CRISPR screen identifies clinically approved immunosuppressants that could treat coronavirus infections

Researchers in Switzerland and Germany have identified host cell factors required for coronavirus replication that could serve as targets for treatment with clinically-approved drugs.

The team found that several autophagy-related genes were common host defense factors required for the replication of both endemic and emerging coronaviruses.

These coronaviruses include the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic.

Autophagy – cellular response to stressors such as hypoxia or infection – involves the recycling of proteins and organelles to maintain homeostasis. Various trafficking pathways enable the transportation of cytoplasmic material to the lysosome, where it is destroyed.

Among the autophagy-related genes were three immunophilins – high affinity-receptor proteins that specifically bind to certain immunosuppressive agents.

Furthermore, inhibition of the immunophilins with the clinically-approved drugs Cyclosporin A and Alisporivir resulted in dose-dependent reduction of coronavirus replication in primary human nasal epithelial cells.

The study was conducted by a team from the Institute of Virology and Immunology in Bern and Mittelhäusern, Switzerland and Ruhr-Universität Bochum in Germany

“Overall, we identified host factors that are crucial for coronavirus replication and demonstrate that these factors constitute potential targets for therapeutic intervention by clinically approved drugs,” writes Volker Thiel and the team.

A pre-print version of the paper is available on the bioRxiv* server, while the article undergoes peer review.

Study: A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets. Image Credit: Meletios Verras / Shutterstock

Three highly pathogenic coronaviruses have emerged over the last two decades

The last two decades have seen the emergence of three highly pathogenic coronaviruses, including the SARS-CoV virus responsible for the 2002-2004 SARS outbreaks, the Middle Eastern respiratory syndrome coronavirus (MERS-CoV) that emerged in 2012 and, most recently, the SARS-CoV-2 virus that causes COVID-19.

The severe risk these outbreaks have posed to human health over a relatively short period has highlighted the importance of developing effective approaches to treating both current coronavirus infections and those that could emerge in the future.

Coronaviruses rely on host dependency factors

Coronaviruses rely on cellular host factors – termed host dependency factors (HDFs) – for viral entry, replication and survival.

“The identification of HDFs is therefore important for understanding essential host-virus interactions required for successful viral replication and providing a framework to guide the development of new pharmacological strategies for the treatment of coronavirus infections,” says Thiel and colleagues.

One hallmark process that occurs during coronavirus replication is extensive virus-induced remodeling of host endomembranes to form double-membrane vesicles (DMVs) that are targeted by viral replication and transcription complexes.

“However, the host factors that are required for the formation of these structures remain elusive,” says the team.

What did the researchers do?

The researchers conducted two independent genome-wide loss-of-function CRISPR screens to identify HDFs required for the replication of both endemic and emerging coronaviruses.

The knockout screens were performed in Huh7 cells infected with the highly pathogenic MERS-CoV and with human coronavirus 229E (HCoV-229E) – a less pathogenic endemic coronavirus that generally only causes mild respiratory symptoms.

Enrichment analysis uncovers host biological networks crucial for CoV replication. (A) Enrichment map summarizing major host biological networks co-opted by CoVs during infection. Gene Ontology (GO) enrichment analysis was performed using hits from both MERS-CoV and HCoV-229E CRISPR screens and filtered to contain conserved representative GO terms and genes. Each node represents an individual GO term and nodes that are functionally related cluster together into a larger network. Node size reflects number of significantly enriched genes in the node and color indicates the CoV screen for which the node was significant.

What did the study find?

The team identified multiple virus-specific and conserved HDFs, including several that are required for replication of SARS-CoV-2.

The study revealed that several autophagy-related genes, including the immunophilins FK506 binding protein 8 (FKBP8), transmembrane protein 41B (TMEM41B), and membrane integral NOTCH2-associated receptor 1 (MINAR1) were common HDFs.

The researchers say that the interaction between autophagy components and coronaviruses in the context of replication has been considered for some time because parts of the autophagy process share similarities with the process of DMV formation.

However, “studies investigating the possible involvement of the early autophagy machinery in the conversion of host membranes into DMVs reached conflicting conclusions,” says Thiel and colleagues.

“Another possibility is that single components of the autophagic machinery may be hijacked by coronaviruses independently of their activity in autophagic processing,” they add.

