neuroinfection, and in neurodegenerative diseases such as alzheimer’s, Parkinson’s, and Huntington’s, oxidative stress is thought to be a principal mechanism in the progression of the disease. (10,11) Examination of HD postmortem tissues has demonstrated an increase in multiple markers of oxidative stress, (12) which suggests that oxidative damage is increased during the course of the disease. Oxidative stress leads to caspase-mediated neuronal cell death and is considered to be a potential cause of observed neuropathological changes. (13)
Antioxidants may play an important role in protecting against a number of human diseases. (14−19) Various studies have shown the role of antioxidants in neuroprotection. (18,20−22) Protopanaxatriol is a plant extract isolated from Panax ginseng mayer and has shown a protective effect against 3-nitropropionic acid (3-NP)-induced oxidative stress in a rat model of HD. (20) Protopanaxtriol restores mitochondrial complex enzyme II and SOD (superoxide dismutase) activity and directly scavenges superoxide anions and hydroxyl radicals. (20) Several plant extracts or secondary metabolites have shown strong antioxidant activity and protection against oxidant-induced damage in the case of neurodegenerative disorders. (14,21,22) Among these plant metabolites is harmine, a plant-derived beta-carboline alkaloid with one indole nucleus and a six-membered pyrrole ring. (23) β-Carboline alkaloids can act as scavengers of ROS. (24−26) Harmine increases superoxide dismutase and catalase activities and decreases carbonyl formation in mitochondria in MPTP-treated mice brains as compared to control. (27) The alkaloid is also able to decrease Cu2+-induced oxidation of low density lipoproteins. (28) Harmine increases hippocampal levels of the brain-derived neurotrophic factor in rat brains, (29) which has been implicated in a number of neurodegenerative disorders. (30) Harmine is also an inhibitor of monoamine oxidase. (31) The alkaloid is a potent ATP-competitive inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylation-regulated kinase 1A), whose overexpression is a risk factor in β-amyloidosis, neurofibrillary degeneration, and a number of malignant conditions. (32)
Studies indicate that the basic cellular machinery is well conserved and aggregation of proteins depends on the conserved pattern of folding, despite the species barrier. (33−35) Many yeast models faithfully recapitulate disease-relevant phenotypes which have been further validated in mammalian systems and human patients. (36) As the huntingtin gene is missing in yeast, HD is modeled in this organism by its heterologous expression. (37) The function of wild-type huntingtin is absent in yeast, so the toxicity of mutant huntingtin is only due to its toxic gain of function. Protein aggregation is associated with elevated levels of oxidative stress. The purpose of the current study was to investigate the mechanism by which harmine, an antioxidant, acts as a neuroprotectant in protein misfolding diseases, using the well-validated yeast model of HD. The constructs used here, pYES2-25Q-htt-EGFP and pYES2-103Q-htt-EGFP, express polyglutamine as FLAG-25Q-htt-EGFP and FLAG-103Q-htt-EGFP, respectively. (37) No separate band for EGFP has been observed on a native gel by us and others, (38,39) indicating that the proteins are expressed as EGFP-fused products and no significant cleaved EGFP is formed. While 25Q-htt-EGFP is seen as diffused fluorescence throughout the cell which is taken to indicate expression of soluble protein, 103Q-htt-EGFP is seen as discrete fluorescent puncta. (37,40) We wanted to study whether harmine has aggregation inhibitory properties or if its role is limited to reduction of oxidative stress, and thus, an indirect effect on protein aggregation.

Results and Discussion

Trehalose Reduces Aggregation of 103Q-htt and Increases Survival of Yeast Cells
Saccharomyces cerevisiae BY4742 cells were transformed with pYES2-25Q-htt-EGFP or pYES2-103Q-htt-EGFP. This strain has Rnq1 protein in the prion form ([RNQ+]), and aggregation-induced toxicity of mutant huntingtin protein carrying longer stretches of polyQ tracts is observed only in yeast cells of this strain. (37) Cells expressing 103Q-htt showed fluorescent puncta which confirmed the formation of aggregates (Figure 1a). (37,40) Trehalose has been used to stabilize proteins under a variety of stress conditions in vitro and in cells. (41−44) The disaccharide has no effect on expression of the wild-type huntingtin fragment, 25Q-htt (Figure S1a–c), (43) and has been shown to have a beneficial effect in cells and animal models of HD. (45) Fluorescence microscopy suggested that the cells in which the expression of 103-htt was induced in the presence of 4% w v–1 trehalose showed diffusible fluorescence as compared to cells which were untreated (Figure 1a). It was observed that in the presence of trehalose, approximately 25% of total cells had diffused expression of 103Q-htt as compared to untreated cells, where it was negligible (Figure 1b). Native PAGE analysis showed a significant increase in the fraction of soluble 103Q-htt when cells were grown in the presence of trehalose (Figures 1c and S2a). Solubilization of 103Q-htt in the presence of trehalose was confirmed by immunoblotting (Figures 1d and S2b).
Figure 1

Figure 1. Trehalose inhibits aggregation of 103Q-htt. (a) Postinduced yeast cells were pelleted down, washed, mounted on glass slides, and viewed under a fluorescence microscope (Nikon E600 Eclipse, Nikon Corporation, Japan). Bar = 10 μm. (b) Number of cells exhibiting diffused fluorescence out of a total 1000 cells in 40 separate, randomly selected fields were counted under a fluorescence microscope. 103Q-htt-EGFP forms discrete intense fluorescent dots. (40) Upon solubilization, these are visible as diffused fluorescence throughout the cell. Cells exhibiting diffused fluorescence were counted to quantify the extent of solubilization. Values shown are the percentage of each set and are mean ± sem of three independent experiments; ***p < 0.001 against untreated cells. (c) Native PAGE analysis of soluble fractions of cell lysates expressing 103Q-htt in the absence and presence of trehalose (4%, w v–1). The gel was scanned with an image scanner (Typhoon Trio, GE Healthcare), using λex 532 nm and λem 610 nm. Lower panel shows densitometric analysis of the bands. Band intensity of 103Q-htt in untreated cells (absence of trehalose) was assigned an arbitrary value of 100%. Values shown are mean ± sem of three independent experiments; ***p < 0.001 against untreated cells. An equal amount of protein was loaded in each well. The Coomassie stained gel is shown in Figure S2a. (d) Western blotting of soluble fractions of cell lysates expressing 103Q-htt in the absence and presence of a trehalose using polyglutamine antibody. Lower panel shows densitometric analysis of the bands. Band intensity of 103Q-htt in untreated cells (absence of trehalose) was assigned an arbitrary value of 100%. Values shown are mean ± sem of three independent experiments; ***p < 0.001 against untreated cells. An equal amount of protein was loaded in each well. The Ponceau S-stained membrane is shown in Figure S2b. (e) Filter retardation assay of cell lysate expressing 103Q-htt in the absence and presence of trehalose. Equal amount of protein was filtered through each slot. Lower panel shows densitometric analysis of the bands. Intensity of dot of 103Q-htt in untreated cells (absence of trehalose) was assigned an arbitrary value of 100%. Values shown are mean ± sem of three independent experiments; *p < 0.05 against untreated cells. (f) Estimation of ROS in cells expressing 25Q-htt and 103Q-htt using DHE (λex 535 nm, λem 635 nm). Values shown are mean ± sem of three independent experiments. (g) Viability of yeast cells expressing 103Q-htt in the absence and presence of trehalose. Values shown are mean ± sem of three independent experiments; ***p < 0.001 against untreated cells.

The intensity of the band for the monomer, that is, 103Q-htt, was found to be ∼1.5-fold higher as compared to cells which were not exposed to trehalose. Being an osmolyte, trehalose stabilizes the protein in its native conformation which leads to decreased aggregation. (41,42)
The effect of trehalose on aggregation of 103Q-htt was further confirmed by filter retardation assay using a cellulose acetate membrane which retains aggregates. (46) Densitometric analysis of dots showed that in the presence of trehalose, the amount of aggregates was reduced by ∼1.2-fold as compared to untreated cells (Figure 1e). Aggregation of proteins is related to increased ROS levels within the cell. (47,48) High levels of ROS in the cell cause modification of functional groups of amino acids which leads to protein aggregation. Addition of trehalose had no effect on the basal level of ROS in cells expressing 25Q-htt (Figure S3a). A significant increase (>3.5-fold) in the fluorescence intensity of 2-hydroxyethidine (2-EOH) was observed in cells expressing 103Q-htt as compared to those expressing wild-type 25Q-htt (Figure 1f), which matched reports in the literature showing a positive correlation between protein aggregation and oxidative stress. (4,43,49,50) In the presence of trehalose, cells expressing 103Q-htt had lower levels of ROS as compared to untreated cells, although the decrease was not significant (Figure 1f).

Aggregation of the mutant huntingtin fragment has been linked to lower survival of yeast cells. (37) Addition of trehalose had no effect on the viability of yeast cells expressing 25Q-htt (Figure S3b). The viability of yeast cells expressing 103Q-htt was significantly higher (∼2-fold) when grown in the presence of trehalose than in its absence (Figure 1g). This correlates well with higher solubilization (Figure 1c,d) and reduced aggregation (Figure 1e) of 103Q-htt observed in the presence of trehalose.
Presence of Harmine Reduces Aggregation of 103Q-htt
Harmine is a phyto-antioxidant and decreases the cellular oxidative stress level by scavenging ROS. (23−26) Addition of harmine had no effect on the expression of 25Q-htt in yeast cells as seen by fluorescence microscopy (Figure S4a), native PAGE (Figure S4b), and western blot analysis (Figure S4c).

Aggregation of 103Q-htt was monitored in yeast cells grown in the presence of different concentrations of harmine, at a fixed concentration (4% w v–1) of trehalose. As mentioned above (Figure 1b–d) and in previous reports, (43) at this concentration, trehalose has an attenuating effect on aggregation of 103Q-htt in yeast cells. Increasing solubilization of 103Q-htt was observed in yeast cells with increasing concentration of harmine along with trehalose as compared to untreated cells or cells grown in the presence of trehalose alone (Figure 2a).
Figure 2

Figure 2. Harmine attenuates aggregation of 103Q-htt in yeast cells. (a) Postinduced yeast cells were pelleted down, washed, mounted on glass slides, and viewed under a fluorescence microscope (Nikon E600 Eclipse, Nikon Corporation, Japan). Bar = 10 μm. (b) Number of cells exhibiting diffused fluorescence out of a total 1000 cells in 40 separate, randomly selected fields were counted under a fluorescence microscope. 103Q-htt-EGFP forms discrete intense fluorescent dots. (40) Upon solubilization, these are visible as diffused fluorescence throughout the cell. Cells exhibiting diffused fluorescence were counted to quantify the extent of solubilization. Values shown are percentage of each set and are mean ± sem of three independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001 against untreated cells (in the absence of trehalose and harmine). (c) Native PAGE analysis of soluble fractions of cell lysates expressing 103Q-htt in the absence and presence of trehalose (4%, w v–1) and different concentrations of harmine. The gel was scanned with an image scanner (Typhoon Trio, GE Healthcare), using λex 532 nm and λem 610 nm. Lower panel shows densitometric analysis of the bands. Band intensity of 103Q-htt in untreated cells (absence of trehalose and harmine) was assigned an arbitrary value of 100%. Values shown are mean ± sem of three independent experiments; *p < 0.05, **p < 0.01 against untreated cells (in the absence of trehalose and harmine). Equal amount of protein was loaded in each well. The Coomassie stained gel is shown in Figure S5a. (d) Western blotting of soluble fractions of cell lysates expressing 103Q-htt in the absence and presence of trehalose (4%, w v–1) and different concentrations of harmine using a polyglutamine antibody. Lower panel shows densitometric analysis of the bands. Band intensity of 103Q-htt in untreated cells (absence of trehalose and harmine) was assigned an arbitrary value of 100%. Values shown are mean ± sem of three independent experiments; **p < 0.01, ***p < 0.001 against untreated cells (in the absence of trehalose and harmine). Equal amount of protein was loaded in each well. The Ponceau S-stained membrane is shown in Figure S5b. (e) Filter retardation assay of cell lysate expressing 103Q-htt in the absence and presence of trehalose (4%, w v–1) and different concentrations of harmine. Equal amount of protein was filtered through each slot. Lower panel shows densitometric analysis of the triplicate dots. Intensity of dot for 103Q-htt in untreated cells (absence of trehalose and harmine) was assigned an arbitrary value of 100%. Values shown are mean ± sem of three independent experiments; **p < 0.01, ***p < 0.001 against untreated cells (absence of trehalose and harmine). (f) Estimation of ROS in cells expressing 103Q-htt using DHE (λex 535 nm, λem 635 nm). Values shown are mean ± sem of three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001 against untreated cells (in the absence of trehalose and harmine). (g) Viability of yeast cells expressing 103Q-htt in the absence and presence of trehalose (4%, w v–1) and different concentrations of harmine. Values shown are mean ± sem of three independent experiments; **p < 0.01, ***p < 0.001 against untreated cells (in the absence of trehalose and harmine). (h) Native PAGE analysis of soluble fractions of cell lysates overexpressing Rnq1-EGFP in the absence and presence of harmine. The gel was scanned with an image scanner (Typhoon Trio, GE Healthcare), using λex 532 nm and λem 610 nm. Lower panel shows densitometric analysis of the bands. Band intensity of Rnq1-EGFP in untreated cells (absence of harmine) was assigned an arbitrary value of 100%. Equal amount of protein was loaded in each well. The Coomassie stained gel is shown in Figure S6. (i) Filter retardation assay of cell lysates overexpressing Rnq1 in the absence and presence of harmine using a Rnq1 antibody. Triplicate dots are shown. Lower panel shows densitometric analysis of the dots. Intensity of dot for Rnq1 in untreated cells (absence of harmine) was assigned an arbitrary value of 100%. Values shown are mean ± sem of three independent experiments; ***p < 0.001 against untreated cells (absence of harmine). (j) Estimation of ROS in cells overexpressing Rnq1 using DHE (λex 535 nm, λem 635 nm). Values shown are mean ± sem of three independent experiments; ***p < 0.001 against untreated cells (in the absence of harmine).

