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In Pursuit of Rare, Subtle, and Fleeting Post-Translational Modifications

Post-translational modifications (PTMs) account for most protein forms, or proteoforms. PTMs have much to tell researchers in the life sciences. But these…



Post-translational modifications (PTMs)—the chemical changes that proteins undergo following biosynthesis—account for most protein forms, or proteoforms. Indeed, according to conservative estimates, there are 1 million proteoforms, 90% of which are believed to be PTM-derived proteoforms. Estimates for the number of proteoforms and the percentage of PTM-derived proteoforms sometimes range quite a bit higher, depending on how the proteoforms are defined.1

The many PTM-derived proteoforms are important because they add greatly to the functional diversity of the proteome. They correspond to dynamic, labile, and often cell- or tissue-specific PTMs that regulate cellular pathways. Presumably, these PTMs have much to tell researchers in the life sciences. But these researchers will have to listen closely. Many PTMs are rare, subtle, and fleeting.

A more fundamental issue is that there are many types of PTMs. Indeed, there are over 400. About 90% of PTMs, however, are of just three types—phosphorylation, acetylation, and ubiquitination. Other major PTM types include glycosylation, succinylation, methylation, malonylation, SUMOylation, palmitoylation, myristoylation, prenylation, and sulfation.2 PTMs can be simple or complex. Examples of the latter include glycosylation and polyubiquitination. These PTMs may result in linear-chain and branched-chain structures.

Although the proteome is vast, it needn’t overwhelm. And although it is full of intricacy, it needn’t bewilder. The proteome may be studied with an expanding array of tools and technologies. Many of these tools and technologies are described in this article.

The resolution of complex proteoforms may exceed the capabilities of ensemble mass spectrometry. An alternative approach involves charge detection. It allows the direct determination of mass values for hundreds to thousands of individual ions in a single spectrum. To commercialize this approach, Thermo Fisher Scientific introduced Direct Mass Technology, which was used to perform the glycoform analysis shown here. (A) Schematic of a protein showing phosphorylation and glycosylation sites. (B) Deconvoluted mass domain spectra. The number of uniquely assigned glycoforms more than doubled.

Accelerating proteoform discovery

Aaron M. Robitaille, PhD, director of marketing, mass spectrometry, Thermo Fisher Scientific, notes that the first PTM was discovered over 100 years ago.3 He adds that it is only now that we are starting to appreciate how many proteoforms exist.

Typically, PTMs are analyzed in both temporal and spatial terms to clarify how they regulate cellular function. According to Robitaille, a classic example is phosphorylation, where the addition or subtraction of phosphate to an amino acid can activate or deactivate a protein. This PTM can serve as a kind of on/off switch.

The sample enrichment requirement prior to analysis to increase detection of low-abundance proteins and/or to reduce sample complexity is a key challenge. “Proteoforms can be transient and rare,” Robitaille notes. “They also can become dysregulated in disease and thus are important therapeutic targets. But small clinical samples such as biopsies present a limitation for enrichment.”

According to Robitaille, mass spectrometry (MS) offers the best sensitivity and specificity to study PTMs. He notes that once a standardized MS assay is developed, it can be deployed globally in multiple settings and produce the same quantitative results.

Robitaille indicates that the generation of Selective Temporal Overview of Resonant Ions (STORI) plots4 can be facilitated by an MS solution called Thermo Scientific Direct Mass Technology mode. Moreover, it can enable precise measurements of heavily modified proteoforms, such as those produced from glycosylation processes that are too complex to resolve using customary methods.

Traditionally, affinity reagents such as antibodies or aptamers must be generated for each specific PTM modification site and verified for specificity, requiring enormous amounts of reagents. In contrast, high-resolution accurate-mass (HRAM) instrumentation enables the characterization of proteoforms in their naïve state as well as the discovery of new PTMs with specific site localization. This fit-for-purpose MS technology is designed specifically to help scale to the level of 6 million proteoforms.

“We are analyzing single proteoforms without the need for enrichment,” Robitaille emphasizes. “The incredible sensitivity shows the heterogeneity of the biological system.” In the future, a range of MS-based assays including enzymatic activity, thermal shift, and protein degradation (PROTAC) studies will potentially address the functional effect of PTMs on protein-based activity.

“MS instruments do not directly measure function,” Robitaille points out. “You need a properly controlled experimental design that can take advantage of the sensitive and specific MS readout to understand function. Many PTMs are understudied due to a lack of enrichment tools or affinity reagents, representing a key area where MS empowered by Thermo Scientific Orbitrap technology and Direct Mass Technology mode can play a major role.”

Grappling with glycobiology

The rise of therapeutic antibodies results in the need to characterize lot-to-lot glycan profiles. “It is well known how glycans influence antibodies’ therapeutic potency,” says Anthony Person, PhD, senior director, Protein Business Unit, Bio-Techne. For example, certain immunoglobulin G1 antibodies rely on the Fc-mediated immune effector function, known as antibody-dependent cellular cytotoxicity (ADCC), as a central way to deplete tumor cells. Core fucosylation on N-glycans on antibody heavy chains directly influences the binding affinity of the antibody’s Fc portion to the FcgRIIIa receptor present on immune effector cells like natural killer cells. Generally, antibodies without this core fucosylation can bind to FcgRIIIa with higher affinity, resulting in higher ADCC and efficacy.