The team says that irrespective of the precise underlying mechanism, the results suggest that FKBP8, TMEM41B, and MINAR1 represent potential therapeutic targets.

CoV HDFs are interactors of the autophagy pathway but do not depend on autophagy for replication. (A) Upon starvation, the mTORC1 complex is blocked and activation of the PI3K complex, as well as the ULK1 complex leads to the initiation of phagophore formation, as an initial step in the autophagy pathway. MERS-CoV and HCoV503 229E top scoring CRISPR knockout screen hits FKBP8, MINAR1, TMEM41B and VMP1 are involved in this early pathway. Furthermore, the ATG8 system containing among others LC3, which is recruited by VPM1 or FBKP8 is necessary for targeting cellular cargo to the autophagosome. PPP3R1 is upregulated and initiates TFEB translocalization to the nucleus, where it catalyzes transcription of ATGs. MERS-CoV or conserved host dependency factors (HDFs) are indicated in respective colors. Inhibitor intervention in this pathway is shown in red.

Targeting the immunophilins with clinically-approved drugs

Next, the researchers showed that inhibition of the immunophilin family with the clinically-approved and well-tolerated drugs Tacrolimus, Cyclosporin A and Alisporivir reduced the replication of MERS-CoV, SARS-CoV, and SARS-CoV-2 in a dose-dependent manner.  

However, the team noted that while Huh7 cells are valuable for studying coronaviruses, they are likely less effective at capturing important aspects of infection than primary human airway epithelial cells.

To address this limitation, the researchers also tested the drugs in primary human nasal epithelial cell cultures.

This revealed that Cyclosporin A and Alisporivir potently inhibited SARS-CoV-2 replication at concentrations known to be achievable and efficacious in patients.

“Overall, the genes and pathways identified in our coronavirus screens expand the current repertoire of essential HDFs required for replication that can be exploited to identify novel therapeutic targets for host-directed therapies against both existing and future emerging CoVs,” writes Thiel and colleagues.

“Together these findings depict a promising path towards the repurposing of Cyclosporin A and Alisporivir as COVID-19 treatment options,” concludes the team.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Thiel V, et al. A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets. bioRxiv, 2021. doi: https://doi.org/10.1101/2021.02.24.432634, https://www.biorxiv.org/content/10.1101/2021.02.24.432634v1

Posted in: Device / Technology News | Medical Research News | Disease/Infection News

Tags: Autophagy, Cell, Coronavirus, Coronavirus Disease COVID-19, CRISPR, Drugs, Gene, Genes, Genome, Hypoxia, Immunology, Knockout, MERS-CoV, Pandemic, Protein, Receptor, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Syndrome, Tacrolimus, Transcription, Virology, Virus

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Written by

Sally Robertson

Sally first developed an interest in medical communications when she took on the role of Journal Development Editor for BioMed Central (BMC), after having graduated with a degree in biomedical science from Greenwich University.

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Lab study of South African SARS-CoV-2 variant and Moderna vaccine: reduced neutralization, but still protective

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic rages on, several virus variants have been emerging with mutations in the structural and non-structural proteins. The SARS-CoV-2 spike protein binds to the host angiotensin-converting enzyme 2 (ACE2) receptor, facilitating viral entry into the host cell. Studies have shown many different mutations in the spike protein over the last twelve months.

The first significant spike protein variant emerged with a mutation from aspartic acid (D) to glycine (G) at position 614, leading to increased viral fitness, replication, and binding to ACE2 and conformational changes within the protein. Several other variants have emerged over the past few months, raising concerns about changes to transmission, nature of the disease, and viral fitness.

When SARS-CoV-2 infects humans, our immune system rapidly responds against the viral spike protein. The receptor-binding motif in the spike protein interacts with the ACE2 receptor and is a key target of neutralization for antibodies. Longitudinal studies have found that the antibodies to the spike protein can remain in the body for at least a year following infection.

The mRNA-1273 vaccine encodes the SARS-CoV-2 spike protein and triggers a potent neutralizing antibody response to the virus that lasts for several months. The B.1.351 variant originated in South Africa has three mutations in the receptor-binding domain and many other mutations in the spike protein, all of which may influence viral binding to the ACE2 receptor and viral resistance to neutralization by antibodies.