At the highest concentration of harmine (25 μg mL–1) with trehalose, approximately 55% of total cells showed diffused fluorescence because of EGFP indicating expression of soluble 103Q-htt as compared to untreated cells, where it is negligible (Figure 2b). Thus, in addition to the disaccharide, the antioxidant was able to solubilize 103Q-htt. Interestingly, at the highest concentration of harmine (25 μg mL–1), the extent of solubilization of 103Q-htt was the same, irrespective of the presence of trehalose. Increased solubilization of 103Q-htt was also seen by native PAGE. The intensity of the band for EGFP fused to 103Q-htt increased with increasing concentration of harmine in the presence of trehalose (Figures 2c and S5a). As mentioned before, at the highest concentration of the antioxidant, solubilization of 103Q-htt was independent of the presence of trehalose. A similar pattern was also observed when solubilization of 103Q-htt was monitored by immunoblotting. Cells which were grown in the presence of the maximum concentration of harmine (25 μg mL–1) along with trehalose showed 3-fold increase in the intensity of the band for soluble 103Q-htt as compared to untreated cells, while this increase in intensity jumped to ∼8-fold for cells grown in the presence of harmine (25 μg mL–1) without trehalose (Figures 2d and S5b).
The effect of harmine on aggregation of 103Q-htt was confirmed by filter retardation assay using a cellulose acetate membrane (Figure 2e). This membrane filters the proteins on the basis of size, allowing only aggregates to be retained on the membrane. (46) Densitometric analysis of dots showed that the amount of 103Q-htt aggregates formed in the presence of harmine and trehalose in treated cells decreased continuously as compared to untreated cells, with the maximum reduction (>2-fold) seen with the highest concentration of harmine in the absence of trehalose (Figure 2e).

Harmine Decreases Oxidative Stress and Increases Cell Viability

Aggregation of proteins is associated with increased oxidative stress in the cell. The mechanism of aggregation-lowering ability of harmine was followed by measuring the generation of ROS in the cell. Addition of harmine in the media had no effect on the basal level of ROS in yeast cells expressing 25Q-htt (Figure S4d). This shows that the activity of harmine as an anti-oxidant is seen only when the level of ROS exceeds a threshold value. Aggregation of 103Q-htt led to increased generation of ROS (Figure 1e) which was reduced when cells were grown in the presence of harmine, in the absence or presence of trehalose (Figure 2f). Reduced aggregation of 103Q-htt and concomitant decrease in oxidative stress resulted in increased survival of yeast cells expressing 103Q-htt in the presence of harmine (Figure 2g). This increase was ∼2-fold at the highest concentration of harmine (25 μg mL–1) in the absence of trehalose. Addition of trehalose and harmine did not show any effect on the viability of yeast cells expressing 25Q-htt (Figure S4e). Thus, in the presence of the antioxidant harmine, aggregation of 103Q-htt was reduced, corresponding with reduced oxidative stress and increased cell viability.
The presence of prions, specifically [RNQ1+], seems to be an essential condition for aggregation-induced proteotoxicity in yeast cells. (37) Hence, the aggregation status of Rnq1 was monitored in yeast cells in the presence of harmine. Yeast cells transformed with pYES2-Rnq1-EGFP were induced to express Rnq1-EGFP in the absence and presence of harmine. Native PAGE analysis of the cell lysate showed that solubilization of Rnq1 increased with increasing concentration of harmine (Figures 2h and S6). Prion formation ([RNQ1+]) was monitored by filter retardation assay. Analysis of the blot showed that in the presence of harmine, the extent of aggregation of Rnq1 was significantly reduced as compared to untreated cells (Figure 2i). Attenuated prion formation ([RNQ1+]) also correlated with reduced intracellular oxidative stress (Figure 2j). Increased prion formation, that is, [RNQ1+], has been directly correlated with aggregation of 103Q-htt in yeast cells due to the “seeding” activity of the prion protein. (37) Decreased formation of prion in the presence of the anti-oxidant harmine may be responsible for the inhibition of aggregation of 103Q-htt observed in this case.
Harmine Has No Effect on Aggregation of the Mutant Huntingtin Fragment in Vitro
The increased ROS level in cells causes oxidative damage to proteins. (4,49) Conversely, the presence of misfolded and aggregated proteins enhances the level of ROS in the cell, primarily by mitochondrial dysfunction. (15,51−54) Reduction in intracellular oxidative stress observed above could be due to decrease in protein aggregation in the presence of harmine. On the other hand, reduced oxidative stress with consequent reduction in oxidative damage to proteins could result in decreased aggregation of 103Q-htt in the presence of harmine. Whether oxidative damage precedes or follows protein aggregation and the step at which harmine intervenes in this process remain to be determined. Hence, we decided to investigate whether harmine has any effect on aggregation of expanded polyglutamine in an extracellular milieu. Aggregation of the polyglutamine tract (in the pathogenic range) occurs in a length-dependent manner. The pattern of aggregation remains unaltered. A change is seen in aggregation kinetics, with longer polyQ stretches exhibiting faster rates of aggregation (37,40,55−58) and higher toxicity. (37,56−58) Because for lengths >72Q, aggregation occurs quite fast (46) and measurement of difference in rates becomes difficult, we selected the well-validated elongated polyQ-containing 51Q-htt system (39,46) to monitor differences in rates aggregation of polyQ in the presence of harmine in vitro.

The mutant huntingtin fragment (GST-51Q-htt) was purified by affinity chromatography, as described before. (46,59) Purification of the protein was followed by SDS-PAGE which showed a band at the expected position (∼50 kDa) (Figure 3a). (46,59) Immunoblotting with the polyglutamine antibody showed a tail (Figures 3b and S7) because of truncated polyglutamine tracts. (39,46) In vitro aggregation of 51Q-htt was monitored by Thioflavin T fluorescence assay. (39,59) Aggregation kinetics showed distinct nucleation, growth (fibrillation), and equilibrium (saturation) stages (Figure 3c) and confirmed that the aggregates formed were of cross β-sheet nature. (46,59,60) No significant difference in the pattern of aggregation of 51Q-htt was seen when the protein was incubated in the presence of harmine (6.25 or 25 μg mL–1) than in its absence.
Figure 3

Figure 3. Harmine has no effect on aggregation of 103Q-htt in vitro. (a) Purification of GST-51Q-htt was carried out by affinity chromatography and followed by SDS-PAGE. Lane 1: molecular weight marker (bovine serum albumin, 65 kDa), lane 2: uninduced cell lysate, lane 3: induced cell lysate, lane 4: flowthrough; lanes 5 and 6: washings, lanes 7 and 8: eluates, and lane 9: dialyzed protein. Protein load was 10 μg in each case. The gel was Coomassie stained. (b) Western blot analysis of purification of GST-51Q-htt; lane 1: cell lysate, lane 2: flowthrough, lanes 2 and 3: washings, lanes 5 and 6: eluted GST-51Q-htt, and lane 7: dialyzed protein. Protein load was 20 μg each lane. The membrane was probed with a polyglutamine antibody followed by an FITC-conjugated antimouse antibody. The Ponceau S-stained membrane is shown in Figure S7. (c) GST-51Q-htt (1 mg mL–1) was incubated at 37 °C. Time-dependent formation of aggregates was monitored by Thioflavin T fluorimetry (λex 440 nm, λem 484 nm). The final concentrations of the protein and the fluorophore were 1.5 and 50 μM, respectively. (d) GST-51Q-htt (1 mg mL–1, 40 mM Tris-HCl buffer, pH 8.0 containing 150 mM NaCl) was incubated at 37 °C at different time intervals. Filter retardation assay using a cellulose acetate membrane (0.2 μm) was carried out at different time intervals, and the amount of aggregates retained was probed with the polyglutamine antibody. The intensity of the dot at the last point of analysis was assigned an arbitrary value of 100%. Triplicate dots are shown for each time point in Figure S8a. (e) GST-51Q-htt (1 mg mL–1, 40 mM Tris-HCl buffer, pH 8.0 containing 150 mM NaCl) was incubated at 37 °C at different time intervals. The incubated protein was filtered through the nitrocellulose membrane and analyzed with the oligomer-specific A11 antibody using a dot-blot assay. The intensity of the dot for the protein incubated alone till 75 h was assigned a value of 100%. Triplicate dots are shown for each time point in Figure S8b. Values shown are mean ± sem of three independent experiments.

The effect of harmine on aggregation of 51Q-htt was further studied by filter retardation assay using the polyglutamine antibody as the probe (Figures 3d and S8a). Unlike Thioflavin T which is an amyloid-specific dye and quantifies the fibrillar aggregates formed (Figure 3c), filter retardation assay quantifies the total amount of aggregates. Comparison of the two curves suggests formation of amorphous aggregates at initial stages followed by fibrillar aggregates. The formation of oligomers during incubation was detected with an oligomer-specific A11 antibody (Figures 3e and S8b). Almost similar patterns were observed in the absence and presence of harmine when the membrane was probed with the A11 antibody. Analysis of both curves suggests that harmine had no effect on either oligomer formation or aggregation of 51Q-htt in vitro.

Thus, it is clear that harmine had no direct effect on aggregation of the mutant huntingtin fragment. Instead, inhibition of aggregation of the mutant huntingtin fragment observed in yeast cells resulted from antioxidation activity of harmine. Harmine scavenges ROS which lowers oxidative damage to the mutant huntingtin fragment. As damage to proteins due to oxidative stress is responsible for protein aggregation in many protein misfolding disorders, (15,51−54) the presence of the antioxidant reduces aggregation of the mutant huntingtin fragment by attenuating oxidative damage and ameliorating aggregation-induced cytotoxicity.

Misfolding and aggregation of proteins have been linked to the development and progression of a number of neurodegenerative and other disorders. A number of epidemiological studies have established correlations between lifestyle choices and progression of disease conditions. For example, coffee drinking and cigarette smoking have been shown to have a negative correlation with disease progression in Parkinson’s disease. (61,62) We have shown that caffeine (63) and nicotine (64) alter the rate of aggregation of α-synuclein and increase the survival of yeast cells expressing α-synuclein, thus providing a mechanistic explanation for the observations. In the present case, the antioxidant harmine is seen to be as good a protein stabilizer under intracellular conditions as the disaccharide trehalose, a known protein stabilizer, although it follows a different mode of action. The level of inhibition of aggregation seen in the presence of harmine and trehalose was marginally lower than that in the presence of the highest concentration of harmine alone. The decreasing level of aggregation of 103Q-htt with increasing concentration of harmine and fixed concentration of trehalose also reflects the importance of the antioxidant in slowing down protein aggregation inside the cell. The presence of harmine lowers the oxidative stress and hence aggregation of oxidatively damaged proteins. Under these conditions, the presence of trehalose does not provide any additional benefit to the cell.
Experimental Section
Materials
S. cerevisiae BY4742 [MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0, (RNQ1+)] is a product of Open BioSystems and was purchased from SAF Labs Pvt. Ltd. Mumbai, India. Harmine, glutathione-agarose matrix, thioflavin T, dihydroethidium (DHE), and the antimouse FITC conjugated antibody were purchased from Sigma-Aldrich, Bengaluru, India. Mouse anti-polyglutamine (polyglutamine expansion disease marker monoclonal antibody, MAB1574) was a product of Chemicon International and was purchased from Millipore (India) Pvt. Ltd., New Delhi, India. A goat anti-Rnq1 antibody was purchased from Santa Cruz, California, USA. The oligomer-specific A11 polyclonal antibody was purchased from Invitrogen Corporation, California, USA. The nitrocellulose membrane (0.2 μm) was purchased from Advanced Microdevices Pvt. Ltd. Ambala Cantt, India. The cellulose acetate membrane was purchased from Sartorius Stedium Biotech, Goettingen, Germany. All other reagents and chemicals used were of analytical grade or higher.

Methods
Expression of 25Q-htt and 103Q-htt in Yeast Cells
S. cerevisiae BY4742 strain was transformed separately with pYES2-25Q-htt-EGFP or pYES2-103Q-htt-EGFP by the lithium acetate–polyethylene glycol (PEG) method. (65) Transformed cells were grown in SC-URA media containing 2% (w v–1) dextrose at 30 °C, 200 rpm till OD600nm 0.6–0.8. Expression of proteins was induced by changing the media to SC-URA media containing 2% (w v–1) galactose, with and without 4% (w v–1) trehalose and different concentrations of harmine for 10 h. Expression of 25Q-htt and 103Q-htt was monitored by fluorescence microscopy (E600 Eclipse microscope, Nikon, Japan) as the proteins were tagged with EGFP. Yeast cells were disrupted using acid-treated glass beads and lysed using lysis buffer (0.05 M Tris, 0.15 M NaCl, 0.002 M DTT, pH 7.5 supplemented with 1 mM PMSF). (66) The lysate was centrifuged at 800g for 10 min, and the supernatant obtained was analyzed for the presence of aggregates using filter retardation assay. This supernatant was further centrifuged at 12,000g for 45 min, and the presence of soluble protein was confirmed by native PAGE and immunoblotting using the polyglutamine-specific antibody. The respective gel and blot were scanned using an image scanner (Typhoon Trio, GE Healthcare). Estimation of the protein content in different samples was carried out by the dye binding method, (67) using bovine serum albumin as the standard protein.
Filter Retardation Assay
The supernatants (obtained after centrifugation of yeast cell lysates at 800g for 10 min) were filtered through a cellulose acetate membrane (0.2 μm pore size) using a dot blot apparatus (Whatman Schleicher & Schuell, UK). For in vitro analysis of the aggregation pattern, affinity purified 51Q-htt was incubated at 37 °C for 95 h. (39) Aliquots (50 μg protein each) were withdrawn at regular intervals and vacuum-filtered through a prewetted cellulose acetate membrane. The membranes were probed with the polyglutamine-specific antibody for detection of aggregates. Aliquots were also filtered through a nitrocellulose membrane and probed with the oligomer-specific A11 polyclonal antibody for detection of oligomers. (39) The membranes were scanned on an image scanner (Typhoon Trio, GE Healthcare). The intensity of dots was quantified using ImageQuant TL software (GE Healthcare). The values were fitted into the equation for a sigmoidal curve (Boltzmann function) using the equation, (39,60)

where yi + mxi is the initial line, yf + mxf is the final line, and x0 is the midpoint of the maximum signal.
Measurement of Oxidative Stress
Intracellular ROS levels were quantified using DHE (dihydroethidium) dye. (68) After the end of the induction period, yeast cells were washed with phosphate-buffered saline, pH 7.4 (PBS), and counted using Neubauer’s chamber. Cells (1 × 107) were aliquoted into a microcentrifuge tube, and DHE (0.01 M, in PBS) was added at a final concentration of 10 μM. The final reaction mixture was made up to 1 mL with PBS and incubated at 37 °C with shaking at 200 rpm for 20 min. The emission intensity of ethidium was recorded at λem 635 nm using λex 535 nm.

