Covid-19 News

    Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants


    https://www.nature.com/articles/s41586-021-03676-z


    Abstract

    Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. While many vaccines have been deployed to date, the continual evolution of the viral receptor-binding domain (RBD) has challenged their efficacy. In particular, emerging variants B.1.1.7 (U.K.), B.1.351 (South Africa) and P.1 (Brazil) have compromised convalescent sera and immunotherapies that received emergency use authorization1–3. One potential alternative to avert viral escape is the use of camelid VHHs or nanobodies, which can recognize epitopes often inaccessible to conventional antibodies4. Here, we isolate anti-RBD nanobodies from llamas and “nanomice” we engineered to produce VHHs cloned from alpacas, dromedaries and camels. We identified two sets of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD-ACE2 interface and fails to neutralize variants carrying E484K or N501Y substitutions. Notably however, group 2 nanobodies retain full neutralization activity against variants when expressed as homotrimers, rivaling the most potent antibodies produced to date against SARS-CoV-2. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2 binding domain, and recognition of conserved epitopes largely inaccessible to human antibodies. Therefore, while new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.

    How the ‘Alpha’ Coronavirus Variant Became So Powerful

    A new study suggests how the variant first identified in Britain hides from the human immune system. Its stealth may be part of its success.


    https://www.nytimes.com/2021/0…vid-alpha-uk-variant.html


    In December, British researchers discovered that a new variant was sweeping through their country. When it arrived in other countries, the variant, now known as Alpha, tended to become more common in its new homes as well. By April, it had become the dominant variant in the United States, and it has remained so ever since.


    Alpha’s swift success has left scientists wondering how the variant conquered the world. A new study points to one secret to its success: Alpha disables the first line of immune defense in our bodies, giving the variant more time to multiply.


    “It’s very impressive,” said Dr. Maudry Laurent-Rolle, a physician and virologist at the Yale School of Medicine who was not involved in the new study. “Any successful virus has to get beyond that first defense system. The more successful it is at doing that, the better off the virus is.”


    The report was posted online on Monday and has not yet been published in a scientific journal.


    Alpha has 23 mutations that set it apart from other coronaviruses. When the variant started to surge in Britain, researchers began inspecting these genetic tweaks to look for explanations as to why it was spreading faster than other variants.

    A lot of researchers focused their attention on the nine mutations that alter the so-called spike protein that covers the coronavirus and allows it to invade cells. One of those mutations helps the virus bind more tightly to cells, potentially improving its chances of a successful infection.


    But other scientists have focused on how Alpha affects the human immune response. Gregory Towers, a virologist at the University College London, and his colleagues grew coronaviruses in human lung cells, comparing Alpha-infected cells with those infected with earlier variants of the coronavirus.


    They found that lung cells with Alpha made drastically less interferon, a protein that switches on a host of immune defenses. They also found that in the Alpha cells, the defensive genes normally switched on by interferon were quieter than in cells infected with other variants.

    Somehow, the immune system’s most important alarm bells were barely ringing in the presence of the Alpha variant. “It’s making itself more invisible,” Dr. Towers said.

    To investigate how Alpha achieved this invisibility, the researchers looked at how the coronavirus replicated inside of infected cells. They found that Alpha-infected cells make a lot of extra copies — some 80 times more than other versions of the virus — of a gene called Orf9b.

    It’s off the chart,” said Nevan Krogan, a molecular biologist at the University of California, San Francisco, and a co-author of the new study.


    In previous research, Dr. Krogan and his colleagues had found that Orf9b makes a viral protein that locks onto a human protein called Tom70. And it just so happens that Tom70 is essential for a cell’s release of interferon in the face of an invading virus.


    Putting all of the evidence together, Dr. Krogan and his colleagues argue that the Alpha variant carries a mutation that forces the production of a lot more Orf9b proteins. Those proteins swarm the human Tom70 proteins, dampening the production of interferon and a full immune response. The virus, protected from attack, has better odds of making copies of itself.


    An infected cell can gradually remove the Orf9b proteins from its Tom70 molecules, however. By about 12 hours after infection, the alarm system starts coming back online. And because of that immune response, Dr. Towers said, “all hell breaks loose.”


    Dr. Towers speculated that when the delayed immune response finally happens, people infected with Alpha have a more robust reaction than they would with other variants, coughing and shedding virus-laden mucus from not only their mouths, but also their noses — making Alpha even better at spreading.


    “What they’re showing makes sense,” Dr. Laurent-Rolle said. But she would like to see more lines of evidence in support of their conclusion. For example, the scientists did not run a standard test to measure the number of Orf9b proteins.

    That’s one thing that could be concerning,” she said. Dr. Krogan said he and his colleagues were developing that test now.


    Dr. Krogan’s team has also started similar experiments on other variants, including the variant first identified in South Africa, known as Beta, and the one first identified in India, known as Delta. The preliminary results surprised them.


    Both Beta and Delta drive down interferon in infected cells. But there’s no sign that they do so by flooding the cells with Orf9b proteins. They may have independently evolved their own tricks for manipulating our immune system.


    “They’re all turning down the immune response in different ways,” Dr. Krogan said.


    Cecile King, an immunologist at the Garvan Institute of Medical Research in Sydney, Australia, who was not involved in the study, said that understanding how the virus was evolving these escapes would help scientists design better vaccines for Covid-19.


    The current crop of vaccines direct the immune system to recognize spike proteins. But studies on people who recover naturally from Covid-19 have shown that their immune systems learn to recognize other viral proteins, including Orf9b.


    A number of researchers are putting together combinations of coronavirus proteins into new vaccines. But they need to take caution, because some of the proteins may actually dampen immunity.


    “It’s quite a tricky enterprise, but becoming more possible as we learn more,” Dr. King said.

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    Quote from Navid

    Don't know how these work, but a datapoint of interest.

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    It is always good to wait for commentary from both sides.