Comparing antibody binding and viral neutralization against two different SARS-CoV-2 variants

Researchers from the US recently compared antibody binding and viral neutralization against 2 SARS-CoV-2 variants that emerged in different parts of the world. The researchers used sera from spike mRNA vaccinated and naturally infected individuals against a circulating B.1 variant and the emerging B.1.351 variant. The study is published on the preprint server bioRxiv*.

Study: Reduced binding and neutralization of infection- and vaccine-induced antibodies to the B.1.351 (South African) SARS-CoV-2 variant. Image Credit: NIAID

EHC-083E (the B.1 variant) belongs to the B.1 PANGO lineage and was isolated in March 2020 from a nasopharyngeal swab of a patient in Atlanta, GA. This variant has the D614G mutation in the viral spike protein. The B.1.351 variant was isolated in November 2020 from an oropharyngeal swab of a patient in KwaZulu-Natal, South Africa. This variant of the virus contains amino acid mutations (L18F, D80A, D215G) within the viral spike protein and deletion at positions 242-244 (L242del, A243del, and L244del), K417N, E484K, N501Y, and D614G.

Neutralizing antibodies for B.1.351 variant are produced early in the infection phase

The researchers observed decreased antibody binding to the B.1.351-derived receptor binding domain of the SARS-CoV-2 spike protein and neutralization power against the B.1.351 variant in sera from both infected and vaccinated individuals. Their longitudinal convalescent COVID-19 cohort assessed the impact on antibody binding to the receptor-binding domain and neutralization across the SARS-CoV-2 variants. Interestingly, most convalescent COVID-19 individuals showed less impact on neutralization against the B.1.351 variant at longer durations post-infection. This showed that neutralizing antibodies for the B.1.351 variant is produced early during infection and last for several months.

Most SARS-CoV-2-infected individuals showed binding and neutralizing titers against the B.1.351 variant in both acute and convalescent sera

According to the observations, most sera samples from acute and convalescent COVID-19 individuals showed antibody binding to the B.1.351-dervied receptor binding domain.  Most samples also showed a neutralizing capacity for the B.1.351 variant, and the effector functions of these neutralizing antibodies might contribute to SARS-CoV-2 infection control.

To summarize, although decreased by a few folds, most SARS-CoV-2 infected individuals showed binding and neutralizing titers against the B.1.351 variant in acute as well as convalescent sera. Moreover, all mRNA-1273 vaccinated individuals still maintained viral neutralization. These findings agree with previous notions that natural infection- and vaccine-induced immunity can offer protection against COVID-19 in the context of the SARS-CoV-2 B.1.351 variant.

“Our results show that despite few fold decrease, most infected individuals showed binding and neutralizing titers against the B.1.351 variant in acute and convalescent sera, and further, all mRNA-1273 vaccinated individuals still maintained neutralization.”

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Reduced binding and neutralization of infection- and vaccine-induced antibodies to the B.1.351 (South African) SARS-CoV-2 variant, Venkata Viswanadh Edara, Carson Norwood, Katharine Floyd, Lilin Lai, Meredith E. Davis-Gardner, William H. Hudson, Grace Mantus, Lindsay E. Nyhoff, Max W. Adelman, Rebecca Fineman, Shivan Patel, Rebecca Byram, Dumingu Nipuni Gomes, Garett Michael, Hayatu Abdullahi, Nour Beydoun, Bernadine Panganiban, Nina McNair, Kieffer Hellmeister, Jamila Pitts, Joy Winters, Jennifer Kleinhenz, Jacob Usher, James B. O’Keefe, Anne Piantadosi, Jesse J. Waggoner, Ahmed Babiker, David S. Stephens, Evan J. Anderson, Srilatha Edupuganti, Nadine Rouphael, Rafi Ahmed, Jens Wrammert, Mehul S. Suthar, bioRxiv, 2021.02.20.432046; doi: https://doi.org/10.1101/2021.02.20.432046, https://www.biorxiv.org/content/10.1101/2021.02.20.432046v1

Posted in: Medical Research News | Disease/Infection News

Tags: ACE2, Amino Acid, Angiotensin, Angiotensin-Converting Enzyme 2, Antibodies, Antibody, Aspartic Acid, Cell, Coronavirus, Coronavirus Disease COVID-19, Enzyme, Glycine, Immune System, Infection Control, Mutation, Pandemic, Protein, Receptor, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Vaccine, Virus

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Susha Cheriyedath

Susha has a Bachelor of Science (B.Sc.) degree in Chemistry and Master of Science (M.Sc) degree in Biochemistry from the University of Calicut, India. She always had a keen interest in medical and health science. As part of her masters degree, she specialized in Biochemistry, with an emphasis on Microbiology, Physiology, Biotechnology, and Nutrition. In her spare time, she loves to cook up a storm in the kitchen with her super-messy baking experiments.