Cell Viability Assay

Postinduction yeast cells were pelleted down, resuspended in 1 mL autoclaved water, and counted using Neubauer’s chamber. Cells (1 × 103) were plated on SC-URA containing 2% dextrose plates, and growth of colonies was observed at 30 °C for 3 days.
Measurement of Uptake of Harmine by Yeast Cells
The amount of harmine taken up by yeast cells was determined by HPLC (SCL-10A VP, Shimadzu, Japan). (69) Yeast cell pellets were thawed on ice, resuspended in 500 μL of 0.5 M trichloroacetic acid, and incubated at room temperature for 1 h. The cell lysates were centrifuged at 12,000g for 30 min at room temperature. The supernatants were collected, and the pellets were resuspended in 500 μL of 0.5 M trichloroacetic acid and incubated at room temperature for 1 h. The suspensions were centrifuged as mentioned above, and the supernatants were pooled and filtered through a 0.2 μm syringe filter. Samples were injected into a C18 Zorbax analysis column (Agilent Technologies, USA), and the eluate was monitored using a photodiode array detector (UV 10A, Shimadzu, Japan) at a flow rate of 1 mL min–1. (69) The mobile phase used was isopropyl alcohol/acetonitrile/water/formic acid in the ratio 100300:0.3 (v/v/v/v), pH 8.6 [adjusted with triethylamine (99%)]. (69)

Aggregation of the Mutant Huntingtin Protein Fragment in Vitro

Competent Escherichia coli BL21 (DE3) cells were transformed with plasmid pGEX-5X1-HDex1-CAG51 and grown at 37 °C in Luria–Bertani media. Protein expression was induced with 1 mM IPTG for 5 h at 37 °C. (46) The mutant huntingtin protein fragment (GST-51Q-htt) was purified by affinity chromatography, as described earlier. (46,59) Purification of 51Q-htt protein was confirmed by SDS-PAGE.
Purified 51Q-htt (1 mg mL–1) was incubated with and without harmine (6.25 and 25 μg mL–1) at 37 °C. Aliquots (25 μg each) were withdrawn at regular time intervals and the aggregation pattern of 51Q-htt was monitored by thioflavin T fluorescence assay. (39,60) Fluorescence intensity of the dye was measured using a spectrofluorimeter (RF-5301PC, Shimadzu) with λex 440 nm and λem 484 nm.
Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b02375.

Coomassie-stained native gels, Ponceau S-staining of immunoblots, microscopy images, native gels, and immunoblots of control (25Q-htt)

Harmine Acts as an Indirect Inhibitor of Intracellular Protein Aggregation

Supplementary informationS1 Harmine acts as an indirect inhibitor of intracellular protein aggregation

Swati Jain, Venkataharsha Panuganti, Sonali Jhaand Ipsita Roy*Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab 160062, India*To whom correspondence to be addressed atTel: 0091-172-229 2061 Fax: 0091-172-221 4692 Email: ipsita@niper.ac.in
Supplementary informationS2FigureS1.Effect of trehalose on the expression of 25Q-htt. (a)Post-induced  yeast cells were  pelleted  down,  washed,  mounted  on  glass  slides  and  viewed  under  a  fluorescence microscope  (Nikon  E600  Eclipse,  Nikon  Corporation,  Japan).  Bar  =  10  μm. (b)Native PAGE analysis of soluble fractions of cell lysates expressing 25Q-htt in the absence and presence of trehalose (4 %, w v-1). The gel was scanned with an image scanner (Typhoon Trio, GE  Healthcare),  using λex532 nm, λem610  nm.  Lower  panel  showsdensitometric analysis of the bands. Band intensity of 25Q-htt in untreated cells (absence of trehalose) was  assigned  an  arbitrary  value  of  100  %.  Values  shown  are  mean±sem  of  three independent  experiments. (c)Western   blotting  of soluble  fractions  of  cell lysates expressing 25Q-htt  in  the  absence  and  presence  of  trehalose  using  polyglutamine antibody. Lower panel showsdensitometric analysis of the bands. Band intensity of 25Q-htt  in  untreated  cells  (absence  of  trehalose)  was  assigned  an  arbitrary  value  of 100  %. Values shown are mean±sem of three independent experiments.

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Corresponding Author
Ipsita Roy - Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab 160062, India;  Orcidhttp://orcid.org/0000-0003-1120-444X; Email: ipsita@niper.ac.in
Authors
Swati Jain - Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab 160062, India
Venkataharsha Panuganti - Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab 160062, India
Sonali Jha - Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab 160062, India
Notes
The authors declare no competing financial interest.
Acknowledgments
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The authors are thankful to Prof. M. Y. Sherman, Boston University School of Medicine, Boston, Massachusetts, USA, for the gift of pYES2-25Q-htt-EGFP and pYES2-103Q-htt-EGFP, Prof. E. Wanker, Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany, for the gift of pGEX-5X1-HDex1-CAG51. pYES2-Rnq1-EGFP was generated by Dr. Ratnika Sethi; the insert (Rnq1) was obtained from pRS315-CUP1-Rnq1-mRFP, which was received from Prof. D. M. Cyr, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA. Partial financial support from the Science and Engineering Research Board and the Department of Biotechnology is gratefully acknowledged.

References
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Keep an eye open:
https://rumble.com/v1qjp2y-died-suddenly-new-covid-19-documentary-from-stew-peters.html
https://rumble.com/v1wcesu-died-suddenly-full-documentary-not-suitable-for-children.html

I went to some of Stew Peters' videos on Bitchute and Rumble and posted the following message.

This Substack post is a good read IMO.
"Died Suddenly" Is Typical Trash from Stew Peters
https://jackanapes.substack.com/p/died-suddenly-is-typical-trash-from

Here's a short quote:
"I completely washed my hands of Stew Peters after his “Watch the Water” snake venom fiasco, and “Died Suddenly” offers no redemption. This is not somebody we can trust. As far as I’m concerned, Stew Peters makes the rest of us look bad, and whether that is on purpose or not is sort of beside the point, because either way, he’s bad for the movement because in the end he discredits us even if he gets some things right. Going forward, he deserves 100% of our derision and 0% of our attention."

"[POSTSCRIPT: Stew recently interviewed Israeli scientist Dr. Shmuel Shapira, who was director of the state’s biological institute. He was injured by the COVID vaccines and then when he went public his reputation was trashed by the establishment. Here is how Stew spun the interview:
https://rumble.com/v1r5lfg-live-israel-makes-secret-deal-with-pfizer-to-murder-the-masses-u.s.-sends-m.html
Needless to say, Dr. Shapira was mortified. He had already put his reputation on the line by speaking out. He publicly denounced the interview and express[ed] his regret for doing it.]

The post mentioned that the first few minutes of the video was apparently designed to make it look like another weird conspiracy video, connecting it to aliens, Alex Jones in a tinfoil hat etc. The fibery blood clots took up a lot of the video and would likely gross out most potential viewers, just like mutilated fetus videos would do for an antiabortion video. If you read the post, there are more explanations of why it seems Stew Peters is not an ally.

Lloyd wrote:I went to some of Stew Peters' videos on Bitchute and Rumble and posted the following message.

This Substack post is a good read IMO.
"Died Suddenly" Is Typical Trash from Stew Peters
https://jackanapes.substack.com/p/died-suddenly-is-typical-trash-from

Here's a short quote:
"I completely washed my hands of Stew Peters after his “Watch the Water” snake venom fiasco, and “Died Suddenly” offers no redemption. This is not somebody we can trust. As far as I’m concerned, Stew Peters makes the rest of us look bad, and whether that is on purpose or not is sort of beside the point, because either way, he’s bad for the movement because in the end he discredits us even if he gets some things right. Going forward, he deserves 100% of our derision and 0% of our attention."

"[POSTSCRIPT: Stew recently interviewed Israeli scientist Dr. Shmuel Shapira, who was director of the state’s biological institute. He was injured by the COVID vaccines and then when he went public his reputation was trashed by the establishment. Here is how Stew spun the interview:
https://rumble.com/v1r5lfg-live-israel-makes-secret-deal-with-pfizer-to-murder-the-masses-u.s.-sends-m.html
Needless to say, Dr. Shapira was mortified. He had already put his reputation on the line by speaking out. He publicly denounced the interview and express[ed] his regret for doing it.]

The post mentioned that the first few minutes of the video was apparently designed to make it look like another weird conspiracy video, connecting it to aliens, Alex Jones in a tinfoil hat etc. The fibery blood clots took up a lot of the video and would likely gross out most potential viewers, just like mutilated fetus videos would do for an antiabortion video. If you read the post, there are more explanations of why it seems Stew Peters is not an ally.

I used to know some coroners back in the day. If they wanted to keep their licenses they had to report in detail anything that would look abnormal --this is what the coroners in Stew's video did.  Stew Peters just reported on it as he found it IMHO. The question is really whether SADS is real or not for some doses and types of the Covid-19 vaccinations? Auto-immune disorders can and do happen after an infection of any aggressive flu. Is Stew Peters' video imagery fake? By the way, I think he was trolling those people who called Covid-19 vaccination injuries "fake" with the AJ video-- like "who is crazier here".  You'll never get a clean presentation on Covid-19 vaccine injuries from the International Press agencies, they cheered on the vaccinations. It is like the founding of the Federal Reserve System, it really was a "conspiracy" that came out only decades later. Truth will out  -- over time at least. Looks like the poster at JackSnapes blog you mentioned is a lecturer at Hebrew University in Israel (hmm...for the 9/11 mentions from Stew...it irks a lot of people if you mention anything outside of the accepted press-university explanation for events. Why? They start to research more in-depth the details.).  

They have vaccine injury lawyers proving cases for damages from influenza vaccines long before SARS-COVID-19 was around. The Guillain-Barre Syndrome is a risk for some people who take a flu vaccination:

https://vaccinelaw.com/lawyer/2016/03/24/Vaccine-Injury/Can-Vaccines-Cause-Auto-Immune-Disorders-%E2%80%93-Providing-Answers-to-Commonly-Asked-Questions_bl24139.htm

They had a reporting and compensation system for it long before Covid-19 (post below from 2016). 9/11 still needs a much deeper investigation but it looked like a hurried cover up of 9/11 with the press full-on to "blame" the Arabs like they went full-on to "blame" the unvaccinated. Bin Laden's dad was a Billionaire working for the Saudi government for decades...(hmm...the press doesn't seem to tell about that too often do they? Why not?). It really comes down to physics as well....were the events on 9/11 "typical" everyday physics? Or were events that day "extraordinary"...pics of the cars told me enough something looked really "weird"? Another "hmm" for that one.

What should I do if I got vaccinated and am now suffering from an immune disorder?

If you or your child began experiencing symptoms of an immune deficiency or autoimmune disorder after receiving a vaccination, you may be entitled to financial compensation under the federal government’s National Vaccine Injury Compensation Program (VICP). The VICP provides individuals who suffer vaccine-related medical conditions the opportunity to obtain no-cost, no-fault compensation without the burden of filing a traditional lawsuit against a hospital, doctor, or pharmaceutical company.

At the Law Offices of Leah V. Durant, PLLC, we provide nationwide legal representation for vaccine claims for immune disorders and other serious vaccine related illnesses.  If you would like more information about how to protect your rights, contact us for a free, no-obligation consultation with our experienced vaccine lawyer.

https://vaccinelaw.com/lawyer/Flu-Vaccine-Attorney-Leah-Durant_cp11731.htm

Interesting appointment by Biden for a new agency:
------------

Wegrzyn Named First ARPA-H Director

Dr. Renee Wegrzyn

President Joe Biden intends to appoint Dr. Renee Wegrzyn as the first director of the Advanced Research Projects Agency for Health (ARPA-H), the agency newly established to drive biomedical innovation that supports the health of all Americans.

In announcing his selection on Sept. 12—the 60th anniversary of President John F. Kennedy’s Moonshot speech—Biden talked about his vision for another American Moonshot: ending cancer as we know it. ARPA-H figures prominently among other initiatives to reach that goal.

Describing Wegrzyn as a leading biomedical scientist and an entrepreneur in synthetic biology with a decade of experience leading multiple biotech projects at the Defense Advanced Research Projects Agency (DARPA), Biden said, “It’s about how to use all the assets we have—all of them. She’s going to bring the legendary DARPA attitude and culture and boldness and risk-taking to ARPA-H to fill a critical need. Discoveries that save lives, change lives, often start at the lab bench. But then those basic research breakthroughs need to be tested, scaled and brought to the clinic. This may require unusual partnerships that may require support to get over many obstacles that exist. That’s what ARPA-H is designed to do, so the advances can reach all Americans sooner. I predict ARPA-H will emerge as a new and exciting member of America’s biomedical ecosystem.”