    First, the media hype that covid is a gain of function result seam to be based on arguments that is really not that strong as indicated in the recent news. I used this discussion to get the pro and cons of it, redit discussion


    Secondly the new mail publishing was really not as damning for Fauci as the republican news commentary make it. But the effect is that a lot of right wingers now go around angry thinking bad bad Fauci. I am of the view that if you collect a lot of mails from essentially anybody you will with some imagination find damning facts that are really just weakly circumstantial. I've seen it happen for our Swedish lead figure regarding the outbreak, and it looks like it is now happens to Fauci.


    As, to me, classic example of these kind of fact, was the cam recording from one of the US counting rooms and there normal operation could with the right commentary become damning facts. That's what you get with pattern matching imagination and fact fishing expeditions.

    In search of preventive strategies: novel high-CBD Cannabis sativa extracts modulate ACE2 expression in COVID-19 gateway tissues


    https://www.aging-us.com/article/202225/text



    Abstract

    With the current COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need for new therapies and prevention strategies that can help curtail disease spread and reduce mortality. The inhibition of viral entry and thus spread is a plausible therapeutic avenue.


    SARS-CoV-2 uses receptor-mediated entry into a human host via the angiotensin-converting enzyme 2 (ACE2), which is expressed in lung tissue as well as the oral and nasal mucosa, kidney, testes and gastrointestinal tract. The modulation of ACE2 levels in these gateway tissues may be an effective strategy for decreasing disease susceptibility.


    Cannabis sativa, especially those high in the anti-inflammatory cannabinoid cannabidiol (CBD), has been found to alter gene expression and inflammation and harbour anti-cancer and anti-inflammatory properties. However, its effects on ACE2 expression remain unknown.


    Working under a Health Canada research license, we developed over 800 new C. sativa cultivars and hypothesized that high-CBD C. sativa extracts may be used to down-regulate ACE2 expression in target COVID-19 tissues. Using artificial 3D human models of oral, airway and intestinal tissues, we identified 13 high-CBD C. sativa extracts that decrease ACE2 protein levels. Some C. sativa extracts down-regulate serine protease TMPRSS2, another critical protein required for SARS-CoV-2 entry into host cells.


    While our most effective extracts require further large-scale validation, our study is important for future analyses of the effects of medical cannabis on COVID-19. The extracts of our most successful novel high-CBD C. sativa lines, pending further investigation, may become a useful and safe addition to the prevention/treatment of COVID-19 as an adjunct therapy.


    Discussion

    The observed down-regulation of ACE2 gene expression by several tested extracts of new C. sativa cultivars is a novel and crucial finding. Our results establish a foundation for further in-depth analyses of the effects of C. sativa on the molecular etiology and pathogenesis of COVID-19 and other viral diseases wherein the viruses use the ACE2 receptor as a molecular gateway. If these results are further confirmed, these high-CBD cannabis extracts can be used to develop prevention strategies directed at lowering ACE2 levels in high-risk gateway tissues. ACE2 level modulation is of particular importance since it appears to change throughout disease progression, and some studies show that ACE2 is essential for lung function in animal models of SARS [18, 19]. It would also be important to test the effects of C. sativa lines on other receptors involved in SARS-CoV-2 entry for their anti-inflammatory potential.


    Furthermore, cannabis has over 100 phytocannabinoids [20], of which the main ones are Δ9-THC and CBD [21]. Cannabis possesses many minor cannabinoids, such as cannabigerol (CBG) and CBN, and numerous terpenes. Terpenes are responsible for variations in scent and may act synergistically with cannabinoids, with the potential to strongly enhance cannabinoid effects. Terpenes and minor cannabinoids are responsible for the ‘entourage effect’ [21], whereby whole plant extracts have more pronounced biological effects than individual cannabinoids. Here, we did not find a strong correlation between CBD levels and the observed down-regulation of ACE2 and TMPRSS2. Hence, we may indeed be observing the entourage effect in action, and the effects of extracts on ACE2 expression may not necessarily be attributed to CBD. In the future, it would be important to identify the cannabinoids and terpenes responsible for these observed effects, although, based on the entourage effect, one could predict that whole flower extracts may be more potent than single compounds [22, 23]. Here, we noted that the effects of extracts were more pronounced than those of CBD or CBN that alone did not affect ACE2 levels (Figure 6). In the future, it would be important to undertake further studies of other isolate cannabinoids and terpenes and determine their biological effects in well-defined conditions, as well as conduct reconstruction experiment to identify the key active ingredients or combinations.


    Moreover, a notable aspect of our study is that, while the molar amounts and ratios of the major cannabinoids (THC and CBD) were similar between the analyzed extracts (Table 1), not all extracts were equally effective. Some produced undesired molecular effects on the levels of the ACE2 gene and protein. This finding emphasizes that different medical cannabis cultivar may have different effects, and each cannabis cultivar has to be analyzed in detail to identify the ones that are the most potent.


    Another intriguing observation is the potential tissue-specificity of cannabis effects. Interestingly, extract #45 upregulated ACE2 expression in EpiOral tissues, but down-regulated it in the EpiIntestinal and EpiAirway tissues. Overall, tissue specificity of cannabis effects needs to be further explored in detail.


    Within this study, extracts were applied via media to model medical delivery, such as local oral cavity applications, encapsulated extracts and dosed oils, and inhalers or nebulizers. Therefore, our results cannot be extrapolated to the effects of cannabis smoking. Moreover, in light of recent findings showing that tobacco smoking increases ACE2 levels and exacerbates the clinical outcomes of COVID-19 [24], the effects of cannabis smoking on the levels of ACE2 expression should be carefully investigated. Interestingly, animal model studies have shown that CBD decreased inflammation and improved lung function in murine models of acute lung injury [25]. In addition, a recent article suggested that CBD can be a useful addition to current COVID-19 protocols [25].


    Most importantly, seven active cultivars have less than 0.3% total THC and therefore can be classified as hemp CBD in Canada and the USA, allowing for easy implementation.