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Three decades-old antibiotics could offer an alternative to opioid-based painkillers

Three decades-old antibiotics administered together can block a type of pain triggered by nerve damage in an animal model, UT Southwestern researchers report. The finding, published online today in PNAS, could offer an alternative to opioid-based painkillers, addictive prescription medications that are responsible for an epidemic of abuse in the U.S.

Over 100 million Americans are affected by chronic pain, and a quarter of these experience pain on a daily basis, a burden that costs an estimated $600 billion in lost wages and medical expenses each year. For many of these patients – those with cancer, diabetes, or trauma, for example – their pain is neuropathic, meaning it's caused by damage to pain-sensing nerves.

To treat chronic pain, prescriptions for opioid painkillers have increased exponentially since the late 1990s, leading to a rise in abuse and overdoses. Despite the desperate need for safer pain medications, development of a new prescription drug typically takes over a decade and more than $2 billion according to a study by the Tufts Center for the Study of Drug Development, explains study leader Enas S. Kandil, M.D., associate professor of anesthesiology and pain management at UTSW.

Seeking an alternative to opioids, Kandil and her UT Southwestern colleagues – including Hesham A. Sadek, M.D., Ph.D., professor of internal medicine, molecular biology, and biophysics; Mark Henkemeyer, Ph.D., professor of neuroscience; Mahmoud S. Ahmed, Ph.D., instructor of internal medicine; and Ping Wang, Ph.D., a postdoctoral researcher – explored the potential of drugs already approved by the Food and Drug Administration (FDA).

The team focused on EphB1, a protein found on the surface of nerve cells, which Henkemeyer and his colleagues discovered during his postdoctoral training nearly three decades ago. Research has shown that this protein is key for producing neuropathic pain. Mice genetically altered to remove all EphB1 don't feel neuropathic pain, he explains. Even mice with half the usual amount of this protein are resistant to neuropathic pain, suggesting EphB1's promise as a target for pain-relieving drugs. Unfortunately, no known drugs inactivate EphB1.

Exploring this angle further, Ahmed used computer modeling to scan a library of FDA-approved drugs, testing if their molecular structures had the right shape and chemistry to bind to EphB1. Their search turned up three tetracyclines, members of a family of antibiotics used since the 1970s. These drugs – demeclocycline, chlortetracycline, and minocycline – have a long history of safe use and minimal side effects, Ahmed says.

To investigate whether these drugs could bind to and inactivate EphB1, the team combined the protein and these drugs in petri dishes and measured EphB1's activity. Sure enough, each of these drugs inhibited the protein at relatively low doses. Using X-ray crystallography, Wang imaged the structure of EphB1 with chlortetracycline, showing that the drug fits neatly into a pocket in the protein's catalytic domain, a key portion necessary for EphB1 to function.

In three different mouse models of neuropathic pain, injections of these three drugs in combination significantly blunted reactions to painful stimuli such as heat or pressure, with the triplet achieving a greater effect at lower doses than each drug individually. When the researchers examined the brains and spinal cords of these animals, they confirmed that EphB1 on the cells of these tissues had been inactivated, the probable cause for their pain resistance. A combination of these drugs might be able to blunt pain in humans too, the next stage for this research, says Kandil.

Unless we find alternatives to opioids for chronic pain, we will continue to see a spiral in the opioid epidemic. This study shows what can happen if you bring together scientists and physicians with different experience from different backgrounds. We're opening the window to something new."