Wegrzyn has professional experience working for two of the institutions that inspired the creation of ARPA-H—DARPA and Intelligence Advanced Research Projects Activity (IARPA). She will be responsible for driving the new agency’s nascent research portfolio and associated budget. The budget is expected to support a broad range of programs to take on challenging health problems in pursuit of high-reward solutions to help everyone.  

“President Biden could not have chosen a better inaugural director for ARPA-H,” said HHS Secretary Xavier Becerra. “With Dr. Wegrzyn at the helm, ARPA-H is poised to drive health innovation and launch bold and ambitious research programs. She will lead us in tackling some of the most pressing health challenges of our time.”

ARPA-H was created earlier this year to push the limits of U.S. biomedical and health research and innovation. Public Law 117-103, which was enacted on Mar. 15, authorized establishment of ARPA-H within HHS. Becerra transferred ARPA-H to NIH on Apr. 14.

On May 25, he formally established ARPA-H as an independent entity within NIH to ensure its ability to operate autonomously and partner across HHS and the wider U.S. government to identify projects that will be transformative and far reaching.

Previously, Wegrzyn served as a vice president of business development at Ginkgo Bioworks and head of Innovation at Concentric by Ginkgo, where she focused on applying synthetic biology to outpace infectious diseases—including Covid-19—through biomanufacturing, vaccine innovation and biosurveillance of pathogens at scale.

Prior to Ginkgo, Wegrzyn was program manager in the Biological Technologies Office at DARPA, where she leveraged the tools of synthetic biology and gene editing to enhance biosecurity, promote public health and support the domestic bioeconomy. Her DARPA portfolio included the Living Foundries: 1000 Molecules, Safe Genes, Preemptive Expression of Protective Alleles and Response Elements and the Detect it with Gene Editing Technologies programs.

Wegrzyn received the Superior Public Service Medal for her work and contributions at DARPA. Prior to joining DARPA, she led technical teams in private industry in the areas of biosecurity, gene therapies, emerging infectious disease, neuromodulation, synthetic biology, as well as research and development teams commercializing multiplex immunoassays and peptide-based disease diagnostics.

Wegrzyn holds doctorate and bachelor’s degrees in applied biology from the Georgia Institute of Technology. She was a fellow in the Center for Health Security Emerging Leaders in Biosecurity Initiative and completed postdoctoral training as an Alexander von Humboldt fellow in Heidelberg, Germany.

https://nihrecord.nih  .gov/2022/10/14/wegrzyn-named-first-arpa-h-director

-----------

Renee Wegrzyn
Director of ARPA-H
Advanced Research Projects Agency for Health (ARPA-H)
Dr. Renee Wegrzyn serves as the first director of the Advanced Research Projects Agency for Health (ARPA-H), appointed on Oct. 11 by President Joseph R. Biden. Previously, Wegrzyn served as a vice president of business development at Ginkgo Bioworks and head of innovation at Concentric by Ginkgo, where she focused on applying synthetic biology to outpace infectious diseases – including COVID-19 – through biomanufacturing, vaccine innovation and biosurveillance of pathogens at scale.Wegrzyn comes to ARPA-H with experience working for two of the institutions that inspired the creation of the agency – the Defense Advanced Research Projects Agency (DARPA) and Intelligence Advanced Research Projects Activity (IARPA).As a program manager in the DARPA Biological Technologies Office, Wegrzyn leveraged the tools of synthetic biology and gene editing to enhance biosecurity, support the domestic bioeconomy and thwart biothreats. Her DARPA portfolio included the Living Foundries: 1000 Molecules, Safe Genes; Preemptive Expression of Protective Alleles and Response Elements (PREPARE); and the Detect it with Gene Editing Technologies (DIGET) programs.Wegrzyn received the Superior Public Service Medal for her work and contributions at DARPA. Prior to joining DARPA, she led technical teams in private industry in the areas of biosecurity, gene therapies, emerging infectious disease, neuromodulation, synthetic biology, as well as research and development teams commercializing multiplex immunoassays and peptide-based disease diagnostics.Wegrzyn served on the scientific advisory boards for the National Academies Standing Committee on Biotechnology Capabilities and National Security Needs, National Academies of Science Board on Army Research and Development, Revive & Restore, Air Force Research Labs, Nuclear Threat Initiative and the Innovative Genomics Institute.

She holds doctoral and bachelor’s degrees in applied biology from the Georgia Institute of Technology, was a fellow in the Center for Health Security Emerging Leaders in Biosecurity Initiative and completed her postdoctoral training as an Alexander von Humboldt Fellow in Heidelberg, Germany.

https://www.synbiobeta .com/speakers/renee-wegrzyn-2

DARPA transitions synthetic biomanufacturing technologies to support national security objectives
by Staff Writers
Washington DC (SPX) Dec 09, 2021

Living Foundries: 1000 Molecules program testing and evaluation partners from three military branches to continue advanced technology development

Launched in 2010, DARPA's Living Foundries program aimed to enable adaptable, scalable, and on-demand production of critical, high-value molecules by programming the fundamental metabolic processes of biological systems to generate a vast number of complex molecules. These molecules were often prohibitively expensive, unable to be domestically sourced, and/or impossible to manufacture using traditional synthetic chemistry approaches. As a proof of concept, DARPA intended to produce 1,000 molecules and material precursors spanning a wide range of defense-relevant applications including industrial chemicals, fuels, coatings, and adhesives.

Divided into two parts - Advanced Tools and Capabilities for Generalizable Platforms (ATCG) and 1000 Molecules - the Living Foundries program succeeded not only in meeting its programmatic goals of producing 1000 molecules as a proof-of-concept, but pivoted in 2019 to expand program objectives to working with military mission partners to test molecules for military applications. The performer teams collectively have produced over 1630 molecules and materials to-date, and more importantly, DARPA is transitioning a subset of these technologies to five military research teams from Army, Navy, and Air Force labs who partnered with the agency on testing and evaluation over the course of the program.

"Biologically-produced molecules offer orders-of-magnitude greater diversity in chemical functionality compared to traditional approaches, enabling scientists to produce new bioreachable molecules faster than ever before," noted Dr. Anne Cheever, Living Foundries program manager. "Through Living Foundries, DARPA has transformed synthetic biomanufacturing into a predictable engineering practice supportive of a broad range of national security objectives."

The Living Foundries teams used a range of technologies and strategies to address significant synthetic biology challenges as outlined below:

+ The Naval Air Warfare Center Weapons Division (NAWCWD) team led by Dr. Ben Harvey in collaboration with Amyris, Inc. and Zymergen developed tools and technologies for producing high-performance chemicals and materials useful in a variety of military applications. NAWCWD converted precursor molecules into high energy density fuels, energetic materials, thermostable polymers, and high-performance composites. These technologies will be further developed through the Office of Naval Research (ONR) Bioengineering and Biomanufacturing Program, the ONR Advanced Energetics Manufacturing Pipeline, The Wright Brothers Institute/AFRL Synthetic Biology Challenge, and the Bioindustrial Manufacturing and Design Ecosystem (BioMADE).

+ United States Army Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) team (Drs. Greg Peterson, Jared DeCoste, and Vipin Rastogi) are developing filters, fabrics, and decontaminating wipes to combat chemical and biological weapons agents using biologically templated materials from the Massachusetts Institute of Technology. This technology will undergo further testing and development at DEVCOM CBC as part of the Defense Threat Reduction Agency multifunctional material program.

+ Air Force Research Laboratory's Materials and Manufacturing Directorate (AFRL RX) team, led by Drs. Nick Godman and Tod Grusenmeyer, produced laser eye protection goggles using bio-derived molecules developed by Zymergen. This technology will be transferred to the Personnel Protection Team in AFRL RX for testing and evaluation.

+ AFRL Aerospace Systems Directorate (AFRL RQ) team (Drs. Oscar Ruiz, Don Phelps, and Paul Wrzesinski) is testing fuels developed by NAWCWD using biologically produced molecules from Amyris, Inc. for use in military aircraft and will work with the Navy on additional development efforts.

+ DEVCOM Army Research Laboratory team (Drs. Joshua Orlicki, Anthony Clay, John La Scala, and Robert Jensen) formulated adhesives for evaluation to potentially attach armor to vehicles and polymers for 3D printing applications using technologies developed by Amyris and Zymergen. ARL will continue development of these technologies.

"Several biologically produced molecules within the 1000 Molecules Program have intriguing molecular characteristics for development of advanced polymers," noted John La Scala, Chief of the Manufacturing Sciences and Technology Branch at DEVCOM Army Research Laboratory. "These unique compounds have potential for use in durable adhesives for armor and other ground vehicle structural applications and high-performance composites for aircraft and missile applications."

All of these technologies transitioned to the military branch they support, and at least one spin-off company has been launched for bio-templated materials for batteries and chemical filtration. Additional industrial partnerships are still being considered in the areas of sustainable fuels, marine epoxies, electronic composites, adhesives and coatings, among others.

"Capability delivery is fundamentally a team sport," said Rear Adm. Scott Dillon, Naval Air Warfare Center Weapons Division commander, "and we need diverse and capable teams from government partners like DARPA, industry, and academic collaborators to ensure our warfighters have the decisive advantage. I'm excited to see where our future partnerships will take us."

https://www.spacedaily
.com/reports/DARPA_transitions_synthetic_biomanufacturing_technologies_to_support_national_security_objectives_999.html

In Conversation with Renee Wegrzyn --The Evolving World of Biosecurity

By Helen Albert -August 16, 2022

Renee Wegrzyn

VP of Business Development
Concentric by Ginkgo
There are few corners of the earth that were not affected by COVID-19 pandemic. Even wealthy countries like the U.S. were left woefully unprepared for the scale and the impact of the SARS-CoV-2 virus, bringing the need for better pandemic preparedness to public attention.

Renee Wegrzyn, vice president of business development at Concentric by Ginkgo, was more prepared than most. Starting out as an applied biologist with a BSc and PhD from Georgia Institute of Technology, she has worked in the biosecurity arena for over a decade.

Prior to starting at Ginkgo Bioworks, Wegrzyn was a program manager at the U.S. Government’s Defense Advanced Research Projects Agency (DARPA) and before starting there she helped advise DARPA and other government agencies by providing scientific and strategic support.

Concentric is a new spin-off from Gingko Bioworks with a particular focus on biosecurity. Already a player in the biosecurity space, the founding of Concentric by Gingko seemed a natural step following on from work done during the pandemic.

Wegrzyn spoke to Inside Precision Medicine senior editor, Helen Albert, about her inspirations, lessons learned from the pandemic, the importance of biosecurity and pathogen surveillance and about her new role as head of innovation at Concentric.



Q: You started your career in academic science. What attracted you to working in industry?

I did my PhD at Georgia Tech, which is officially in applied biology. So even though I was working on molecular genetics, and working with a model system, the outlook was always how is it going to be implemented in the real world. This was something that was taught to me from a very early stage in my career, and I didn’t really know any different.

I did a postdoc in academia as well and I brought this knowledge to industry. I worked with various diagnostic companies really applying some of that knowledge on protein folding and protein folding disorders. I really focused on those types of challenges, but taking that fundamental knowledge and innovation of ‘what do we know about protein folding that can help us develop a totally new type of diagnostic?’ That was really connecting my academic work to real products and was an exciting thing that that I just wanted to keep doing, whether it was as a funder, at DARPA, or here at Concentric working with our customers and the ecosystem.



Q: What made you want to work at DARPA and in biosecurity?

I had never heard of DARPA, actually, when I was first in industry, but the CEO of the small company that I worked for used to be a DARPA program manager. He started to tell me about this amazing place called DARPA, where you were very well resourced, but were able to take bets and do high risk research. It didn’t always pan out, but when it did was usually very high reward and could really move the stake in the ground in whatever field that was.

When I saw the writing on the wall that maybe my company wouldn’t be around forever, I actually then interviewed with the different consulting companies that were advising DARPA. Once I became an advisor to DARPA, I took on a fellowship and I was part of the inaugural fellowship class of the emerging leaders in biosecurity initiative.

At the time, the Center for Biosecurity, which is now the Center for Health Security, at Johns Hopkins University, was a very small program, there were maybe 20 of us in the first year. People who were in this ecosystem of biology and national security wanted to have a program to say, ‘Okay, what does that mean in the context of Washington and governments?’ and so that was really eye opening.

Biosecurity still is a small community, but it was even smaller then. That program gave us access to leaders in the Pentagon, leaders in the White House, leaders in health and human services, and access to how they were thinking about engineered biology and national security, and that just blew my mind. I found out there’s this whole other world and this created a sense of urgency for me.



Q: Why is biosecurity important?

A great analogy is personal computers and distributed computation. The internet was pushed out very early and quickly with incredible gains, but it wasn’t until about 20 years later that the vulnerabilities posed by cyber threats were really recognized. Then we started to try to implement cybersecurity and we’ve been trying to catch up for decades now. We’re still in very early stages for engineered biology, but I think now there’s an urgency to build biosecurity into programs.

I think the last two years has shown us why we need biosecurity. I would say that if you asked me that question two years ago, I feel like I would have had to convince people, but the one silver lining from this pandemic is that people get this is incredibly powerful biology. In this case, a natural threat that actually had major economic and travel consequences and a massive death toll.

Ginkgo’s biosecurity business
Concentric by Ginkgo, Ginkgo’s biosecurity business, sequences positive samples [Kris eng/Ginkgo Bioworks]
But I think it has also brought a realization that there are going to be other threats that come forward, like agricultural threats, for example. Happening at the same time as the COVID 19 pandemic is an outbreak of high path, avian influenza in the United States. African swine fever, where we don’t have a medical countermeasure or a vaccine, there’s an outbreak in the Caribbean. And the solution to deal with that, of course, is to cull those pigs that have been infected. And that would be devastating if that came to the United States.

We can’t be reactive to things like that, because the reaction causes massive consequences. We want to be proactive and recognize and detect those threats. Whether they’re natural or engineered, many of the same systems are going to be used to respond to them. And so, I would argue very strongly that that we can’t move forward without biosecurity.