    However, our study has limitations. Our initial experiments were conducted using two replicates per condition. While it is feasible to conduct a differential expression study with only two replicates, the capability to detect true differentially expressed genes (DEGs) is reduced. Indeed, a recent study by Lamarre et al. (2018) [26] analyzed the impact of the number of replicates on the number of DEGs. For a library size of 15–20 million reads, which is consistent with that of the current study, Lamarre et al. detected 14,000 DEGs. The DEG number increased by about 1,000 with every increase in the number of replicates, and the DEG increase rate slowed down after five replicates were reached. A power analysis presented in the same study showed that, with a sequencing depth of 15–20 million reads, one would expect to detect 80%–90% DEGs with a two-replicate design, depending on the level of expression (low, medium, high). Lamarre et al.’s specificity and sensitivity analysis, which compared four different DEG detection tools (including DESeq2, which was used in the present study), showed a 40%–50% true positive rate (TPR), 0.2%–2% false-positive rate (FPR) and 0.01 FDR [26]. Moreover, Lamarre and colleagues performed their analyses using individual organisms, as opposed to the 3D tissues in our case. We expect the biological variance between 3D tissue samples to be lower compared with separate plants or animals, resulting in a further reduction of the replicate number required to reach the same statistical power [26]. However, we do acknowledge the limited statistical power of our experiment; the addition of replicates would expand the list of DEGs and enhance the ability to detect significant genes at fewer fold changes.


    To substantiate our data we, therefore, conducted additional experiments using EpiAirway, EpiAirway FT, and EpiOral tissues with three replicates per group, and further confirmed the effectiveness of several novel cannabis cultivars in modulation of the ACE2 expression. Nonetheless, future studies are needed to establish the precise mechanisms of action of high-CBD cannabis extracts, their tissue specificity, and the effects on ACE2, given the new knowledge on the tissue and disease stage specificity of ACE 2 expression.


    Our original experiments were designed to screen the biological activities of novel cannabis extracts in 3D human tissue models. They allowed us to pinpoint the important effects of cannabis on the levels of ACE2 and TMPRSS2 expression. Further studies are needed and being undertaken to link the levels of ACE2 and inflammation. Indeed, higher ACE2 expression after SARS-CoV-2 infection is correlated with a cytokine surge in patients [8], which is thought to occur via activation of the NLRP3 inflammasome [27]. ACE2 levels are affected by age [14], and ACE2 blockers have an anti-aging potential [28]. Furthermore, linking ACE2 levels, inflammation and aging is extremely important, as aging is an inflammation-related condition. Inflamm-aging, a certain level of chronic inflammation that develops with advanced age, increases the rate of biological aging and underlies numerous age-related diseases [29]. Inflamm-aging and macroph-aging, a related phenomenon that involves senescence-related changes in macrophages, and their contributions to COVID-19 need to be further investigated [30].

    Scientists Discover Exactly How COVID-19 Wreaks Havoc on Human Lungs


    https://scitechdaily.com/scien…havoc-on-human-lungs/amp/


    Scientists at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory have published the first detailed atomic-level model of the SARS-CoV-2 “envelope” protein bound to a human protein essential for maintaining the lining of the lungs. The model showing how the two proteins interact, just published in the journal Nature Communications, helps explain how the virus could cause extensive lung damage and escape the lungs to infect other organs in especially vulnerable COVID-19 patients. The findings may speed the search for drugs to block the most severe effects of the disease.


    “By obtaining atomic-level details of the protein interactions we can explain why the damage occurs, and search for inhibitors that can specifically block these interactions,” said study lead author Qun Liu, a structural biologist at Brookhaven Lab. “If we can find inhibitors, then the virus won’t cause nearly as much damage. That may give people with compromised health a much better chance for their immune systems to fight the virus successfully.”

    Scientists discovered the details and developed the molecular model using one of the new cryo-electron microscopes at Brookhaven Lab’s Laboratory for BioMolecular Structure (LBMS), a new research facility built with funding from New York State adjacent to Brookhaven’s National Synchrotron Light Source II (NSLS-II).


    “LBMS opened last summer ahead of schedule because of its importance in the battle against COVID-19,” said Sean McSweeney, director of LBMS and a coauthor on the paper. “LBMS and NSLS-II offer complementary protein-imaging techniques and both are playing important roles in deciphering the details of proteins involved in COVID-19. This is the first paper published based on results from the new facility.”


    Liguo Wang, scientific operations director of LBMS and another coauthor on the paper, explained that “cryo-electron microscopy (cryo-EM) is particularly useful for studying membrane proteins and dynamic protein complexes, which can be difficult to crystallize for protein crystallography, another common technique for studying protein structures. With this technique we created a 3-D map from which we could see how the individual protein components fit together.”


    “Without cryo-EM, we couldn’t have gotten a structure to capture the dynamic interactions between these proteins,” Liu said.


    Triggering lung disruption

    The SARS-CoV-2 envelope protein (E), which is found on the virus’s outer membrane alongside the now-infamous coronavirus spike protein, helps to assemble new virus particles inside infected cells. Studies published early in the COVID-19 pandemic showed that it also plays a crucial role in hijacking human proteins to facilitate virus release and transmission. Scientists hypothesize that it does this by binding to human cell-junction proteins, pulling them away from their usual job of keeping the junctions between lung cells tightly sealed.

    That interaction can be good for the virus, and very bad for humans–especially elderly COVID-19 patients and those with pre-existing medical conditions,” Liu said.

    When lung cell junctions are disrupted, immune cells come in to try to fix the damage, releasing small proteins called cytokines. This immune response can make matters worse by triggering massive inflammation, causing a so-called “cytokine storm” and subsequent acute respiratory distress syndrome.


    Also, because the damage weakens the cell-cell connections, it might make it easier for the viruses to escape from the lungs and travel through the bloodstream to infect other organs, including the liver, kidneys, and blood vessels.