The cell culture step is central to having the desired glycan profiles on recombinant proteins. Commonly, immature N-glycans, like high-mannose N-glycans, lack terminal sialyation and galactosylation and are more easily cleared in vivo, resulting in lower antibody efficacy. High-mannose N-glycans result from suboptimal CHO or HEK293 cell culture parameters.

Direct Fluorescent Glycan Labeling illustration
Bio-Techne used a direct fluorescent glycan labeling technique to characterize differences in the N- and O-glycans on the recombinant SARS-CoV-2 spike proteins made in CHO cells, in HEK293 cells, in Sf21 insect cells, and in Tn5 insect cells. The company determined that the technique could serve as a quick way to obtain a global assessment of glycosylation patterns. Differences in patterns for the spike proteins from different host cell types were taken to suggest that patterns for the spike proteins from COVID-19 patients could be different as well.

“Although MS analysis is still the gold standard for characterization, glycan profiling can be infeasible,” Person warns. “We produce over 6,000 research-grade, non-antibody protein products and do not have the ability to run them all through MS due to the low throughput and high cost.”

Recently developed benchtop techniques rely on Bio-Techne’s catalog of glycobiology enzymes and substrates to footprint both N– and O-linked glycans. “We can use neuraminidase to remove sialic acid or FUCA1 to remove fucose followed by fucosyltransferases and sialyltransferases to add fluorescently labeled sugars to glycans,” Person relates. “Labeled recombinant proteins can then be run on SDS-PAGE gels, and differences in sialylation, galactosylation, or fucosylation can be easily visualized to compare glycan footprints.”

The approach was used to investigate differences in glycosylation patterns of SARS-CoV-2 spike proteins made in insect, CHO, and HEK293 production hosts.

The original SARS-CoV-2 canonical spike protein comprising the receptor binding domain (RBD) that was produced in insect hosts (Sf21 or Tn5) possessed generally high-mannose, immature N-glycans with more immature core-1 O-glycans. CHO host-produced RBD proteins showed N-glycans that were terminally galactosylated without terminal sialylation with core-1 O-glycans. The most mature RBD glycan profiles were seen on the HEK293 host-produced proteins that were both terminal sialylated on N-glycans and also displayed core-2 O-glycans.

“The finding that the original SARS-CoV-2 spike protein contains 22 N-glycosylation sites emphasizes glycobiology’s complexity,” Person declares.

Controlling microheterogeneity

“The importance of PTMs, particularly glycosylation, has been established in biopharmaceutical manufacturing,” says Nan Lin, PhD, director, New Technologies and Service Applications, Cytiva. Variants of glycosylation, together with variants in charge and size, are among the key aspects of recombinant protein microheterogeneity.

Controlling microheterogeneity to advance PTM control has become essential in implementing quality by design (QbD) and in meeting biosimilarity criteria. In recent years, industrial and academic researchers have made significant progress in controlling and modulating glycosylation via media and process optimization. “Glycosylation modulation,” Lin remarks, “has gained more focus not only in fed-batch but also in perfusion processes in recent years.”

Current methods are mainly based on rather lengthy workflows—collecting samples, purification, and analysis. High-throughput, low-sample-volume, and low-protein-quantity PTM analytical methods remain the challenge.

“High-throughput analytical methods, coupled with high-throughput cell culture platforms such as microbioreactors would enable assessment of PTMs in very early steps in cell line and process development,” Lin suggests. “Online/at-line analyses in an integrated, fully automated system has become increasingly discussed and developed for online PTM data monitoring.”

Numerous chip- and kit-based rapid glycosylation analysis methods are available and make good tools for bioprocess development, especially where analytical capabilities are not readily available.

“Several research groups have proposed to characterize global cellular PTM characterization, including glycosylation, phosphorylation, and ubiquitination, as part of understanding and controlling cell physiology,” Lin notes. “Such groundbreaking work would provide effective cell engineering targets and process monitoring endpoints in the future.”

Emerging biopharmaceutical molecules, including Fc-fusion and bispecific proteins, have gained presence in many pharmaceutical companies’ development pipelines. Many new molecules have unique structures and PTMs required by mechanisms of action.

“To be able to control PTMs of the new molecules early in development will be critical for future QbD processes,” Lin stresses. “Novel host cell lines for producing therapeutic proteins are on the horizon. Human origin or glycoengineered CHO host cell lines may be able to produce more human-like glycosylation patterns, and to achieve better glycosylation control.”

Exploring new ways to study low-abundance PTMs

“In vertebrates, the investigative focus has primarily been on serine, threonine, and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other, ‘noncanonical’ amino acids also regulates critical aspects of cell biology,” reports a scientific team led by Claire E. Eyers, PhD, professor of biological mass spectrometry at the University of Liverpool.