Q: Why did you leave DARPA and what attracted you to come to work for Gingko?

DARPA is a term appointment organization. One of the things I think that gets people so motivated at DARPA is because the day you start your clock is ticking and your countdown timer is going down. You’re going to need to leave the organization, typically within three to five years. At the end of the day, I was technically on the books for four and a half years.

I started at Ginkgo during the pandemic in August 2020, but that was not the first time I encountered the company. I’ve known Gingko for a very long time. Even before the pandemic it was deeply vested in biosecurity. I think the founders and their early team members understood that this powerful engineered biology technology also needs biosecurity. Key members of that founding team and early folks like Patrick Boyle, were often in Washington at meetings and policy discussions around biosecurity too, because they saw we needed to be talking about this now.

Gingko has an annual conference called Ferment, where they bring together people in their ecosystem. I participated a few years ago as a speaker, talking about some of the work that we were doing at DARPA thinking about the future of engineered biology and genome editing and how industry was going to play a role. It was great to participate in that event a few years ago. When it came time to having to look at what’s next, Gingko, for me, was a really natural fit.

I love engineered biology. And I see a future where that really solves so many of the world’s challenges from health to climate, but also paired with thinking about biosecurity, so it was just a pretty natural transition from the work I had been passionate about at DARPA.



Q: Why did Gingko decide to found Concentric?

Early in the pandemic, our CEO and leadership team made the decision to stop everything and just acknowledge this is a biological event of our lifetimes. This is very powerful biology. They said ‘we’ve talked a lot about biosecurity as a company, well, now it’s time to do something.’ Jason Kelly opened up access to Ginkgo’s foundry to people that needed it, whether it was an engineered antibody, or something along the lines of raw materials for mRNA vaccines. It was a very public discussion of some of the work that came out of using the foundry.

The other piece that built out of that, which ultimately became Concentric, was testing at scale. Ginkgo’s really good at high throughput and scale. We thought about how to do that outside the building for testing. A team got together and started to work on that problem. We built business partnerships with labs across the country to help deliver testing, but what we designed was a validated, pooled classroom test. We wanted to help schools get open, we saw that as an urgent unmet need.

We made a pooled classroom test where you do one molecular test [PCR] for a classroom, that’s more cost efficient at the per student level. Most of the time it’s negative, but if there is a positive, then we can do a deconvolution of that test. That is what Concentric really was focused on for a long time was K12 testing.

Once that was operating at scale, we took a step back and saw what we built here is actually a platform. Not unique to SARS-CoV-2, but we could do other pathogens, we could also do other settings.

The first thing that we did in another setting was bringing that lab network and scaled testing and pooled testing to an airport. We brought in our partners Express Check, who had access to those airports and could collect those samples on the ground, and then started a partnership with the CDC, who could help direct us and we shared that genomic surveillance with them.



Q: What influence has the pandemic had on the take-up of biosecurity and pathogen surveillance measures?

There is, I think, a greater understanding of what it is and why it’s important. But I think we have to balance that with a little bit of testing fatigue that we’re seeing. One of the ways that we’re dealing with that is passive monitoring. And what I mean by passive is something that’s happening in the background, I don’t have to be taking a swab in your nose every week, but passive monitoring, like wastewater testing, or air monitoring.

Maybe for some time, all we will need is passive monitoring. If we start to see an uptick in infections, we can transition you to a program where we can start to do more regular testing, if you should need it. That flexibility moving forward is going to be really key to ensure that you’re delivering the right solution for the right situation that you’re facing.



Q: What’s next for Concentric once the pandemic is officially over?

We can have an end to end offering for a given entity, which I think is really critical. It’s just easy to work with us because we’ve filled in all those gaps for folks. We’ve recently announced, ‘okay, we’re in airports, we’re in schools, we have this testing platform, let’s start to layer on new threats.’ Monkeypox, of course, has been in the news as a as a growing epidemic. And we’re ramping up our capability to allow us to be able to look at other sample types like wastewater, but then also look at other pathogens.

That’s really our vision for the future. Fast forward 5, 10, 15 years from now, we’re able to test for many, many different things, in different types of environments and samples, so that we can really be ready for what’s next. Otherwise, we’re going to continue to be surprised and then have to play catch up. With the muscle of the Gingko foundry behind us, we have the ability to make new sensors, new tests that we want to then bring forward and push into this scaled marketplace that we’ve developed.

We had the airport program in place during the Delta surge. When the first case of Omicron popped up in South Africa, we just did a small pivot and said, ‘Hey, we’re going to start picking up more flights from Africa and Europe because that’s where we’re seeing the first cases.’ This allowed us to be in position to pick up the first U.S. cases of BA.2 and BA.3, we didn’t have to start a new program from scratch. It was a small tweak, so that we could be responsive, but not have to rebuild. That was a really exciting proof of concept. That’s what we’d like to repeat as we go forward, not only domestically but something that we’re working to build out internationally as well.

How an Airport Nail Salon Became the Frontline of US Covid Surveillance

XpresSpa, Ginkgo Bioworks and the CDC have teamed up to hunt for Covid variants — and prepare for future pandemics

By Riley Griffin
June 5, 2022 at 8:00 AM CDT
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As the pandemic engulfed the world in March 2020, no one was thinking much about getting a manicure. So XpresSpa Group Inc., an airport chain that offers mani-pedis and massages to travelers, closed all 50 of its locations. To survive the next two years, it would have to pivot. It turned to the most obvious next market: Covid testing.

Through a partnership with the Centers for Disease Control and Prevention and synthetic biology firm Ginkgo Bioworks Inc., XpresSpa launched a surveillance operation to hunt for new and emerging Covid variants among international travelers. Over the last eight months, the trio has tested tens of thousands of passengers arriving from more than 15 countries around the globe. Despite rules that require travelers to produce a negative Covid result in order to enter the US, the collaboration routinely finds both positive cases and new mutations.

In a world in which the virus knows no borders, Ezra Ernst, the chief executive officer of the spa’s testing business, XpresCheck, calls this “a form of border protection.”

The program has become a rare example of government and private industry working together productively during the pandemic, so much so that the project has expanded and is being discussed as a way to monitor different biological threats in the future.

relates to How an Airport Nail Salon Became the Frontline of US Covid Surveillance
Ginkgo’s biosecurity business, Concentric by Ginkgo, sequences positive samples.Source: Kris Cheng/Ginkgo Bioworks
Global travel has made Covid all but impossible to contain. Some of the earliest cases in the US and Europe can be traced back to passengers who brought the virus via airplane from China. New mutations have repeatedly cropped up since then, racing around the globe within weeks, if not days. Realizing that shutting down borders wasn’t going to stop Covid, many countries have focused on real-time surveillance like this to prepare for the inevitable.

For much of the pandemic, the US was woefully behind in mutation hunting. Countries such as Israel, UK and Japan had already been sequencing viral genomes in travel hubs. More recently, the Biden administration signaled plans to invest in these efforts, granting the CDC $1.75 billion from the American Rescue Plan to expand genomic sequencing capabilities now and in coming years.  

The CDC-funded airport surveillance project differs from other countries’ in that it relies on the voluntary participation of travelers. XpresCheck’s airport staff offer free Covid tests to international passengers at baggage claim and in taxi lines in New York City, New Jersey, San Francisco and Atlanta. Those who opt in take a PCR test on the spot and receive an at-home kit to test again a few days later.

Swabs are sent to Boston-based Ginkgo’s network of more than 60 labs, which pool as many as 25 samples together for testing. In the first week of January, amid a holiday surge in travel and cases, 46% of those pooled tests turned out to be positive. During a week in April, 15% of the pools were positive. Through positive viral genomes, the program has so far identified the entry of several omicron subvariants into the US.

“If you ask me if we are ready for the next pandemic, my answer is no, we are not,” said Renee Wegrzyn, who heads innovation at Concentric by Ginkgo, the company’s biosecurity business. “But we’ve gotten better at this pandemic.”

Snapshot of Covid on International Flights
From April 18-24, the program identified BA.2, BA.2.9, BA.4 and other indeterminable omicron sub-lineages.


Sources: Concentric by Ginkgo, XpresCheck

The project was set in motion more than year ago when Ernst reached out to government officials about using the airport testing business as part of the government’s pandemic response. The timing was fortuitous: the CDC happened to already be looking for ways to improve its virus detection systems. Most of the mutation-hunting work in the first year of the pandemic had been done via a patchwork of efforts at universities and hospitals rather than through a federally-coordinated (or funded) network of labs.

The CDC signed off on a $2 million, 8-week proof-of-concept program last August in which XpresCheck would partner with Ginkgo at three US airports where they would focus on seven regular flights arriving from India, where delta had spurred a surge. Then came omicron. The CDC greenlit an expansion of the program in November to quickly identify the new variant. Two samples taken at Newark airport within days of the project’s expansion unearthed the first US case of subvariant BA.2 and the first North American case of BA.3 – 43 days before it was reported elsewhere in the continent.

“Everyone was looking for omicron and we were positioned to do it,” said Cindy Friedman, chief of the CDC’s Travelers’ Health Branch. “The system worked, full stop.”

Hunting for Variants
Omicron subvariant BA.2 was the most prevalent Covid mutation detected among international travelers between April 18-24.


Sources: Concentric by Ginkgo, XpresCheck

The CDC sees the program as critical to the future of US biosurveillance. Identifying a new variant or subvariant can help inform prevention measures and develop better-performing treatments, Friedman said. And so the health agency more than doubled its investment to $5.6 million in January. The companies are expanding into more US airports and piloting new projects, like removing “blue juice”—a friendly term for toilet water—off of airplanes in their hunt for Covid mutations and perhaps eventually other viral diseases like monkeypox.

Many countries are starting to shut down such projects. Israel, which had previously sent positive Covid samples obtained at airports to be sequenced, lifted its testing requirements in May. Friedman said she sees the US collaboration all the more important as other nations roll back sequencing efforts. “I don't see this airport surveillance project going away anytime soon,” she said.

XpresCheck and Ginkgo are also in active discussions with other countries to expand the service abroad. As the crisis phase of the pandemic has faded, companies boosted by it have struggled to keep their share prices high. Since the partners announced they'd identified BA.3 on December 13, Ginkgo's shares are down about 65%, while XpresSpa's have fallen about 57%. Growing their surveillance services, they hope, will provide a boost.

Beyond the business potential, Taj Azarian, a genomic epidemiologist at the University of Central Florida, sees airport monitoring as a critical long-term public health tool. “This approach would have been valuable in the past with other diseases,” he said, pointing to cross-border spread of Zika and tuberculosis. The end goal, according to Azarian, would be to broaden airport surveillance capabilities so that countries can detect other pathogens and never-before-seen threats.

The CDC, XpresCheck and Ginkgo share that vision. Ginkgo is already developing new sequencing panels that can simultaneously evaluate a sample against multiple pathogens, such as the flu and measles.

(more at link: https://www.bloomberg  .com/news/articles/2022-06-05/xpresspa-and-ginkgo-bioworks-are-hunting-for-new-covid-variants-at-airports )

MAY 22, 2012 2:58 PM

Darpa, Venter Launch Assembly Line for Genetic Engineering

The military-industrial complex just got a little bit livelier. Quite literally. That's because Darpa, the Pentagon's far-out research arm, has kicked off a program designed to take the conventions of manufacturing and apply them to living cells. Think of it like an assembly line, but one that would churn out modified biological matter -- man-made organisms -- instead of cars or computer parts.

The program, called "Living Foundries," was first announced by the agency last year. Now, Darpa's handed out seven research awards worth $15.5 million to six different companies and institutions. Among them are several Darpa favorites, including the University of Texas at Austin and the California Institute of Technology. Two contracts were also issued to the J. Craig Venter Institute. Dr. Venter is something of a biology superstar: He was among the first scientists to sequence a human genome, and his institute was, in 2010, the first to create a cell with entirely synthetic genome.

"Living Foundries" aspires to turn the slow, messy process of genetic engineering into a streamlined and standardized one. Of course, the field is already a burgeoning one: Scientists have tweaked cells in order to develop renewable petroleum and spider silk that's tough as steel. And a host of companies are investigating the pharmaceutical and agricultural promise lurking -- with some tinkering, of course -- inside living cells.

But those breakthroughs, while exciting, have also been time-consuming and expensive. As Darpa notes, even the most cutting-edge synthetic biology projects "often take 7+ years and tens to hundreds of millions of dollars" to complete. Venter's synthetic cell project, for example, cost an estimated $40 million.

Synthetic biology, as Darpa notes, has the potential to yield "new materials, novel capabilities, fuel and medicines" -- everything from fuels to solar cells to vaccines could be produced by engineering different living cells. But the agency isn't content to wait seven years for each new innovation. In fact, they want the capability for "on-demand production" of whatever bio-product suits the military's immediate needs.

To do it, Darpa will need to revamp the process of bio-engineering -- from the initial design of a new material, to its construction, to its subsequent efficacy evaluation. The starting point, and one that agency-funded researchers will have to create, is a library of "modular genetic parts": Standardized biological units that can be assembled in different ways -- like LEGO -- to create different materials.

Once that library is created, the agency wants researchers to come up with a set of "parts, regulators, devices and circuits" that can reliably yield various genetic systems. After that, they'll also need "test platforms" to quickly evaluate new bio-materials. Think of it as a biological assembly line: Products are designed, pieced together using standardized tools and techniques, and then tested for efficacy.

The process, once established, ought to massively accelerate the pace of bio-engineering -- and cut costs. The agency's asking researchers to "compress the biological design-build-test cycle by at least 10X in both time and cost," while also "increasing the complexity of systems that can be designed and executed."