    “In this scenario, most damage would occur in patients with more viruses and more E proteins being produced,” Liu said. And this could become a vicious cycle: More viruses making more E proteins and more cell-junction proteins being pulled out, causing more damage, more transmission, and more viruses again. Plus, any existing damage, such as lung-cell scarring, would likely make it harder for COVID patients to recover from the damage.


    “That’s why we wanted to study this interaction–to understand the atomic-level details of how E interacts with one of these human proteins to learn how to interrupt the interactions and reduce or block these severe effects,” Liu said.


    From specks to blobs to map to model

    The scientists obtained atomic-level details of the interaction between E and a human lung-cell-junction protein called PALS1 by mixing the two proteins together, freezing the sample rapidly, and then studying the frozen sample with the cryo-EM. The electron microscopes use high-energy electrons to interact with the sample in much the same way that regular light microscopes use beams of light. But electrons allow scientists to see things at a much smaller scale due to their extremely short wavelength (100,000 times shorter than that of visible light).


    The first images didn’t look like much more than specks. But image-processing techniques allowed the team to select specks that were actual complexes of the two proteins.

    We used two-dimensional averaging and started to see some structural features that are shared among these particles. Our images showed the complex from different orientations but at fairly low resolution,” Liu said. “Then we use computational tools and computation infrastructure at Brookhaven’s Computational Science Initiative to perform three-dimensional reconstructions. These give us a 3-D model–an experimental map of the structure.”


    With an overall resolution of 3.65 Angstroms (the size of just a few atoms), the map had enough information about the unique characteristics of the individual amino acids that make up the two proteins for the scientists to fit the known structures of those amino acids into the map.


    “We can see how the chain of amino acids that makes up the PALS1 protein folds to form three structural components, or domains, and how the much smaller chain of amino acids that makes up the E protein fits in a hydrophobic pocket between two of those domains,” Liu said.


    The model provides both the structural details and an understanding of the intermolecular forces that allow E proteins deep within an infected cell to wrench PALS1 from its place at the cell’s outer boundary.


    “Now we can explain how the interactions pull PALS1 from the human lung-cell junction and contribute to the damage,” Liu said.


    Implications for drugs and evolution

    “This structure provides the foundation for our computational science colleagues to run docking studies and molecular dynamics simulations to search for drugs or drug-like molecules that might block the interaction,” said John Shanklin, chair of Brookhaven Lab’s Biology Department and a coauthor on the paper. “And if they identify promising leads, we have the analytical capabilities to rapidly screen through such candidate drugs to identify ones that might be key to preventing severe consequences of COVID-19.”


    Understanding the dynamics of this protein interaction will also help scientists track how viruses like SARS-CoV-2 evolve.


    “When the virus protein pulls PALS1 out of the cell junction, it could help the virus spread more easily. That would provide a selective advantage for the virus. Any traits that increase the survival, spread, or release of the virus are likely to be retained,” Liu said.


    The longer the virus continues to circulate, the more chances there are for new evolutionary advantages to arise.


    “This is one more reason it is so essential for us to identify and implement promising therapeutics,” Liu said. “In addition to preventing the most severe infections, drugs that effectively treat COVID-19 will keep us ahead of these mutations.”


    Reference: 8 June 2021, Nature Communications.

    DOI: 10.1038/s41467-021-23533-x


    This research was funded by Brookhaven National Laboratory’s COVID-19 Laboratory Directed Research and Development (LDRD) fund. LBMS is supported by the DOE Office of Science (BER), NSLS-II is a DOE Office of Science user facility, supported by the Office of Science (BES).

    Mysterious Brain Disease Spreading in Canada; a Side Effect of Covid-19 Vaccine?


    https://amp.ibtimes.sg/mysteri…ct-covid-19-vaccine-57982


    Doctors in Canada are baffled by the death of six persons and infections in 48 others caused by an unknown neurological disease. New Brunswick Health Minister Dorothy Shephard said on Thursday that an expert committee will lead the ongoing investigation to find the cause of the deadly and mysterious brain disease.


    "The discovery of a potentially new and unknown syndrome is scary," Shephard said during a media conference further adding, "I know that New Brunswickers are concerned and confused about this potential neurological syndrome."


    The public health department in the province investigating the cases has observed that 48 people were struck by a mix of symptoms including insomnia, impaired motor function, and hallucinations, such as visions of the dead.

    Brain Disease Conspiracy Theories

    As doctors and neurologists are redoubling their efforts to decipher the clues for the unknown disease, there are new conspiracy theories blaming the infection in human brains on cellphone network towers, fracking, and even the hurriedly-developed Covid-19 vaccines.


    These are some features of the mystery that have stumped Canada's medical establishment and attracted the attention of the world's top neurologists. Many residents of the New Brunswick province on picturesque Canada's Atlantic coast have fallen ill due to the disease in the past six years.


    Unknown Brain Disease Linked to Geography?

    The first case of the disease dates back to 2015. But the news of the unknown syndrome came to light on March 5 as the province's deputy chief medical officer of health, Dr. Cristin Muecke, sent a memo to various medical associations in New Brunswick mentioning the unknown disease.

    Reportedly, most of the existing patients at the time were living in and around the Moncton, N.B., area and the Acadian Peninsula in the northeastern part of New Brunswick. However, it is unclear if the syndrome is linked to geography as there isn't any hard evidence.


    Who is Gabrielle Cormier?

    Gabrielle Cormier is one of the youngest patients out of the 48 infected by the mysterious disease. She has been afflicted with extreme fatigue, involuntary jerking movements, memory lapses, and hallucinations due to the brain disease.