“Eyers’ use of strong anion exchange-mediated phosphoproteomics to detect noncanonical phosphorylation indicated that the number of unique noncanonical phosphosites is approximately one-third of the number of observed canonical phosphosites,” comments Cristina Martin-Granados, PhD, research area scientific lead, Cell Signaling, Abcam.5 Although the numbers presented by Eyers and colleagues need to be firmed up, they are already encouraging researchers to consider broadening their views of the phosphorylation landscape.

According to Martin-Granados, low-abundance PTMs can make detection by antibody-based approaches difficult. Enrichment of a specific PTM by immunoprecipitation or by ion exchange, immobilized metal ion affinity, or immunoaffinity chromatography can help overcome low-stoichiometry challenges.

She emphasizes that dynamic and frequently transient PTMs require stringent positive and negative experimental controls if results are to be correctly interpreted. Since many PTMs are the aftermath of enzymatic reactions, uncontrolled enzymatic activity in sample processing can affect them.

“Antibodies are essential tools for detection and enrichment,” Martin-Granados remarks. “The challenge in developing highly specific antibodies with exquisite binding affinity is the small size of the PTM chemical moieties, similarities in some chemical structures, and poor antigenicity. Polyclonal antibodies present serious drawbacks in delivering reproducible and reliable data. To avoid these drawbacks, it may be necessary to switch to highly functional, reliable, and renewable recombinant monoclonals.”

Computational methods for predicting PTMs are attracting considerable attention. For example, AlphaFold, an artificial intelligence platform, made the news when it demonstrated its prowess in predicting unsolved protein structures from amino acid sequences. However, as Martin-Granados explains, AlphaFold2 does not yet consider the impact of PTMs on protein structure. Fortunately, databases on protein PTMs and predictive computational tools are available.

Proximity ligation assay (PLA), an antibody-dependent, fast, and highly sensitive immunoassay technology, can be used to detect PTMs, allowing for identification of several PTMs in a specific target.6,7 PLA technology can be used in combination with immunocytochemistry/immunofluorescence analysis and fluorescence immunohistochemistry analysis to study protein localizations and validate potential biomarkers for clinical diagnostic testing.

Genetic code expansion enables site-specific placement of functionally masked unnatural amino acids into proteins. These unnatural amino acids may remain inert until they are activated by a signal of some kind, that is, by a particular kind of illumination or by a small molecule. This provides rapid and temporal external control over addition and removal of PTMs, eliminating compensatory mechanisms that arise by slow conventional approaches, and it is expected to significantly increase the understanding of transient, dynamic PTM events.8

“Sensitivity and the dynamic range for detection of low-abundance PTMs need improvement as well as accurate, quantitative methods for studying dynamics and for analysis of less-well-known or newly identified PTMs,” Martin-Granados adds. “The relevance of multisite, cooperative PTMs highlights the need to develop strategies for full-spectrum identification of PTMs.”

Meeting characterization challenges

Previously, PTMs were thought of in binary terms—as either activating or inactivating, based on the particular modification and pathway. “Now, we understand to a greater degree how subtle changes in PTMs can modulate activity and signals,” says Carl Ascoli, PhD, chief science officer, Rockland Immunochemicals.

H3K9me2 Antibod
Rockland Immunochemicals asserts that its histone H3 K9me2 antibody is highly specific for K9Me2, and that the antibody’s sensitivity goes to the low picomolar range. The company adds that even when the antibody is present in high concentrations, it avoids cross-reactions with any other modifications of K9 while maintaining high sensitivity.

Although PTMs of proteins increase the functional diversity of the proteome to adapt to rapid changes in the environment, this diversity places strain on the availability of detection and characterization antibodies to certain modifications.

“Consider two characterization challenges: those involving ubiquitination, and those involving epigenetics,” Ascoli suggests. “Ubiquitination is a complex, multistep, reversible process that is highly regulated by the sequential action of very specific enzymes that add or remove ubiquitin. These enzymes may add monoubiquitin, polyubiquitin, and branched-chain ubiquitin at one or more targets sites on a given protein. This complexity makes understanding the machinery of ubiquitin in the laboratory a difficult process.”

“Epigenetic modifications, such as PTMs on histones, can be numerous, diverse, subtle, and transient, and they can work in combination,” Ascoli continues. Different cellular processes are affected depending on whether a particular amino acid is modified by a single, double, or triple methylation event, or whether it is unmethylated. Subtle variations in PTMs can result from changes due to environment, diet, disease, stress, or drug therapy.

Immunoassays using specific antibodies to PTMs generated in context with the surrounding amino acids on an enzyme are valuable tools for detection and characterization whether experiments are conducted in vitro or with biological systems involving immunofluorescence microscopy of organoids or whole animal in vivo imaging.

Antibodies against PTMs
Antibodies against post-translational modifications (PTMs) can be used in a wide range of in vitro and in vivo studies. For example, they can be used to identify and purify protein-protein, protein-DNA, and/or protein-RNA complexes containing proteins subject to particular PTMs. To develop modification-specific PTM antibodies, Rockland Immunochemicals relies on proprietary technology. The company also offers antibody selection tips that encompass preparation, production, and validation procedures.