No doubt, Darpa's making some big asks of the scientists tasked with this research. And not everyone's convinced they'll pull it off. "The biology will fight them," Daniel Drell, a program manager with the U.S. Department of Energy, predicted last year. Which suggests it might be a few years, at least, before Darpa's bio-creations try to fight us.

https://www.wired .com/2012/05/living-foundries/

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Overview
The J. Craig Venter Institute (JCVI) has been involved in viral sequencing and analysis for more than 25 years, and coronavirus research for more than 15. This work has aided human health through vaccine development; improved food security by protecting crops and livestock; and provides critical data and analysis to researchers around the world. The Institute has also pioneered a synthetic biology platform which is now integral to fighting current and emerging viral threats.


Watch JCVI founder, Dr. J. Craig Venter, explain the importance of philanthropic donations to the Institute’s history of major breakthroughs.

Following is a summary of our various research projects on the novel coronavirus, SARS-CoV-2.

SARS-CoV-2 Research

SARS-CoV-2 Clones & Reverse Genetic System
Sanjay Vashee, PhD and Lauren Oldfield, PhD are collaborating with investigators at the University of Maryland to use synthetic genomics to develop a SARS-CoV-2 infectious clone. They are pairing this with a rapid, modular reverse genetic system to assess genomic variants identified in the wealth of global sequencing data, develop and test vaccine candidates, and generate needed reagents, including fluorescent and tagged virus strains.

Funding: JCVI


Data, Tools, and Machine Learning
Richard Scheuermann, PhD is leading a team of investigators at JCVI in response to the COVID-19 outbreak. Through his NIH-sponsored Bioinformatics Resource Center project, his team released a dedicated public web portal for the SARS-CoV-2 virus during the early stages of the outbreak to ensure data and tools are accessible to frontline researchers worldwide. Additionally, in collaboration with investigators from La Jolla Institute for Immunology, Dr. Scheuermann and his team provided machine learning methods to help complete the first analysis that identified potential targets for human immune responses to SARS-CoV-2 infection. This information is crucial to the design and evaluation of diagnostics and vaccine candidates.

Funding: National Institute of Allergy and Infectious Diseases

Surveillance Testing for SARS-CoV-2
To improve workplace safety, JCVI has instituted surveillance testing, led by Richard Scheuermann, PhD, of pooled nasal swab samples with an in-house validated RT-qPCR assay using the primers/probes developed by the US Centers for Disease Control and Prevention. Set up to combat the lack of fast, inexpensive, and reliable testing infrastructure, this weekly, bicoastal testing program was added as a layer of risk mitigation to our COVID-19 workplace policy that also includes masking, physical distancing, staying home when sick, and daily temperature screening. JCVI employees can also opt-in to a research study to compare SARS-CoV-2 detection in paired self-collected nasal swabs and saliva samples.

Funding: Private donors and JCVI


Exploiting the SARS-CoV-2 "Spike"
Gene Tan, PhD is leading efforts to develop SARS-CoV-2 spike glycoprotein pseudotyped viruses for receptor binding and antibody neutralization assays.

Funding: The Board of Trustees of the Leland Stanford Junior University


Salivary Serological & Immune Testing
Marcelo Freire, PhD is leading efforts in the development of a salivary serological and immune testing. Monitoring saliva immunity provides non-invasive molecular information, essential for diagnostics, and response to therapy during the pandemic. His laboratory is comparing COVID-19 patients’ blood and salivary immunoglobulins levels to find better ways to evaluate immune response to SARS-CoV-2.

Funding: Private donors and JCVI
https://www.jcvi .org/research/coronavirus-research

SARS-CoV-2 Defense Mechanisms
Through an IARPA-funded collaboration with investigators at Harvard Wyss Institute, John Glass, PhD is using in vitro cell culture systems to assess the capacity of SARS-CoV-2 proteins to inhibit antiviral mechanisms of the human innate immune system that would otherwise prevent viral replication in those cells.

Funding: Intelligence Advanced Research Projects Activity

COVID-19 Serology Testing
Informatics
JCVI is developing critical resources for the evaluation of antibody reactivity for serology testing. From an informatics perspective, the use of comparative genomics methods and predictive machine learning algorithms are being used to identify the key determinants for immune system recognition and to monitor the impact of virus evolution in immune system evasion. The bioinformatics analyses will aid in developing critical resources for the evaluation of antibody reactivity for serology testing.

Lab
In the laboratory, viral surface proteins are being generated for use as ELISA antigens, pseudotyped recombinant viruses are being constructed for neutralizing assays, and candidate vaccine constructs are being synthetized to define the immunogenicity of the viral proteins and to understand the basis of immune response and protection against SARS-CoV-2.

Note I don't agree with everything at this site but it captures a lot of relevant details in this article. The links at the bottom worth checking out:
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https://rightsidetoday.com/8-ways-spike-protein-harms-the-body-and-how-to-remove-it/

8 Ways Spike Protein Harms the Body, and How to Remove It

When the pandemic first started to spread, people’s understanding of the spike protein was very limited. It was thought that the spike protein only played the role of invading our cells by binding to the ACE (angiotensin-converting enzyme) receptors on our cell walls. However, scientists have slowly discovered that the effects of this protein are multifaceted, and it interacts with other cellular tissues beside the ACE2 receptors.
8 Ways the Spike Protein Harms the Body

During the two-plus years of the COVID-19 pandemic, many studies have analyzed the effects of the spike protein from different aspects, and have discovered that it is harmful to the human body in several ways, including:

1. Damaging the lung cells (including the pulmonary alveoli and pulmonary endothelial cells);
2. Damaging the mitochondria and DNA structures;
3. Damaging cardiovascular cells;
4. Increasing the risk of blood clots;
5. Damaging brain cells;
6. Promoting inflammation;
7. Suppressing immunity;
8. Increasing the risk of cancer

We will go into the details of each of these points.
S Proteins Can Affect Multiple Organs

When the virus enters the human body, the spike proteins will affect multiple organs in different ways. Studies have shown that many organ cells can be affected by spike proteins, such as those in the heart, brain, and cardiovascular system. In addition, a paper published in 2021 in the bioRxiv preprint repository states that the S proteins cause:

   the Type 1 catalytic receptors in the kidneys to increase in kidney cell tissues, and these types of receptors can become hosts for the virus, making the kidneys more susceptible to viral infection
   cells in the small intestine to stimulate a large amount of L-SIGN (liver/lymph node-specific intracellular adhesion molecules-3 grabbing non-integrin) receptors to defend against pathogens. However, this causes a reaction that eventually makes the small intestine more susceptible to viral infection. A similar situation can also occur in other organs, such as the kidneys and duodenum (the first section of the small intestine)
   the amount of DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin) receptors in the lungs to increase, which may cause inflammatory symptoms in the lungs

In addition, spike proteins can cause different degrees of oxidation of the organs, leading to more cells suffering premature death and putting the body in a hyperoxidized state, which may further increase the risk of cancer.

A new study published in the Journal of the American Heart Association found that spike proteins also have a direct effect on lung function.

When spike proteins are present in the human body, the pulmonary alveolar cell walls in the lungs will begin to thicken and solidify, and lung functions will decline. The pulmonary alveoli are the tiny, balloon-shaped air sacs that expand and shrink in our lungs as we breathe.

The spike proteins will also affect the function of cellular mitochondria in the lungs. Mitochondria are the power plants of our cells, and hence the body’s energy base.
Spike Proteins Damage Mitochondria, Possibly Causing Long COVID

Under normal circumstances, mitochondria in cells are tubular cellular power stations responsible for synthesizing energy.

When the spike proteins stimulate our pulmonary alveolar cells or endothelial cells (which line our blood vessels and lymphatic vessels), the mitochondrial structure will change dramatically and become heavily fragmented, and the number of tubular mitochondria will be greatly reduced. As the cells in the alveoli or endothelial tissues become damaged, they no longer produce energy efficiently, which is likely to cause the cells to enter a state of premature decline and death.

Scientists have hypothesized long COVID may be caused by this damage to the mitochondria. One of the major symptoms of this chronic form of COVID-19 is fatigue. This may be due to the fact that the cellular mitochondrial are damaged or dying, resulting in a lack of energy at the most essential level of our body.

Mitochondrial damage in different cells can also bring about different symptoms. If the mitochondrial function of lung cells are damaged, then the pulmonary alveoli’s ability to expand and contract will be weakened, oxygen absorption will be poor, and the body’s metabolic level will also be reduced. This also makes one more prone to fatigue.
Damage to the Cardiovascular System and Blood Clots

The cardiac and the myocardial systems of the heart contain a very important type of cell: the pericardial cells, which are outside the endothelial cells of blood vessels and are usually combined with endothelial cells to help blood vessels transmit different signals.

A study published in Clinical Science discovered that when the SARS-CoV-2 virus enters the body, the spike proteins will bind to the CD147 receptors on the surface of the pericardial cells, making them more likely to shed from the surface of the endothelial cells. This will affect some of the functions of the endothelial cells and accelerate the death of the vascular wall cells.

In addition, spike proteins themselves can directly stimulate pericardial cells to produce more pro-inflammatory factors that can damage the myocardium and cause blood clots.

Spike proteins can also induce thrombosis, which is when blood clots block veins. Another experiment published in the BioRxiv preprint repository investigated how platelets would change after being stimulated by spike proteins.

The experiment compared the SARS-CoV-2 virus spike protein with another viral protein called VSV (Vesicular stomatitis virus), and found that more platelets were induced to clot in the presence of the spike proteins.
Spike Proteins Impair Immunity

When the human body is infected by a coronavirus like COVID-19, the immune system recognizes the spike protein as an invader and the innate immune system and acquired immune system get to work. Cytokines are released to signal the area to defend. In short, the impact of spike proteins on the immune systems is comprehensive. This is also shown in a paper published in the journal Leukemia.

For instance, there are 11 types of toll-like receptors in the innate immune system, and the seventh type of these toll-like receptors can recognize the RNA of single-stranded viruses. The mRNA (messenger RNA) that enters the body after COVID-19 infection or vaccination is also single-stranded, and immune cells will recognize the virus’s RNA and attack it. In the presence of spike proteins, the toll-like receptor expression quantity may increase in response to the viral attack, and complementary immune cells may release more interferons in response to different viral variants.

If the amount of viral spike proteins in the body is too high, they may over-activate the expression of interferons, which may even induce the body’s immune systems to attack its own cells.
Do Vaccine Spike Proteins Stay in the Body?

As we know, the COVID-19 vaccines have incorporated an expression mechanism for the viral spike proteins, which triggers our immune systems to respond to the virus. But the question remains, how long do the spike proteins from the vaccines remain in the body?

The spike proteins are divided into two parts: S1 and S2, with S1 remaining in the blood and S2 bound to the cell membranes.

A study published in Clinical Infectious Diseases discovered that the S1 proteins would appear in humans soon after the first dose of the Moderna vaccine, and that some people would still have intact spike proteins two weeks after the first dose of the vaccine.

The finding that intact spike proteins were still present in people two weeks after vaccination was beyond expectations.

Another study published in the Journal of Immunology found that S2 proteins could still be detected four months after the second dose of the Pfizer vaccine.

However, the harm of spike proteins is related to their amount. The aforementioned side effects are all based on in vitro studies and animal models, and the relatively serious damage occurred only when the amount of spike proteins was large.

If the viral mRNA in the vaccine only appeared in small amounts in the muscles and didn’t enter the blood or organs in large amounts, then these serious side effects would not occur. That means clearing out these spike proteins can limit their potential negative effects in the body.
How to Get Spike Proteins Out of the Body

If someone develops Long COVID-19 syndrome after infection or vaccination, they may wonder what medicines can be used to expel the spike proteins and relieve their symptoms. The World Council for Health (WCH) has made several recommendations for nutrients and medications.

1. Nutrients that may help improve symptoms include:

Vitamin C
Vitamin D
Omega 3
Quercetin
Melatonin
Zinc

These are all nutrients helpful in boosting the immune system, thereby helping the body remove spike proteins.

2. Common medications used to improve symptoms:

   Aspirin
   Antihistamines
   Steroids
   Colchicine
   Mast cell stabilizers
   Ivermectin

3. Plant Extracts

Some plant extracts in nature can also help detoxify the body, including:

   Selfheal extract
   Pine needle extract
   Dandelion leaf extract
   Rheum emodin

Some of these ingredients, such as the shikimic acid contained in pine needles, have antioxidant properties that can reduce oxidized free radicals in the body and provide a detoxifying effect.

The above suggested medicines are not cures, but they can help boost the body’s immunity and balance the body’s immune mechanism, which is helpful in the overall fight against the virus.

It is important to emphasize that everyone’s situation is different. So talk to your physician before taking the medications recommended by the WCH to make sure they are suitable.

References

https://www.biorxiv.org/content/10.1101/2021.07.07.451411v1.full.pdf

https://www.ahajournals.org/doi/epub/10.1161/CIRCRESAHA.121.318902

https://pubmed.ncbi.nlm.nih.gov/34807265/

https://www.nature.com/articles/s41375-021-01332-z

https://www.biorxiv.org/content/10.1101/2021.12.14.472668v2.full.pdf

https://academic.oup.com/cid/article/74/4/715/6279075

https://www.jimmunol.org/content/207/10/2405

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https://wmcresearch.substack.com/p/spike-protein-induced-cytokines-and

Abstract

The COVID-19 pandemic necessitated the rapid production of vaccines aimed at the production of neutralizing antibodies against the COVID-19 spike protein required for the corona virus binding to target cells. The best well-known vaccines have utilized either mRNA or an adenovirus vector to direct human cells to produce the spike protein against which the body produces mostly neutralizing antibodies. However, recent reports have raised some skepticism as to the biologic actions of the spike protein and the types of antibodies produced. One paper reported that certain antibodies in the blood of infected patients appear to change the shape of the spike protein so as to make it more likely to bind to cells, while other papers showed that the spike protein by itself (without being part of the corona virus) can damage endothelial cells and disrupt the blood-brain barrier. These findings may be even more relevant to the pathogenesis of long-COVID syndrome that may affect as many as 50% of those infected with SARS-CoV-2. In COVID-19, a response to oxidative stress is required by increasing anti-oxidant enzymes. In this regard, it is known that polyphenols are natural anti-oxidants with multiple health effects. Hence, there are even more reasons to intervene with the use of anti-oxidant compounds, such as luteolin, in addition to available vaccines and anti-inflammatory drugs to prevent the harmful actions of the spike protein.