    More evidence for the liars and deniers and vaccine warriors


    Ivermectin and outcomes from Covid-19 pneumonia: A systematic review and meta-analysis of randomized clinical trial studies


    https://onlinelibrary.wiley.com/doi/full/10.1002/rmv.2265


    Summary

    Ivermectin is an FDA-approved drug for a parasitic disease that has broad antiviral activity. This study aims to analyse the efficacy of ivermectin in improving the Covid-19 outcomes. We systematically searched the PubMed, Europe PMC and ClinicalTrials.gov database using specific keywords related to our aims until 10th May 2021. All published randomized clinical trial studies on Covid-19 and ivermectin were retrieved. The quality of the study was assessed using Jadad scale assessment tool for clinical trial studies. Statistical analysis was done using Review Manager 5.4 software. A total of 19 studies with 2768 Covid-19 patients were included in this meta-analysis. This meta-analysis showed that ivermectin was associated with reduction in severity of Covid-19 (RR 0.43 [95% CI 0.23–0.81], p = 0.008), reduction of mortality (RR 0.31 [95% CI 0.15–0.62], p = 0.001), higher negative RT-PCR test results rate (RR 1.23 [95% CI 1.01–1.51], p = 0.04), shorter time to negative RT-PCR test results (mean difference [MD] −3.29 [95% CI −5.69, −0.89], p = 0.007), higher symptoms alleviations rate (RR 1.23 [95% CI 1.03−1.46], p = 0.02), shorter time to symptoms alleviations (MD −0.68 [95% CI −1.07, −0.29], p = 0.0007) and shorter time to hospital discharge (MD −2.66 [95% CI −4.49, −0.82], p = 0.004). Our study suggests that ivermectin may offer beneficial effects towards Covid-19 outcomes. More randomized clinical trial studies are still needed to confirm the results of our study.


    DISCUSSION

    According to our pooled analysis, ivermectin was discovered to have an association with a higher negative RT-PCR test results rate, shorter time to negative RT-PCR test results, higher symptoms alleviations rate, shorter time to symptoms alleviations, shorter time to hospital discharge and reduction in the severity and mortality from Covid-19. Our subgroup analysis also showed that the benefits of ivermectin therapy in reducing the severity and mortality outcomes from Covid-19 were more prominent when administered into mild to moderate patients, compared with in severe patients. On the other side, the benefits of ivermectin therapy in increasing negative RT-PCR test results rate, shortening time to negative RT-PCR test results, increasing symptoms alleviations rate and shortening time to symptoms alleviations were more apparent in severe patients when compared with in mild to moderate patients.


    A few arguments might explain these findings. The sequestration of the SARS-CoV-2 viral nucleocapsid protein (NCP) into the host nucleus through the nuclear-pore-complex is a vital step in viral pathogenesis and defence against host immune response.48 Ivermectin can selectively inhibit the host importin α/β transporter protein which decreases translocation (shuttling) of SARS-CoV-2 NCP from the cytoplasm to the nucleus, alteration of NCP distribution will lead to viral propagation disruption and survival.23 The in vitro study by Caly et al.23 has proved that giving ivermectin in one dose was able to reduce the viral RNA load by 99.98% at 48 h and replication of an Australian isolate of SARS-CoV-2 in Vero/hSLAM cells by 5000-folds. Therefore, it has potent effects in altering disease progression and spread. These in vitro findings were further supported with the results from a double-blind, placebo-controlled, randomized clinical trial study, showing that patients who received ivermectin 400 μg/kg single dose have a lower median viral load at Day 4 (161,000 vs. 493,500 copies/ml) and Day 7 post-treatment (1018 vs. 23,550 copies/ml). The differences were found, rising from a threefold decrease on the fourth day to about 18-fold lower on the seventh day when compared with placebo.49 Second, the pathophysiologic process which underlies severe Covid-19 involves hyperinflammatory response and accumulation of cytokines, called a cytokine storm. A meta-analysis study has demonstrated that severe Covid-19 patients tend to produce higher cytokine levels such as interleukin-6 (IL-6), IL-8, IL-10 and tumour necrosis factor-α (TNF-α), in comparison to non-severe cases.50 On the other side, an anti-inflammatory effect was also demonstrated in ivermectin, both in vivo and in vitro studies. Ivermectin can reduce the IL-1, IL-6, TNF-α production and suppressing lipopolysaccharide-induced nuclear factor-kappa B translocation.51 The suppression of mucus due to hypersecretion in the respiratory tract, the reduction of immune cell recruitment, and a decrease in the production of cytokines and immunoglobulin E/immunoglobulin G1 in bronchoalveolar lavage of experimental mice, were found as a consequence of 2 mg/kg of ivermectin administration.52 These findings suggest that ivermectin has an anti-inflammatory effect on the lung tissue, besides at the systemic level, which might help to reduce the severity and prevent mortality from Covid-19.


    This study has several limitations. First, significant heterogeneities were found on most of the outcomes of interests included in this study. This was probably caused by the difference in the given ivermectin doses and the medications used as a standard of care or placebo. Second, the total number of patients included in this study was relatively small because at this time, ivermectin is still considered as new repurposed drug for Covid-19 where early trials still show conflicting results and there is still no meta-analysis study to support its efficacy, therefore it may be difficult to collect the participants and receiving their consent to participate in the trials. Third, we include some pre-print studies to minimize the risk of publication bias; however, the authors have made exhaustive efforts to ensure that only sound studies were included, and we expect that most of those studies currently available in pre-print form will eventually be published and that we will identify them through ongoing electronic literature surveillances. We hope that this study can give further insight into the management of Covid-19 patients.


    5 CONCLUSION

    Our meta-analysis of randomized clinical trial studies indicates that ivermectin administration had an association with favourable outcomes of Covid-19, compromising of higher rate of negative RT-PCR test results, shorter time to negative RT-PCR test results, higher rate of symptoms alleviations, shorter time to symptoms alleviations, shorter time to hospital discharge, reduction in the severity and mortality rate from Covid-19. This study suggests that ivermectin may be the potential therapeutic agents for the managements of Covid-19 to give better outcomes for the patients. However, more randomized clinical trial studies are still necessary and encouraged to be done for confirming the results of our study. Finally, ivermectin should be considered as an essential drug for future Covid-19 therapy models.