According to Ascoli, antibodies to AKT pS473, MEK pT386, or STAT3 pY705 have been invaluable in elucidating regulatory pathways and exploring the efficacy of drugs in both benchtop and biological experiments. He adds, however, that PTMs are more diverse and abundant than well-characterized antibodies.

Understanding the function of PTMs requires a more universal detection strategy. For instance, it may be necessary to rely on physicochemical detection methods such as MS analysis. Methods that look at entire proteins may be especially useful. Variations that increase accuracy and add context to the MS data include MALDI-MS, ESI-MS, and tandem MS.

“Detection or analysis methods that are based on protein binding directly or indirectly to DNA or RNA are of interest and will continue to evolve,” Ascoli predicts. “While assays like CUT&RUN, CUT&TAG, and more recent variations called TIP-seq and MULTI-CUT&TAG are themselves dependent on antibodies that bind to the protein that binds nucleic acids, the information that these assays provide is much richer in context.”



1. Aebersold R, Agar JN, Amster IJ, et al. How many human proteoforms are there? Nat. Chem. Biol. 2018; 14: 206–214. DOI: 10.1038/nchembio.2576.

2. Ramazi S, Zahiri J. Post-translational modifications in proteins: resources, tools and prediction methods. Database (Oxford) 2021; 2021: baab012. DOI: 10.1093/database/baab012.

3. Levene P, Alsberg C. The cleavage products of vitellin. J. Biol. Chem. 1906; 2: 127–133.

4. Kafader JO, Beu SC, Early BP, et al. STORI Plots Enable Accurate Tracking of Individual Ion Signals. J. Am. Soc. Mass Spectrom. 2019; 30: 2200–2203. DOI: 10.1007/s13361-019-02309-0.

5. Hardman G, Perkins S, Brownridge PJ, et al. Strong anion exchange-mediated phosphoproteomics reveals extensive human non-canonical phosphorylation. EMBO J. 2019; 38: e100847. DOI: 10.15252/embj.2018100847.

6. Tong QH, Tao T, Xie LQ, Lu HJ. ELISA–PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications. Biosens. Bioelectron. 2016; 80: 385–391. DOI: 10.1016/j.bios.2016.02.006.

7. de Oliviera FMS, Mereiter S, Lönn P, et al. Detection of post-translational modifications using solid-phase proximity ligation assay. N. Biotechnol. 2018; 45: 51–59. DOI: 10.1016/j.nbt.2017.10.005.

8. Zhou W, Deiters A. Chemogenetic and optogenetic control of post-translational modifications through genetic code expansion. Curr. Opin. Chem. Biol. 2021; 63: 123–131. DOI: 10.1016/j.cbpa.2021.02.016.

The post In Pursuit of Rare, Subtle, and Fleeting Post-Translational Modifications appeared first on GEN - Genetic Engineering and Biotechnology News.

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Chronic stress and inflammation linked to societal and environmental impacts in new study

From anxiety about the state of the world to ongoing waves of Covid-19, the stresses we face can seem relentless and even overwhelming. Worse, these stressors…



From anxiety about the state of the world to ongoing waves of Covid-19, the stresses we face can seem relentless and even overwhelming. Worse, these stressors can cause chronic inflammation in our bodies. Chronic inflammation is linked to serious conditions such as cardiovascular disease and cancer – and may also affect our thinking and behavior.   

Credit: Image: Vodovotz et al/Frontiers

From anxiety about the state of the world to ongoing waves of Covid-19, the stresses we face can seem relentless and even overwhelming. Worse, these stressors can cause chronic inflammation in our bodies. Chronic inflammation is linked to serious conditions such as cardiovascular disease and cancer – and may also affect our thinking and behavior.   

A new hypothesis published in Frontiers in Science suggests the negative impacts may extend far further.   

“We propose that stress, inflammation, and consequently impaired cognition in individuals can scale up to communities and populations,” explained lead author Prof Yoram Vodovotz of the University of Pittsburgh, USA.

“This could affect the decision-making and behavior of entire societies, impair our cognitive ability to address complex issues like climate change, social unrest, and infectious disease – and ultimately lead to a self-sustaining cycle of societal dysfunction and environmental degradation,” he added.

Bodily inflammation ‘mapped’ in the brain  

One central premise to the hypothesis is an association between chronic inflammation and cognitive dysfunction.  

“The cause of this well-known phenomenon is not currently known,” said Vodovotz. “We propose a mechanism, which we call the ‘central inflammation map’.”    

The authors’ novel idea is that the brain creates its own copy of bodily inflammation. Normally, this inflammation map allows the brain to manage the inflammatory response and promote healing.   

When inflammation is high or chronic, however, the response goes awry and can damage healthy tissues and organs. The authors suggest the inflammation map could similarly harm the brain and impair cognition, emotion, and behavior.   