Keywords: ACE2; antibodies; blood vessels; blood-brain barrier; coronavirus; endothelial cells; receptor; spike protein.
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https://wmcresearch.org/the-spike-protein-as-metatastic-cancer/
THE SPIKE PROTEIN AS METATASTIC CANCER
May 29, 2021

The Spike Protein of SARS-CoV-2 has been shown to interact with tumor suppressor p53. p53 mutations are the most common genetic alterations found in cancers and are observed in >50% of all tumors. From the approximately 200 different single mutations already described in p53, several residues are considered as hotspots, including R248, R175, G245, R273, R249, and R282 (Petitjean et al. 2007). All of these residues are found in p53's core domain, which is responsible for its interactions with DNA. For this reason, most of these mutants are incapable of exerting the wild-type (WT) level of transcriptional activity (Bullock et al. 1997). The most frequent effect of p53 mutations is loss-of-function (LoF); however, GoF effects, such as increased migration, invasion, and metastasis, have also been observed. Mutations in the p53 gene occur in more than half of human cancers and often result in altered transcriptional activities.
spikemetatastic2

METATASTIC CANCER AND PRION DISEASE

Recently discovered characteristics of the tumor suppressor p53 include its prion-like properties and cellular uptake mechanisms, which are related to its GoF and are associated with tumor formation and malignancy.

The implications are nothing short of cataclysmic.
spikemetatastic3
Referenced/Related Papers

S2 Subunit of SARS-nCoV-2 Interacts with Tumor Suppressor Protein p53 and BRCA: an In Silico Study

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324311/

Aggregation and Prion-Like Properties of Misfolded Tumor Suppressors: Is Cancer a Prion Disease?

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5046694/
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Front Immunol

. 2021 Jan 28;11:621441.
doi: 10.3389/fimmu.2020.621441. eCollection 2020.
Cytokine Signature Induced by SARS-CoV-2 Spike Protein in a Mouse Model

Abstract

Although COVID-19 has become a major challenge to global health, there are currently no efficacious agents for effective treatment. Cytokine storm syndrome (CSS) can lead to acute respiratory distress syndrome (ARDS), which contributes to most COVID-19 mortalities. Research points to interleukin 6 (IL-6) as a crucial signature of the cytokine storm, and the clinical use of the IL-6 inhibitor tocilizumab shows potential for treatment of COVID-19 patient. In this study, we challenged wild-type and adenovirus-5/human angiotensin-converting enzyme 2-expressing BALB/c mice with a combination of polyinosinic-polycytidylic acid and recombinant SARS-CoV-2 spike-extracellular domain protein. High levels of TNF-α and nearly 100 times increased IL-6 were detected at 6 h, but disappeared by 24 h in bronchoalveolar lavage fluid (BALF) following immunostimulant challenge. Lung injury observed by histopathologic changes and magnetic resonance imaging at 24 h indicated that increased TNF-α and IL-6 may initiate CSS in the lung, resulting in the continual production of inflammatory cytokines. We hypothesize that TNF-α and IL-6 may contribute to the occurrence of CSS in COVID-19. We also investigated multiple monoclonal antibodies (mAbs) and inhibitors for neutralizing the pro-inflammatory phenotype of COVID-19: mAbs against IL-1α, IL-6, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and inhibitors of p38 and JAK partially relieved CSS; mAbs against IL-6, TNF-α, and GM-CSF, and inhibitors of p38, extracellular signal-regulated kinase, and myeloperoxidase somewhat reduced neutrophilic alveolitis in the lung. This novel murine model opens a biologically safe, time-saving avenue for clarifying the mechanism of CSS/ARDS in COVID-19 and developing new therapeutic drugs.

Keywords: COVID-19; SARS-CoV-2; acute respiratory distress syndrome; cytokine storm syndrome; murine model.

Copyright 2021 Gu, Zhao, Jin, Song, Zhi, Zhao, Ma, Zheng, Wang, Liu, Xin, Han, Li, Dong, Liu and Dong.
https://pubmed.ncbi.nlm.nih.gov/33584719/

==================================


Naunyn Schmiedebergs Arch Pharmacol

. 2021 Mar;394(3):561-567.
doi: 10.1007/s00210-020-02035-5. Epub 2021 Jan 4.
NF-κB signalling as a pharmacological target in COVID-19: potential roles for IKKβ inhibitors
Mahesh Kandasamy  1   2
Affiliations

   PMID: 33394134
   PMCID: PMC7780215
   DOI: 10.1007/s00210-020-02035-5

Free PMC article
Abstract

Coronavirus disease 2019 (COVID-19) has been characterized by lymphopenia as well as a proinflammatory cytokine storm, which are responsible for the poor prognosis and multiorgan defects. The transcription factor nuclear factor-κB (NF-κB) modulates the functions of the immune cells and alters the gene expression profile of different cytokines in response to various pathogenic stimuli, while many proinflammatory factors have been known to induce NF-κB signalling cascade. Besides, NF-κB has been known to potentiate the production of reactive oxygen species (ROS) leading to apoptosis in various tissues in many diseases and viral infections. Though the reports on the involvement of the NF-κB signalling pathway in COVID-19 are limited, the therapeutic benefits of NF-κB inhibitors including dexamethasone, a synthetic form of glucocorticoid, have increasingly been realized. Considering the fact, the abnormal activation of the NF-κB resulting from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection might be associated with the pathogenic profile of immune cells, cytokine storm and multiorgan defects. Thus, the pharmacological inactivation of the NF-κB signalling pathway can strongly represent a potential therapeutic target to treat the symptomatology of COVID-19. This article signifies pharmacological blockade of the phosphorylation of inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ), a key downstream effector of NF-κB signalling, for a therapeutic consideration to attenuate COVID-19.

Keywords: COVID-19; IKKβ inhibitors; Inflammation; NF-kB; SARS-CoV-2.

Naunyn Schmiedebergs Arch Pharmacol

. 2021 Mar;394(3):561-567.
doi: 10.1007/s00210-020-02035-5. Epub 2021 Jan 4.
======================



==================================
Harmine is an in-vivo cytokine suppressor.

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Biochem Biophys Res Commun

. 2017 Jul 29;489(3):332-338.
doi: 10.1016/j.bbrc.2017.05.126. Epub 2017 May 24.
Harmine is an inflammatory inhibitor through the suppression of NF-κB signaling
Xin Liu  1 , Mingxia Li  1 , Si Tan  1 , Changhong Wang  2 , Shengjie Fan  3 , Cheng Huang  4
Affiliations

   PMID: 28551404
   DOI: 10.1016/j.bbrc.2017.05.126


Abstract

Harmine is a major constituent in a hallucinogenic botanical mixture ayahuasca and medical plant Peganum harmala L. The plant is used for various illnesses and exhibits anti-inflammatory activity. However, the active constituents remain unclear. Here, we screened the seven alkaloids in P. harmala for their anti-inflammatory activity using an nuclear factor-κB (NF-κB) reporter assay. We found that harmine and harmol could inhibit NF-κB transactivity. As the most abundant compound, harmine inhibited tumor necrosis factor-α (TNF-α)- and lipopolysaccharides (LPS)-induced NF-κB transactivity and nuclear translocation in mouse macrophage RAW 264.7 cells. The mRNA and protein levels of NF-κB downstream inflammatory cytokines also reduced. In an LPS-challenged mouse model, harmine markedly averted inflammatory damage of the lung, and decreased serum TNF-α, interleukin-1β (IL-1β) and IL-6 levels. Our data indicate that harmine may exert the anti-inflammatory effect by inhibition of the NF-κB signaling pathway and harmine is probably responsible for the anti-inflammatory effects of P. harmala.

Keywords: Harmin; IL-1β; IL-6; Inflammation; NF-κB; Peganum harmala L.; TNF-α.

Copyright 2017 Elsevier Inc. All rights reserved.

===================

The potential of glycyrrhizin and licorice extract in combating COVID-19

Licorice contains leading natural active agents, promising as a basis for developing novel antiviral agents.The most common way to extract the active ingredients of licorice root is by hot water or an ethanol / water mixture, however, other solvents are used to prepare licorice roots extracts ( Tian et al., 2008 ).

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https://www.businessinsider.com/iceland-has-a-bizarre-obsession-with-licorice-heres-why-2017-4?op=1

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https://www.sciencedirect.com/science/article/pii/S2667031321000257

===============

The first settlers to arrive on Iceland’s shores were Scandinavian, and had no shortage of cold-weather expertise. They quickly learned, however, that even hardy winter crops could barely take hold in the volcanic soil, sulphuric waters, and treacherously long winters. Grain was hard to grow, and was abandoned completely during the “little ice age” that began in the 14th century. Icelanders had such a hard time cultivating carbs, they took to buttering dried fish like it was bread and harvesting mosses and seaweed to make up for the dearth of starch.
Atlas Obscura Trips
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Flowers and bees couldn’t flourish, so neither could honey. Trading ships had difficulty making it ashore in the icy Atlantic, so imports were unreliable. Licorice, on the other hand, did not need to flower to be viable—the edible portion is the root, which contains a compound 30–50 times sweeter than sucrose. In lieu of other sugar, this flavor began to predominate in the chilly climes of northern Europe, and Icelanders, too, came to rely on its strong flavor to satisfy their cravings.

But it also served functions beyond sugar fix. Considered a highly effective mucokinetic (a drug that clears mucus from the airways), licorice has been relied on by Icelandic pharmacists for centuries to combat the respiratory ailments frequently afflicting inhabitants of the subarctic, perpetually damp island. The pharmacists added it to their bespoke cough syrups and lozenges and served them to everyone from sick children to fishermen—a practice that lasted well into the 20th century, according to Icelandic food journalist Ragnar Egilsson.

The effects of the climate on public health (and the subsequent licorice cures) do not stop there. For centuries, local produce was practically nonexistent and as a result, the bowels of the Icelandic people were in need of some help. Luckily for them, licorice root doesn’t just thin nasal fluids. The plant, found in modern-day aperient teas, is known for its laxative properties, a trait that surely comes in handy in a country where fermented shark carcass and lamb hot dogs are the cuisine de rigueur.

All of this explains why early Icelanders had to seek alternative sweets, but it doesn’t explain why their palates haven’t broadened as international trade has expanded and American candy proliferation has reached near-global saturation. To be fair, Icelanders can pick up American candy at their corner store now, but as recently as the late 1990s this was not the case.

https://www.atlasobscura.com/articles/iceland-licorice

Keep in mind licorice root should only be taken when feeling sick. Too much can raise blood pressure, cause Edema and reduce cortisol too much for some people. It can help increase ATP levels a lot, hence, extra energy which also gives the body the ability to fight influenza infections -- i.e, infections that can reduce ATP levels (energy) in the body. People sleep a lot with low ATP levels and respiratory blockage:
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The Mechanism for Licorice (Glycyrrhiza glabra) Induced Swelling and Edema

Summary:

   Licorice (Glycyrrhiza glabra) contains an ingredient called glycyrrhizin, also called glycyrrhizic acid, and has been used in the treatment of stomach ulcers, bronchitis, sore throat and even viral hepatitis.
   Unfortunately, licorice ingestion can lead to excess mineralocorticoid activity that is manifested by suppressed renin levels, sodium retention, hypervolemia, hypokalemia, hypertension and edema.
   Licorice is a known inhibitor of the type 2 isoenzyme of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD2) and thus prevents to the inactivation of cortisol.
   The increased cortisol is available to bind to the mineralocorticoid receptor in the kidney resulting in the reabsorption of sodium and water that leads to increases in intravascular volume and capillary hydrostatic pressures that contribute to edema formation.


Editor-in-Chief: Anthony J. Busti, MD, PharmD, FNLA, FAHA
Last Reviewed: August 2015
Explanation

   Licorice (Glycyrrhiza glabra) contains an ingredient called glycyrrhizin or glycyrrhizic acid and has been used in the treatment of stomach ulcers, bronchitis, sore throat and even viral hepatitis.1  It is available in a number of dosage forms that include powdered forms, capsules, tablets and liquid extracts.1  Unfortunately, licorice ingestion can lead to excess mineralocorticoid activity that is manifested by suppressed renin levels, sodium retention, hypokalemia, hypertension and edema.1-5  As it relates to edema, it can be caused one or more of the following: things known to influence vasculature oncotic pressures, vasomotor tone of the veins, permeability of the capillary membranes, lymphatic flow and/or intravascular volume.   Of these various biologic mechanisms, the change in intravascular volume appears to be the major contributor to the increased risk in patients developing swelling or edema when taking licorice supplements.

   What is the mechanism by which the Glycyrrhiza glabra in licorice can increase the intravascular volume?
   The normal physiology for sodium-water retention is largely influenced by the expression of mineralocorticoids.  While aldosterone is regarded as the main hormone binding to mineralocorticoid receptors involved in the regulation of sodium reabsorption and potassium excretion in the distal renal tubules of the kidney, cortisol also binds to this receptor with the same binding affinity as aldosterone.  Interestingly, even though cortisol blood concentrations tend to be greater than aldosterone concentrations, the effect of aldosterone dominates in terms of regulating sodium and water reabsorption and blood volume.  

   If cortisol concentrations are greater than aldosterone and both have equal affinity for the mineralocorticoid receptor, why doesn't cortisol have a greater influence on the overall mineralocorticoid activity?
   The type 2 isoenzyme of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD2) is normally involved in regulating corticosteroid specificity in the gastrointestinal tract, kidney and salivary glands (see figure 1).6  Cortisol (not aldosterone) is metabolized by 11 beta-HSD2 to cortisone, which does not bind to either the mineralocorticoid or glucocorticoid receptor.  Without this inactivation of cortisol, there would be mineralocorticoid excess.  In fact, there is an inherited disease called syndrome of apparent mineralocorticoid excess in which the mineralocorticoid receptor is overly activated thereby causing hypokalemia and hypervolemia due to the excessive reabsorption of sodium and water at the expense of potassium excretion.  This is the same effect created by licorice use.