    Quote from stefan

    First, the media hype that covid is a gain of function result seam to be based on arguments that is really not that strong as indicated in the recent news.

    CoV-19 is the result of gain of research. Almost all experts from gen tech labs agree with this. There is no explanation possible following a natural evolution as some intermediate steps would never form/replicate/spread in the known hosts.


    Quote from Fm1

    Many residents of the New Brunswick province on picturesque Canada's Atlantic coast have fallen ill due to the disease in the past six years.

    Normally this is food related. Fungus/bacteria on anything you yet. This is very old news.

    I would like a commentary about what Thuxleynew says about publication bias, else this is interesting. I'm after arguments and counter arguments and found this link that discusses why India is recovering and is against the hypothesis that it is due to medication. I do not see that they are dishonest, they seam to be open about it and think that there is not enough proof yet. Well see, the study you linked seam to indicate that there is an effect. Actually maybe an even better strategy than taking Ivermectin, is to go vegan, a study taken up by the news here say that vegans have 75% less risk of getting a tough covid. But sure as hell this is a correlation and no proof, maybe vegans care about health more then other people, doing proper social distancing, masks etc and get a small dose of covid when they catch it.

    Cascading from SARS-CoV-2 to Parkinson’s Disease through Protein-Protein Interactions


    https://www.mdpi.com/1999-4915/13/5/897/htm


    Abstract

    Extensive extrapulmonary damages in a dozen of organs/systems, including the central nervous system (CNS), are reported in patients of the coronavirus disease 2019 (COVID-19). Three cases of Parkinson’s disease (PD) have been reported as a direct consequence of COVID-19. In spite of the scarce data for establishing a definitive link between COVID-19 and PD, some hypotheses have been proposed to explain the cases reported. They, however, do not fit well with the clinical findings reported for COVID-19 patients, in general, and for the PD cases reported, in particular. Given the importance of this potential connection, we present here a molecular-level mechanistic hypothesis that explains well these findings and will serve to explore the potential CNS damage in COVID-19 patients. The model explaining the cascade effects from COVID-19 to CNS is developed by using bioinformatic tools. It includes the post-translational modification of host proteins in the lungs by viral proteins, the transport of modified host proteins via exosomes out the lungs, and the disruption of protein-protein interaction in the CNS by these modified host proteins. Our hypothesis is supported by finding 44 proteins significantly expressed in the CNS which are associated with PD and whose interactions can be perturbed by 24 host proteins significantly expressed in the lungs. These 24 perturbators are found to interact with viral proteins and to form part of the cargoes of exosomes in human tissues. The joint set of perturbators and PD-vulnerable proteins form a tightly connected network with significantly more connections than expected by selecting a random cluster of proteins of similar size from the human proteome. The molecular-level mechanistic hypothesis presented here provides several routes for the cascading of effects from the lungs of COVID-19 patients to PD. In particular, the disruption of autophagy/ubiquitination processes appears as an important mechanism that triggers the generation of large amounts of exosomes containing perturbators in their cargo, which would insult several PD-vulnerable proteins, potentially triggering Parkinsonism in COVID-19 patients.

    Keywords: COVID-19; Parkinson’s disease; protein–protein interactions; exosomes; molecular mechanisms; post-translational modifications


    Discussion

    Among the VPs, Rab7A and NUP62 (p62) are the ones having the largest number of potentially perturbed interactions. These proteins interact with four perturbators each. In the case of Rab7A, the perturbators are Rab1A, Rab8A, Vps11, and Vps39. Rab7A is a relatively small protein (208 amino acids) which is mainly found in late endosomes and which has been recognized as the only lysosomal Rab protein in the Rab GTPase superfamily [83]. It increases the degradation of α-syn aggregates whose presence in the brain is one of the main features of PD [83,84]. Rab7A reduces the proportion of cells with α-syn particles, as well as the amount and toxicity of α-syn. Therefore, the overexpression of Rab7A has been found beneficial on PD [83,84]. The mechanism by which Rab7A drops α-syn in cells is mediated by autophagy, and requires the autophagosome maturation, which consists on the fusion of autophagosomes and lysosomes to form autolysosomes. The last step allows autophogosomes to obtain hydrolytic enzymes indispensable for subsequent autophagic degradation. In fact, Rab7A is involved in governing several cellular processes, such as early-to-late endosome transition, biogenesis of lysosomes, transport of autophagosomes to endosomes/lysosomes, and vacuole formation. A crucial step in the early-to-late endosome maturation is the Rab5-Rab7 switch which is mediated by the complex Mon1-Ccz1. This complex interacts with Vps18 and Vps11 to recruit and activate Rab7A on early endosomes, which allows the conversion to late endosomes. As it is known, Vps11 interacts with SARS-CoV-2 protein Orf3A, which may modify it as to avoids the Vps11-Rab7A interaction and so impeding the “cleaning” mechanism of the last on α-syn in brain cells. In a recent study, Miao et al. [85] have found that Orf3a does not interact with any of the autophagy proteins. However, they proved that autophagy is inhibited in SARS-CoV-2 infected cells. Their findings indicate that such inhibition occurs through the interaction of Orf3a with the HOPS complex, consisting of Vps11, Vps16, Vps18, and Vps33A, and the HOPS-specific subunits Vps39 and Vps41. In particular, they reported the sequestration of Vps39 by Orf3a causing accumulation of the HOPS protein on late endosomes/lysosomes. Both proteins, Vps11 and Vps39, which are found here as perturbators of PD VPs, may result in the inhibition of autophagy, thus resulting in PD. Other potential mechanism also exist, such as via the inhibition of the biogenesis of the phagophore, which depends on the functionality of Rab1a (see Reference [83] and refs. therein). We should remark here that macroautophagy is cross-linked with exosome biogenesis [86]. For instance, when autophagy or lysosomal misfunction prevent degradation of protein aggregates there is an increase in exosome release to alleviate proteoxomic stress [86]. This explain our mechanism of cascade from viral proteins to CNS effects via exosome-mediated transport of perturbators. It is worth mentioning here that the autophagy-exosome crosstalk has been found to play a role in neurodegenerative diseases like PD.