Accelerated spread of stress and inflammation online   

A second premise is the spread of chronic inflammation from individuals to populations.  

“While inflammation is not contagious per se, it could still spread via the transmission of stress among people,” explained Vodovotz.   

The authors further suggest that stress is being transmitted faster than ever before, through social media and other digital communications.  

“People are constantly bombarded with high levels of distressing information, be it the news, negative online comments, or a feeling of inadequacy when viewing social media feeds,” said Vodovotz. “We hypothesize that this new dimension of human experience, from which it is difficult to escape, is driving stress, chronic inflammation, and cognitive impairment across global societies.”   

Inflammation as a driver of social and planetary disruption  

These ideas shift our view of inflammation as a biological process restricted to an individual. Instead, the authors see it as a multiscale process linking molecular, cellular, and physiological interactions in each of us to altered decision-making and behavior in populations – and ultimately to large-scale societal and environmental impacts.  

“Stress-impaired judgment could explain the chaotic and counter-intuitive responses of large parts of the global population to stressful events such as climate change and the Covid-19 pandemic,” explained Vodovotz.  

“An inability to address these and other stressors may propagate a self-fulfilling sense of pervasive danger, causing further stress, inflammation, and impaired cognition in a runaway, positive feedback loop,” he added.  

The fact that current levels of global stress have not led to widespread societal disorder could indicate an equally strong stabilizing effect from “controllers” such as trust in laws, science, and multinational organizations like the United Nations.   

“However, societal norms and institutions are increasingly being questioned, at times rightly so as relics of a foregone era,” said Prof Paul Verschure of Radboud University, the Netherlands, and a co-author of the article. “The challenge today is how we can ward off a new adversarial era of instability due to global stress caused by a multi-scale combination of geopolitical fragmentation, conflicts, and ecological collapse amplified by existential angst, cognitive overload, and runaway disinformation.”    

Reducing social media exposure as part of the solution  

The authors developed a mathematical model to test their ideas and explore ways to reduce stress and build resilience.  

“Preliminary results highlight the need for interventions at multiple levels and scales,” commented co-author Prof Julia Arciero of Indiana University, USA.  

“While anti-inflammatory drugs are sometimes used to treat medical conditions associated with inflammation, we do not believe these are the whole answer for individuals,” said Dr David Katz, co-author and a specialist in preventive and lifestyle medicine based in the US. “Lifestyle changes such as healthy nutrition, exercise, and reducing exposure to stressful online content could also be important.”  

“The dawning new era of precision and personalized therapeutics could also offer enormous potential,” he added.  

At the societal level, the authors suggest creating calm public spaces and providing education on the norms and institutions that keep our societies stable and functioning.  

“While our ‘inflammation map’ hypothesis and corresponding mathematical model are a start, a coordinated and interdisciplinary research effort is needed to define interventions that would improve the lives of individuals and the resilience of communities to stress. We hope our article stimulates scientists around the world to take up this challenge,” Vodovotz concluded.  

The article is part of the Frontiers in Science multimedia article hub ‘A multiscale map of inflammatory stress’. The hub features a video, an explainer, a version of the article written for kids, and an editorial, viewpoints, and policy outlook from other eminent experts: Prof David Almeida (Penn State University, USA), Prof Pietro Ghezzi (University of Urbino Carlo Bo, Italy), and Dr Ioannis P Androulakis (Rutgers, The State University of New Jersey, USA). 

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Four Years Ago This Week, Freedom Was Torched

Four Years Ago This Week, Freedom Was Torched

Authored by Jeffrey Tucker via The Brownstone Institute,

"Beware the Ides of March,” Shakespeare…



Four Years Ago This Week, Freedom Was Torched

Authored by Jeffrey Tucker via The Brownstone Institute,

"Beware the Ides of March,” Shakespeare quotes the soothsayer’s warning Julius Caesar about what turned out to be an impending assassination on March 15. The death of American liberty happened around the same time four years ago, when the orders went out from all levels of government to close all indoor and outdoor venues where people gather. 

It was not quite a law and it was never voted on by anyone. Seemingly out of nowhere, people who the public had largely ignored, the public health bureaucrats, all united to tell the executives in charge – mayors, governors, and the president – that the only way to deal with a respiratory virus was to scrap freedom and the Bill of Rights. 

And they did, not only in the US but all over the world. 

The forced closures in the US began on March 6 when the mayor of Austin, Texas, announced the shutdown of the technology and arts festival South by Southwest. Hundreds of thousands of contracts, of attendees and vendors, were instantly scrapped. The mayor said he was acting on the advice of his health experts and they in turn pointed to the CDC, which in turn pointed to the World Health Organization, which in turn pointed to member states and so on. 

There was no record of Covid in Austin, Texas, that day but they were sure they were doing their part to stop the spread. It was the first deployment of the “Zero Covid” strategy that became, for a time, official US policy, just as in China. 

It was never clear precisely who to blame or who would take responsibility, legal or otherwise. 