   Licorice is a known inhibitor of 11 beta-HSD2 and thus prevents the inactivation of cortisol, thereby causing a state of excess mineralocorticoid activity or pseudohyperaldosteronism.2-6  The increase in mineralocorticoid activity results in greater sodium and water reabsorption at the expense of potassium excretion. This will eventually manifest as an increase in hydrostatic and overall blood pressures thereby resulting in the development of edema.

   The Details for Those Interested:
       How does cortisol binding to the mineralocorticoid receptor in the distal renal tubule increase sodium and water reabsorption at the expense of potassium excretion?  The increased cortisol resulting from licorice use increases gene expression and availability of several enzymes.  The first of these is the Na+ ion permease enzyme, which allows for a greater number of sodium ions to cross from the lumen to the inside of the renal tubular cell.   Next is Na+/K+ATPase on the basal-lateral side of the renal tubular cell which acts to transfer the increased cytosolic Na+ into the peritubular fluid resulting in a lowering of the intracellular electronegativity.  Lastly, there is an increase in citrate synthase activity within the mitochondria for the purpose of increasing the number of ATP available to fuel the increase in Na+/K+ATPase activity on the basal-lateral side of the renal tubular cell.7-9

   Conclusion
   Excess mineralocorticoid activity and resulting increases in blood volume are clearly the main mechanisms by which licorice causes both edema and hypertension.  In fact, the increase in blood pressure and development of hypertension can be significant and last several weeks before returning to baseline despite the discontinuation of the licorice supplementation.2,4 Given the documented increases in edema and blood pressure, the use of licorice supplements should be taken into consideration for any patient with an unexplained increase in blood pressure, worsening of hypertension that is being adequately treated with antihypertensive medications and/or worsening of previously controlled heart failure. There is some evidence that spironolactone (Aldactone) may confer some benefit on blood pressure; however, stopping the licorice would be the preferred recommendation for patients.10

https://www.ebmconsult.com/articles/licorice-Glycyrrhiza-glabra-edema-swelling-mechanism-side-effect

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https://my.clevelandclinic.org/health/articles/21205-respiratory-system

The respiratory system has many functions. Besides helping you inhale (breathe in) and exhale (breathe out), it:

   Allows you to talk and to smell.
   Warms air to match your body temperature and moisturizes it to the humidity level your body needs.
   Delivers oxygen to the cells in your body.
   Removes waste gases, including carbon dioxide, from the body when you exhale.
   Protects your airways from harmful substances and irritants.

Anatomy
What are the parts of the respiratory system?

The respiratory system has many different parts that work together to help you breathe. Each group of parts has many separate components.

Your airways deliver air to your lungs. Your airways are a complicated system that includes your:

   Mouth and nose: Openings that pull air from outside your body into your respiratory system.
   Sinuses: Hollow areas between the bones in your head that help regulate the temperature and humidity of the air you inhale.
   Pharynx (throat): Tube that delivers air from your mouth and nose to the trachea (windpipe).
   Trachea: Passage connecting your throat and lungs.
   Bronchial tubes: Tubes at the bottom of your windpipe that connect into each lung.
   Lungs: Two organs that remove oxygen from the air and pass it into your blood.

From your lungs, your bloodstream delivers oxygen to all your organs and other tissues.

Muscles and bones help move the air you inhale into and out of your lungs. Some of the bones and muscles in the respiratory system include your:

   Diaphragm: Muscle that helps your lungs pull in air and push it out.
   Ribs: Bones that surround and protect your lungs and heart.

When you breathe out, your blood carries carbon dioxide and other waste out of the body. Other components that work with the lungs and blood vessels include:

   Alveoli: Tiny air sacs in the lungs where the exchange of oxygen and carbon dioxide takes place.
   Bronchioles: Small branches of the bronchial tubes that lead to the alveoli.
   Capillaries: Blood vessels in the alveoli walls that move oxygen and carbon dioxide.
   Lung lobes: Sections of the lungs — three lobes in the right lung and two in the left lung.
   Pleura: Thin sacs that surround each lung lobe and separate your lungs from the chest wall.

Some of the other components of your respiratory system include:

   Cilia: Tiny hairs that move in a wave-like motion to filter dust and other irritants out of your airways.
   Epiglottis: Tissue flap at the entrance to the trachea that closes when you swallow to keep food and liquids out of your airway.
   Larynx (voice box): Hollow organ that allows you to talk and make sounds when air moves in and out.

Conditions and Disorders
What conditions affect the respiratory system?

Many conditions can affect the organs and tissues that make up the respiratory system. Some develop due to irritants you breathe in from the air, including viruses or bacteria that cause infection. Others occur as a result of disease or getting older.

Conditions that can cause inflammation (swelling, irritation and pain) or otherwise affect the respiratory system include:

   Allergies: Inhaling proteins, such as dust, mold, and pollen, can cause respiratory allergies in some people. These proteins can cause inflammation in your airways.
   Asthma: A chronic (long-term) disorder, asthma causes inflammation in the airways that can make breathing difficult.
   Infection: Infections can lead to pneumonia (inflammation of the lungs) or bronchitis (inflammation of the bronchial tubes). Common respiratory infections include the flu (influenza) or a cold.
   Disease: Respiratory disorders include lung cancer and chronic obstructive pulmonary disease (COPD). These illnesses can harm the respiratory system’s ability to deliver oxygen throughout the body and filter out waste gases.
   Aging: Lung capacity decreases as you get older.
   Damage: Damage to the respiratory system can cause breathing problems.

Care
How can I keep my respiratory system healthy?

Being able to clear mucus out of the lungs and airways is important for respiratory health.

To keep your respiratory system healthy, you should:

   Avoid pollutants that can damage your airways, including secondhand smoke, chemicals and radon (a radioactive gas that can cause cancer). Wear a mask if you are exposed to fumes, dust or other types of pollutants for any reason.
   Don't smoke.
   Eat a healthy diet with lots of fruits and vegetables and drink water to stay hydrated
   Exercise regularly to keep your lungs healthy.
   Prevent infections by washing your hands often and getting a flu vaccine each year.

When should I call a healthcare provider about an issue with my respiratory system?

Contact your provider if you have breathing trouble or pain. Your provider will listen to your chest, lungs, and heartbeat and look for signs of a respiratory issue such as infection. To see if your respiratory system is working as it should, your healthcare provider may use imaging tests such as a CT scan or MRI. These tests allow your provider to see swelling or blockages in your lungs and other parts of your respiratory system. Your provider may also recommend pulmonary function tests, which will include spirometry. A spirometer is a device that can tell how much air you inhale and exhale. See your doctor for regular checkups to help prevent serious respiratory conditions and lung disease. Early diagnosis of these issues can help prevent them from becoming severe.

More on Harmine. It is a bio-photon receptor. If taken with curcumin which helps bind Ultra-violet light, it is very powerful against most infections including cancers-heart infections. If they had a Harmine vape with it and curcumin-CBDs-ATP enhancers...it could really set things straight, of course it would have to be tested so be careful is you do any bio-hacking with this:
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Harmine is an effective therapeutic small molecule for the treatment of cardiac hypertrophy
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Article
Published: 30 March 2021

Harmine is an effective therapeutic small molecule for the treatment of cardiac hypertrophy

Jie Huang, Yang Liu, Jia-xin Chen, Xin-ya Lu, Wen-jia Zhu, Le Qin, Zi-xuan Xun, Qiu-yi Zheng, Er-min Li, Ning Sun, Chen Xu & Hai-yan Chen

Acta Pharmacologica Sinica volume 43, pages 50–63 (2022)Cite this article

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Abstract

Harmine is a β-carboline alkaloid isolated from Banisteria caapi and Peganum harmala L with various pharmacological activities, including antioxidant, anti-inflammatory, antitumor, anti-depressant, and anti-leishmanial capabilities. Nevertheless, the pharmacological effect of harmine on cardiomyocytes and heart muscle has not been reported. Here we found a protective effect of harmine on cardiac hypertrophy in spontaneously hypertensive rats in vivo. Further, harmine could inhibit the phenotypes of norepinephrine-induced hypertrophy in human embryonic stem cell-derived cardiomyocytes in vitro. It reduced the enlarged cell surface area, reversed the increased calcium handling and contractility, and downregulated expression of hypertrophy-related genes in norepinephrine-induced hypertrophy of human cardiomyocytes derived from embryonic stem cells. We further showed that one of the potential underlying mechanism by which harmine alleviates cardiac hypertrophy relied on inhibition of NF-κB phosphorylation and the stimulated inflammatory cytokines in pathological ventricular remodeling. Our data suggest that harmine is a promising therapeutic agent for cardiac hypertrophy independent of blood pressure modulation and could be a promising addition of current medications for cardiac hypertrophy.
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Introduction

Cardiac hypertrophy is an adaptive response to the overload of heart work and also a common pathological process in many cardiovascular diseases such as hypertension, aortic stenosis, and ischemic injury [1, 2]. Through hypertrophic growth, the heart reduces stress and oxygen consumption of ventricular wall and improves contractile function [3]. Cardiac hypertrophy is characterized by cellular hypertrophy, increased protein synthesis, and high arrangement of sarcomeres in cardiomyocytes [4]. Initially, the symptoms often come with hypertrophy of the left ventricle, increase of ventricular wall thickness, and shrinkage of the ventricular chamber. With progress of the disease, it is prone to develop chronic heart failure, fatal arrhythmia, and even sudden cardiac death [5, 6].

Current treatments for cardiac hypertrophy commonly target the symptoms by prescribing medications to alleviate symptoms so that cardiac systolic and diastolic function can be partially improved. These include β-blockers, calcium antagonists, angiotensin-converting enzyme (ACE) inhibitors, and other medicines [7]. However, these drugs often do not play a radical role and only bring about 10%–20% regression of left ventricular mass in patients [8, 9]. If pharmacological treatment fails in patients with severe outflow tract stenosis, surgery is needed to remove the hypertrophic interventricular septum [10]. Cardiac hypertrophy is closely related to various forms of heart failure and has become a risk factor in cardiovascular disease [11]. Therefore, there is an urgent need for developing new drugs and treatment strategies for cardiac hypertrophy.

Many studies have focused on the involvement of inflammation which is a prominent hallmark in the development of cardiac hypertrophy [12, 13]. When the heart is subjected to long-term/chronic stress, the balance between pro-inflammatory and anti-inflammatory response is disturbed, with a shift to the activation of pro-inflammatory cytokines [14,15,16]. Pro-inflammatory cytokines may cause cardiac hypertrophy through activating the downstream of signal transduction mediators (such as p38 and ERK) and transcription factors (such as nuclear factor kappa B (NF-κB)). Both cardiac hypertrophy and inflammatory signaling cascades share a common downstream transcription target of NF-κB and cAMP response element [17]. Therefore, regulation of immune response to the anti-inflammatory signaling cascade may provide a potential therapeutic target for cardiac hypertrophy [18].

Harmine is a β-carboline alkaloid widely existing in natural plants and can be extracted from Banisteria caapi and Peganum harmala L [19, 20]. As a precious herbal medicine in Chinese Traditional Medicine (TCM), harmine shows a wide range of pharmacological activities, including anti-inflammatory [21], antitumor [22, 23], anti-depressant [24], antioxidant properties [25], as well as anti-leishmanial [26], antibacterial [27], and antiviral bioactivities [28, 29]. In cardiovascular research, previous studies reported that harmine reduced systemic arterial blood pressure and peripheral vascular resistance [30]. Harmine also showed a vasorelaxant effect by inhibiting the L-type voltage-dependent Ca2+ channels in endothelium-intact aortic rings in a dose-dependent manner [31]. Karaki et al. found that harmine inhibited contraction of vascular and intestinal muscles by inhibiting calcium channels [32]. He et al. reported that harmine could improve myocardial infarction (MI)-induced heart injury by upregulating the Dyrk1a-ASF-CAMKIIδ pathway [33]. These studies indicate that harmine has a potential pharmacological effect on the cardiovascular system. However, whether harmine has a therapeutic effect on cardiac hypertrophy has not been reported yet.

Here, we firstly explored the protective effect of harmine on cardiac hypertrophy in spontaneously hypertensive rats (SHRs). We found that harmine significantly alleviated cardiac hypertrophy in a blood pressure-independent manner in vivo. We further established a human cardiac hypertrophy cell model by using human embryonic stem cell (hESC)-derived cardiomyocytes induced by norepinephrine (NE). Our data validated that harmine also attenuated the agonist-induced hypertrophic response of cardiomyocytes in vitro. And the protective effect of harmine on reducing pathological hypertrophy may partially rely on inhibiting NF-κB phosphorylation and thereby suppressing inflammatory response. Our study showed that harmine effectively improved the phenotypes of hypertrophy both in SHRs and in NE-induced hypertrophy of hESCs-derived cardiomyocytes, indicating that harmine could be a promising therapeutic agent for cardiac hypertrophy.

https://www.nature.com/articles/s41401-021-00639-y

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Harmine as a Luciferase compound:

https://en.wikipedia.org/wiki/Luciferase

This is your blood and brain using it. Helps with proper DNA-RNA formation. With curcumin it helps radiate energy in the body for transcription factors for cells that cytokines can disturb -- hence in-vivo better cell creation.

Further studies on the antiviral activity of harmine, a photoactive beta-carboline alkaloid
J B Hudson, E A Graham, R Fong, L L Hudson, G H Towers

   PMID: 3024190 DOI: 10.1111/j.1751-1097.1986.tb04696.x

https://doi.org/10.1111/j.1751-1097.1986.tb05586.x

https://onlinelibrary.wiley.com/doi/10.1111/j.1751-1097.1986.tb04696.x