    Let us now return to the example provided in Figure 1 which involved the interaction of the viral nucleocapsid N protein with host G3BP1 and the interaction of this with NUP62. We will now explain this mechanism at the molecular level with the help of Figure 4. First, we should remark that SARS-CoV-2, similarly to other virus, inhibits autophagy in human host cells [85,87,88,89]. This is schematically represented in Figure 41a. It has been shown by Hyun et al. [90] that this inhibition causes accumulation of NUP62 and protein aggregates (Figure 41b), with a subsequent delayed migration of ubiquitinated substrates to the proteasomes (Figure 41c). NUP62 is a 520-amino acids protein associated with the nuclear envelope, which is found here to be perturbed by NUP88, NUTF2, RAE1, and G3BP1. We should notice that the interaction NUP62-G3BP1 was not among those reported in the database STRING (see Methods), but it is reported in a recent work by Anasimov et al. in 2019 [91]. These four perturbators interact with SARS-CoV-2 proteins Nsp9, Nsp15, Orf6, and N, respectively [65]. The protein NUP62 is involved in the formation of autophagosomes and its defective function or altered modulation is known to be associated with the pathogenesis of PD [90,91,92,93,94]. Indeed, NUP62 is known to induce protein aggregation and aggresome formation, which, in the case of aggregate formation of α-syn, produces PD.

    On the other hand, Nabeel-Shah et al. [95] have reported that SARS-CoV-2 N protein sequesters G3BP1 and G3BP2 through its strong physical interaction with these proteins, which attenuate stress granule (SG) formation (Figure 42a). It is known that N protein is a multifunctional protein which is involved in many aspects of viral life cycle [96,97,98]. Anisonov et al. [91] have found that G3BP1 (Ras GTPase-activating protein binding site 1) inhibits ubiquitinated protein aggregations induced by NUP62 (Figure 42b). That is, G3BP1 is a negative regulator of protein aggregation, such that depletion of G3BP1 stimulates NUP62-induced aggregation of α-syn and aggresome formation (Figure 42c). It is important to remark here that ubiquitination plays a role, together with the inibition of the autophagy, in the selective incorporation of exosomal proteins, which may explain the formation and excessive liberation of exosomes containing perturbator cargoes as hypothesized in this work (Figure 41d). The mechanism is completed by the G3BP2 attenuation of G3BP1 inhibitory effect by competing with the G3BP1-NUP62 interaction (Figure 42b). Then, because N protein targets G3BP1 and G3BP2 for SG formation, by means of strong PPI, the ubiquitinated oligomers are not complexed into aggregates (formed anyway due to autophagy inhibition). These ubiquitinated oligomers are known to be toxic to the cells [91].

    Therefore, both pathways may end up in PD. The inhibition of autophagy by the formation of ubiquitinated protein aggregates and the excessive formation of exosomes which may contain perturbators, and the sequestration of G3BP1 and G3BP2 by the viral N protein may also triggers NUP62-induced aggregation of α-syn. It should be remarked that G3BP1 is also a perturbator for the PD-VPs USP10 [91], GiGYF2 [99], EIF4G1 [100], and TIAL1 [101]. The first protein (USP10), which promotes protein aggregation and aggresome formation, interacts with NUP62 increasing NUP62-induced protein aggregation [91]. Anisonov et al. [91] also found that USP10 is inhibited by G3BP1 in a mechanism similar to that for NUP62, which indicates an analogous cascading route from SARS-CoV-2 to PD for this protein. For the other proteins, it is possible that other mechanisms are involved. For instance, a mutation situated in the GYF domain of GIGYF2 which disrupt the ligand-binding abilities of this domain, has been found to be associated with PD [99]. In the case of EIF4G1, it was linked to PD in a recent study after some conflicting results on its role in familial Parkinsonism [100]. Finally, TIAL1 has been found with significant alterations in motor cortex of postmortem brain donors with PD [101].

    The most abundant PTM that viral proteins can induce in host proteins is their phosphorylation. Bouhaddou et al. [66] have reported that 40 out of the 332 host proteins interacting with SARS-CoV-2 are significantly differentially phosphorylated upon infection by SARS-CoV-2 (for statistical criteria see Bouhaddou et al. [66]). Among these 40 significantly differentially phosphorylated host proteins, there are 6 identified here as perturbators of PD-VPs. They are: MARK2, HDAC2, LARP7, PRKACA, PRKAR2A, and PRKAR2B. In total, these proteins are found to perturb 10 PD-VPs, namely: BRAF, EZH2, FOX01, LIN28A, MAP2, MAPT, MTA1, PRKACA, PRKACB, and PRKAR1B. From these genes, PRKACB is perturbed by three proteins: PRKACA, PRKAR2A, and PRKAR2B, which interact with the viral proteins Nsp13, Nsp14, and Nsp15. PRKACB is a member of the serine/threonine protein kinase family known to mediate signaling via cyclic adenosine monophosphate, which has been found to be downregulated in patients with PD [102]. Therefore, it is plausible that the differential phosphorylation of the perturbators of this protein, PRKACA, PRKAR2A, and PRKAR2B, produces its downregulation and may trigger Parkinsonism. Notice that one of these perturbators, PRKACA, which is significantly expressed in both the lungs and the CNS, has also been reported as potentially implicated in PD by modulating MAPK and insulin signaling pathways [103]. It is perturbed by two perturbators: PRKAR2A, PRKAR2B. The rest of the PD-VPs mentioned before are perturbed by only one perturbator. Several of these PD-VPs, apart from PRKACB, have been reported to be downregulated or having lost their functions in PD. These are the cases of FOXO1 [104], LIN28A [105], and MAP2 [106]. Thus, it is plausible that they are affected by the significant PTM of their perturbators, as in the case of PRKACB. The protein EZH2 [107] has been found to be involved in proteosomal degradation of α-syn and alteration on its levels has been implicated in PD, possibly due to similar reasons as for the case of NUP62. The implication of MAPT [108,109,110], which is known as tau protein, is well known for its implication in Alzheimer disease, but it has also been found associated with PD. MTA1 is an upstream modulator of tyrosine hydroxylase and has been found to play a significant role in PD pathogenesis [111]. Finally, BRAF is known to play a role in neuronal survival and maturation. Its connection with PD has been linked through its interaction with RIT2, which is a PD risk factor in Asian and Caucasian cohorts [112]. The remaining 27 PD-VPs are perturbed by only one perturbator each.