This Friday evening press conference in Austin was just the beginning. By the next Thursday evening, the lockdown mania reached a full crescendo. Donald Trump went on nationwide television to announce that everything was under control but that he was stopping all travel in and out of US borders, from Europe, the UK, Australia, and New Zealand. American citizens would need to return by Monday or be stuck. 

Americans abroad panicked while spending on tickets home and crowded into international airports with waits up to 8 hours standing shoulder to shoulder. It was the first clear sign: there would be no consistency in the deployment of these edicts. 

There is no historical record of any American president ever issuing global travel restrictions like this without a declaration of war. Until then, and since the age of travel began, every American had taken it for granted that he could buy a ticket and board a plane. That was no longer possible. Very quickly it became even difficult to travel state to state, as most states eventually implemented a two-week quarantine rule. 

The next day, Friday March 13, Broadway closed and New York City began to empty out as any residents who could went to summer homes or out of state. 

On that day, the Trump administration declared the national emergency by invoking the Stafford Act which triggers new powers and resources to the Federal Emergency Management Administration. 

In addition, the Department of Health and Human Services issued a classified document, only to be released to the public months later. The document initiated the lockdowns. It still does not exist on any government website.

The White House Coronavirus Response Task Force, led by the Vice President, will coordinate a whole-of-government approach, including governors, state and local officials, and members of Congress, to develop the best options for the safety, well-being, and health of the American people. HHS is the LFA [Lead Federal Agency] for coordinating the federal response to COVID-19.

Closures were guaranteed:

Recommend significantly limiting public gatherings and cancellation of almost all sporting events, performances, and public and private meetings that cannot be convened by phone. Consider school closures. Issue widespread ‘stay at home’ directives for public and private organizations, with nearly 100% telework for some, although critical public services and infrastructure may need to retain skeleton crews. Law enforcement could shift to focus more on crime prevention, as routine monitoring of storefronts could be important.

In this vision of turnkey totalitarian control of society, the vaccine was pre-approved: “Partner with pharmaceutical industry to produce anti-virals and vaccine.”

The National Security Council was put in charge of policy making. The CDC was just the marketing operation. That’s why it felt like martial law. Without using those words, that’s what was being declared. It even urged information management, with censorship strongly implied.

The timing here is fascinating. This document came out on a Friday. But according to every autobiographical account – from Mike Pence and Scott Gottlieb to Deborah Birx and Jared Kushner – the gathered team did not meet with Trump himself until the weekend of the 14th and 15th, Saturday and Sunday. 

According to their account, this was his first real encounter with the urge that he lock down the whole country. He reluctantly agreed to 15 days to flatten the curve. He announced this on Monday the 16th with the famous line: “All public and private venues where people gather should be closed.”

This makes no sense. The decision had already been made and all enabling documents were already in circulation. 

There are only two possibilities. 

One: the Department of Homeland Security issued this March 13 HHS document without Trump’s knowledge or authority. That seems unlikely. 

Two: Kushner, Birx, Pence, and Gottlieb are lying. They decided on a story and they are sticking to it. 

Trump himself has never explained the timeline or precisely when he decided to greenlight the lockdowns. To this day, he avoids the issue beyond his constant claim that he doesn’t get enough credit for his handling of the pandemic.

With Nixon, the famous question was always what did he know and when did he know it? When it comes to Trump and insofar as concerns Covid lockdowns – unlike the fake allegations of collusion with Russia – we have no investigations. To this day, no one in the corporate media seems even slightly interested in why, how, or when human rights got abolished by bureaucratic edict. 

As part of the lockdowns, the Cybersecurity and Infrastructure Security Agency, which was and is part of the Department of Homeland Security, as set up in 2018, broke the entire American labor force into essential and nonessential.

They also set up and enforced censorship protocols, which is why it seemed like so few objected. In addition, CISA was tasked with overseeing mail-in ballots. 

Only 8 days into the 15, Trump announced that he wanted to open the country by Easter, which was on April 12. His announcement on March 24 was treated as outrageous and irresponsible by the national press but keep in mind: Easter would already take us beyond the initial two-week lockdown. What seemed to be an opening was an extension of closing. 

This announcement by Trump encouraged Birx and Fauci to ask for an additional 30 days of lockdown, which Trump granted. Even on April 23, Trump told Georgia and Florida, which had made noises about reopening, that “It’s too soon.” He publicly fought with the governor of Georgia, who was first to open his state. 

Before the 15 days was over, Congress passed and the president signed the 880-page CARES Act, which authorized the distribution of $2 trillion to states, businesses, and individuals, thus guaranteeing that lockdowns would continue for the duration. 

There was never a stated exit plan beyond Birx’s public statements that she wanted zero cases of Covid in the country. That was never going to happen. It is very likely that the virus had already been circulating in the US and Canada from October 2019. A famous seroprevalence study by Jay Bhattacharya came out in May 2020 discerning that infections and immunity were already widespread in the California county they examined. 

What that implied was two crucial points: there was zero hope for the Zero Covid mission and this pandemic would end as they all did, through endemicity via exposure, not from a vaccine as such. That was certainly not the message that was being broadcast from Washington. The growing sense at the time was that we all had to sit tight and just wait for the inoculation on which pharmaceutical companies were working. 

By summer 2020, you recall what happened. A restless generation of kids fed up with this stay-at-home nonsense seized on the opportunity to protest racial injustice in the killing of George Floyd. Public health officials approved of these gatherings – unlike protests against lockdowns – on grounds that racism was a virus even more serious than Covid. Some of these protests got out of hand and became violent and destructive. 

Meanwhile, substance abuse rage – the liquor and weed stores never closed – and immune systems were being degraded by lack of normal exposure, exactly as the Bakersfield doctors had predicted. Millions of small businesses had closed. The learning losses from school closures were mounting, as it turned out that Zoom school was near worthless. 

It was about this time that Trump seemed to figure out – thanks to the wise council of Dr. Scott Atlas – that he had been played and started urging states to reopen. But it was strange: he seemed to be less in the position of being a president in charge and more of a public pundit, Tweeting out his wishes until his account was banned. He was unable to put the worms back in the can that he had approved opening. 

By that time, and by all accounts, Trump was convinced that the whole effort was a mistake, that he had been trolled into wrecking the country he promised to make great. It was too late. Mail-in ballots had been widely approved, the country was in shambles, the media and public health bureaucrats were ruling the airwaves, and his final months of the campaign failed even to come to grips with the reality on the ground. 

At the time, many people had predicted that once Biden took office and the vaccine was released, Covid would be declared to have been beaten. But that didn’t happen and mainly for one reason: resistance to the vaccine was more intense than anyone had predicted. The Biden administration attempted to impose mandates on the entire US workforce. Thanks to a Supreme Court ruling, that effort was thwarted but not before HR departments around the country had already implemented them. 

As the months rolled on – and four major cities closed all public accommodations to the unvaccinated, who were being demonized for prolonging the pandemic – it became clear that the vaccine could not and would not stop infection or transmission, which means that this shot could not be classified as a public health benefit. Even as a private benefit, the evidence was mixed. Any protection it provided was short-lived and reports of vaccine injury began to mount. Even now, we cannot gain full clarity on the scale of the problem because essential data and documentation remains classified. 

After four years, we find ourselves in a strange position. We still do not know precisely what unfolded in mid-March 2020: who made what decisions, when, and why. There has been no serious attempt at any high level to provide a clear accounting much less assign blame. 

Not even Tucker Carlson, who reportedly played a crucial role in getting Trump to panic over the virus, will tell us the source of his own information or what his source told him. There have been a series of valuable hearings in the House and Senate but they have received little to no press attention, and none have focus on the lockdown orders themselves. 

The prevailing attitude in public life is just to forget the whole thing. And yet we live now in a country very different from the one we inhabited five years ago. Our media is captured. Social media is widely censored in violation of the First Amendment, a problem being taken up by the Supreme Court this month with no certainty of the outcome. The administrative state that seized control has not given up power. Crime has been normalized. Art and music institutions are on the rocks. Public trust in all official institutions is at rock bottom. We don’t even know if we can trust the elections anymore. 

In the early days of lockdown, Henry Kissinger warned that if the mitigation plan does not go well, the world will find itself set “on fire.” He died in 2023. Meanwhile, the world is indeed on fire. The essential struggle in every country on earth today concerns the battle between the authority and power of permanent administration apparatus of the state – the very one that took total control in lockdowns – and the enlightenment ideal of a government that is responsible to the will of the people and the moral demand for freedom and rights. 

How this struggle turns out is the essential story of our times. 

CODA: I’m embedding a copy of PanCAP Adapted, as annotated by Debbie Lerman. You might need to download the whole thing to see the annotations. If you can help with research, please do.

*  *  *

Jeffrey Tucker is the author of the excellent new book 'Life After Lock-Down'

Tyler Durden Mon, 03/11/2024 - 23:40

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Fauci Deputy Warned Him Against Vaccine Mandates: Email

Fauci Deputy Warned Him Against Vaccine Mandates: Email

Authored by Zachary Stieber via The Epoch Times (emphasis ours),

Mandating COVID-19…



Fauci Deputy Warned Him Against Vaccine Mandates: Email

Authored by Zachary Stieber via The Epoch Times (emphasis ours),

Mandating COVID-19 vaccination was a mistake due to ethical and other concerns, a top government doctor warned Dr. Anthony Fauci after Dr. Fauci promoted mass vaccination.

Coercing or forcing people to take a vaccine can have negative consequences from a biological, sociological, psychological, economical, and ethical standpoint and is not worth the cost even if the vaccine is 100% safe,” Dr. Matthew Memoli, director of the Laboratory of Infectious Diseases clinical studies unit at the U.S. National Institute of Allergy and Infectious Diseases (NIAID), told Dr. Fauci in an email.

“A more prudent approach that considers these issues would be to focus our efforts on those at high risk of severe disease and death, such as the elderly and obese, and do not push vaccination on the young and healthy any further.”