    Finally, we would like briefly to consider the role played by some of the most important perturbators found here. There are three perturbators, G3BP1, OS9, and SCCPDH, which perturb 5 PD-VPs each. We have previously considered G3BP1, so that we focus now on OS9 and SCCPDH. Osteosarcome 9 (OS9) is a component of the endoplasmic reticulum (ER)-associated degradation (ERAD) machinery [113]. The main function of ERAD is to retrotranslocate to the cytoplasm the unfolded or malfolded proteins which are retained in the ER, such that they can be degraded by the proteosome. In particular, OS9 is responsible for the recognition of unfolded proteins to which it binds to. Failing such recognition will leave unfolded/malfolded proteins in the ER which may expose their hydrophobic amino acids, increasing their tendency to form protein aggregates. Therefore, there are many reports linking ER stress and neurodegenerative diseases, such as PD (see Reference [113] and refs. therein). OS9 forms a complex with SARS-CoV-2 Orf8 protein [65]. This somehow enigmatic 121-amino acids protein has a relatively high number of disulfide bridges, which points out to its residence in the ER [114]. Thus, this joint habitability of Orf8 and OS9 in the ER may increase the chances of modification of the host protein impeding the realization of its important function. This PTM of OS9 may also modify its interaction with AMFR [115], P4HB [116], SEL1L [117], SYVN1 [117], and VCP [118], which are found here as PD-VPs. For instance, SEL1L [117] has been suggested to be the ERAD tuning receptor, which selectively captures OS9–by means of a PPI–for constitutive clearance from the ER. Thus, there are many routes linking OS9 disruption by SARS-CoV-2 with PD. The other major perturbator of PD-VPs, SCCPDH, is targeted by viral Nsp7 [65]. There is not much information about the possible role that this host protein could have in PD. However, we have found that 3 of the five PD-VPs that interact with it appear upregulated/significantly-increased in blood (APP [119] and SERPINE1 [120]) or CSF (TGFB1 [121]) of PD patients. The other two PD-VPs are neuroprotective for PD (HGF [122] and TIMP1 [123]). Therefore, we may suggest two potential alternative mechanisms involving the interactions of SCCPDH with PD-VPs. The two perturbators interacting with 4 PD-VPs each, PRKACA and VPS39, were already analyzed here. Other possible mechanistic links can be extracted from the rest of perturbators and their PD-VPs, which, in general, can serve for proposing and validating research hypothesis related to the potential impact of COVID-19 on CNS, in general, and with Parkinsonism, in particular.

    6. Conclusions

    Here, we provide a mechanism explaining how the damages produced by the interactions of SARS-CoV-2 proteins with human proteins expressed in the lungs can cascade to affect proteins mainly expressed in the CNS, which trigger the development of PD. This mechanism is based on the modern paradigm stating that diseases are mainly produced by the disruption of PPI networks. In this case, we consider that some proteins expressed significantly in the lungs can be post-translationally modified by SARS-CoV-2 proteins. These proteins can act as perturbators of PD-VPs if they can be encapsulated in exosomes which then navigate up to the CSN. We have identified 24 perturbators, damaged in the lungs by SARS-CoV-2 proteins, which may disrupt the normal functioning of 44 PD-VPs triggering Parkinsonism. Several mechanisms are devised here for the cascading of effects from the lungs to PD. We have found that interfering with autophagy/ubiquitination processes triggers the generation of large amounts of exosomes containing perturbators in their cargoes, which would insult several PD-VPs. These findings offer a great opportunity for testing several hypothesis about the potential CNS damage of SARS-CoV-2 and possibly other virus, as well as to confirm some mechanisms involving new potential biomarkers for PD.

    Quote from Fm1

    Why did you take the vaccine ? Short term rct is now in question and no long term rct for vaccine. Seems your logic comes back to bite you

    I ahve no idea what you are talking about.


    The real world UK data from areas affected by the now prevalent Delta variant (which is increasing 70%/week) show the vaccine (2 shots) reduces hospitalisation and death by a factor of 10 or more.


    The delta variant is some 40% more deadly than the alpha variant, which itself is more deadly than original. so we are talking about maybe 2% overall - and at my age etc that probably means around 2%, also.


    In addition long COVID seems to have a rate 5% more than the death rate.


    So we have, if a catch COVID which given the anticipated rates later this year in UK and the high transmissability of Delta seems quite likely:


    death 2%, long COVID 10% (without vaccine).

    death 0.2%, long COVID 1%. (with vaccine).


    The figures for vaccine effectiveness against Delta are not yet accurate - I hope reduction in risk is more than 10X.


    But it is clearly large because in areas with high Delta infection the hospitalisation rate remains low, and nearly all of those (95%) have not been double vaccinated. Given that > 50% of population, and >>50% of the older population, have been double vaccinated you can see from this very clearly that the vaccine is a life saver.


    FM1 - have a misunderstood you? I really don't understand what you are saying...

    Yes, you misunderstood. You were referring to RCT from treatments and said without, you wouldn't take one, so without long term RCT, you took the vaccine, just an observation.

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