Dr. Anthony Fauci, ex-director of the National Institute of Allergy and Infectious Diseases (NIAID. in Washington on Jan. 8, 2024. (Madalina Vasiliu/The Epoch Times)

Employing that strategy would help prevent loss of public trust and political capital, Dr. Memoli said.

The email was sent on July 30, 2021, after Dr. Fauci, director of the NIAID, claimed that communities would be safer if more people received one of the COVID-19 vaccines and that mass vaccination would lead to the end of the COVID-19 pandemic.

“We’re on a really good track now to really crush this outbreak, and the more people we get vaccinated, the more assuredness that we’re going to have that we’re going to be able to do that,” Dr. Fauci said on CNN the month prior.

Dr. Memoli, who has studied influenza vaccination for years, disagreed, telling Dr. Fauci that research in the field has indicated yearly shots sometimes drive the evolution of influenza.

Vaccinating people who have not been infected with COVID-19, he said, could potentially impact the evolution of the virus that causes COVID-19 in unexpected ways.

“At best what we are doing with mandated mass vaccination does nothing and the variants emerge evading immunity anyway as they would have without the vaccine,” Dr. Memoli wrote. “At worst it drives evolution of the virus in a way that is different from nature and possibly detrimental, prolonging the pandemic or causing more morbidity and mortality than it should.”

The vaccination strategy was flawed because it relied on a single antigen, introducing immunity that only lasted for a certain period of time, Dr. Memoli said. When the immunity weakened, the virus was given an opportunity to evolve.

Some other experts, including virologist Geert Vanden Bossche, have offered similar views. Others in the scientific community, such as U.S. Centers for Disease Control and Prevention scientists, say vaccination prevents virus evolution, though the agency has acknowledged it doesn’t have records supporting its position.

Other Messages

Dr. Memoli sent the email to Dr. Fauci and two other top NIAID officials, Drs. Hugh Auchincloss and Clifford Lane. The message was first reported by the Wall Street Journal, though the publication did not publish the message. The Epoch Times obtained the email and 199 other pages of Dr. Memoli’s emails through a Freedom of Information Act request. There were no indications that Dr. Fauci ever responded to Dr. Memoli.

Later in 2021, the NIAID’s parent agency, the U.S. National Institutes of Health (NIH), and all other federal government agencies began requiring COVID-19 vaccination, under direction from President Joe Biden.

In other messages, Dr. Memoli said the mandates were unethical and that he was hopeful legal cases brought against the mandates would ultimately let people “make their own healthcare decisions.”

“I am certainly doing everything in my power to influence that,” he wrote on Nov. 2, 2021, to an unknown recipient. Dr. Memoli also disclosed that both he and his wife had applied for exemptions from the mandates imposed by the NIH and his wife’s employer. While her request had been granted, his had not as of yet, Dr. Memoli said. It’s not clear if it ever was.

According to Dr. Memoli, officials had not gone over the bioethics of the mandates. He wrote to the NIH’s Department of Bioethics, pointing out that the protection from the vaccines waned over time, that the shots can cause serious health issues such as myocarditis, or heart inflammation, and that vaccinated people were just as likely to spread COVID-19 as unvaccinated people.

He cited multiple studies in his emails, including one that found a resurgence of COVID-19 cases in a California health care system despite a high rate of vaccination and another that showed transmission rates were similar among the vaccinated and unvaccinated.

Dr. Memoli said he was “particularly interested in the bioethics of a mandate when the vaccine doesn’t have the ability to stop spread of the disease, which is the purpose of the mandate.”

The message led to Dr. Memoli speaking during an NIH event in December 2021, several weeks after he went public with his concerns about mandating vaccines.

“Vaccine mandates should be rare and considered only with a strong justification,” Dr. Memoli said in the debate. He suggested that the justification was not there for COVID-19 vaccines, given their fleeting effectiveness.

Julie Ledgerwood, another NIAID official who also spoke at the event, said that the vaccines were highly effective and that the side effects that had been detected were not significant. She did acknowledge that vaccinated people needed boosters after a period of time.

The NIH, and many other government agencies, removed their mandates in 2023 with the end of the COVID-19 public health emergency.

A request for comment from Dr. Fauci was not returned. Dr. Memoli told The Epoch Times in an email he was “happy to answer any questions you have” but that he needed clearance from the NIAID’s media office. That office then refused to give clearance.

Dr. Jay Bhattacharya, a professor of health policy at Stanford University, said that Dr. Memoli showed bravery when he warned Dr. Fauci against mandates.

“Those mandates have done more to demolish public trust in public health than any single action by public health officials in my professional career, including diminishing public trust in all vaccines.” Dr. Bhattacharya, a frequent critic of the U.S. response to COVID-19, told The Epoch Times via email. “It was risky for Dr. Memoli to speak publicly since he works at the NIH, and the culture of the NIH punishes those who cross powerful scientific bureaucrats like Dr. Fauci or his former boss, Dr. Francis Collins.”

Tyler Durden Mon, 03/11/2024 - 17:40

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