Enas S. Kandil, M.D., Associate Professor, Anesthesiology and Pain Management, UT Southwestern

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UT Southwestern Medical Center

Posted in: Medical Science News | Medical Research News | Pharmaceutical News

Tags: Anesthesiology, Animal Model, Antibiotic, Cancer, Cardiology, Chronic, Chronic Pain, Crystallography, Diabetes, Drugs, Education, heat, Medicine, Minocycline, Molecular Biology, Nerve, Neuropathic Pain, Neuroscience, Opioids, Pain, Pain Management, pH, Prescription Drug, Protein, Receptor, Research, Tetracycline, Trauma, X-Ray

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COVID-19 vaccine candidate shows potential against SARS-CoV-2 and potential future zoonotic coronaviruses

Over the last two decades, three major outbreaks of highly pathogenic coronaviruses have occurred. The third is the ongoing coronavirus disease 2019 (COVID-19) pandemic that has claimed well over 2.46 million human lives so far, in a little over a year from its onset. Without any targeted, safe and effective antivirals to prevent or treat the infection by the causative pathogen, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), population immunity via mass vaccination seems to be the only way out – as complex and expensive as the process is likely to be.

Study: SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys. Image Credit: Numstocker / Shutterstock

Pan-group 2b CoV vaccine

A new study, released on the bioRxiv* preprint server, sheds light on the threat posed by future zoonotic coronaviruses to make similar leaps across species barriers to infect human beings and cause other pandemics. The goal would appear to be a vaccine capable of inducing not limited immunity against SARS-CoV-2 alone, but one that can elicit broadly neutralizing antibody and cellular immune responses against a range of other betaCoVs.

This includes existing SARS-related coronaviruses (SARSr-CoVs) in humans, as well as those that are now circulating in animals.

The first evidence that this could be so came from the observation that SARS-CoV caused the production of cross-neutralizing antibodies against many betacoronaviruses (betaCoVs). This proof-of-concept drove the search for a vaccine that would induce neutralizing antibodies against multiple group 2b Sarbecoviruses.

Cross-neutralizing antibodies

Cross-neutralizing antibodies always target the viral receptor-binding domain (RBD) via a specific epitope. The RBD can be rendered more immunogenic by using a multimeric form. One way to achieve this is by using nanoparticles to mount arrays of RBD proteins, creating a virus-like particle (VLP).

Vaccines have been shown to successfully induce cross-neutralizing antibodies against pseudoviruses expressing CoV antigens in mouse studies. The current study describes a non-human primate (NHP) study that explores the cross-neutralizing ability of a SARS-CoV-2 vaccine based on multimeric SARS-CoV-2 RBD-bearing nanoparticles.

RBD-conjugated nanoparticle vaccine

The RBD-conjugated nanoparticle vaccine comprises 24 RBD protomers on a sortase-ferritin platform for the sake of versatility. This bound not only to the human host cell receptor, the angiotensin-converting enzyme 2 (ACE2), which is thought to be the SARS-CoV-2 entry receptor, but also to potent anti-RBD neutralizing antibodies. These include DH1041, DH1042, DH1043, DH1044, and DH1045.

All these antibodies bind to epitopes within the receptor-binding motif, within the RBD. However, antibodies that bound to epitopes outside the RBD were not able to bind the RBD-bearing nanoparticle. In contrast, it did show binding to the cross-neutralizing antibody DH1047.

This vaccine was assessed by a three-dose regimen, administered at four-week intervals, in a non-human primate (NHP) study. The vaccine was found to result in high plasma levels of antibodies to the SARS-CoV-2 RBD and to the stabilized spike protein.

The antibodies completely blocked the ACE2 binding site on the spike protein after two doses of vaccine and partially blocked the binding of the RBD antibody DH104.

SARS-CoV-2 receptor binding domain (RBD) sortase conjugated nanoparticles (scNPs) elicits extremely high titers of SARS-CoV-2 pseudovirus neutralizing antibodies. a. SARS-CoV-2 RBD nanoparticles were constructed by expressing RBD with a C-terminal sortase A donor sequence (blue and red) and a Helicobacter pylori ferritin nanoparticle with N737 terminal sortase A acceptor sequences (gray) on each subunit (top left). The RBD is shown in blue with the ACE2 binding site in red. The RBD was conjugated to nanoparticles by a sortase A (SrtA) enzyme conjugation reaction (top right). The resultant nanoparticle is modeled on the bottom left. Nine amino acid sortase linker is shown in orange. Two dimensional class averages of negative stain electron microscopy images of actual RBD nanoparticles are shown on the bottom right. b. Antigenicity of RBD nanoparticles determined by biolayer interferometry against a panel of SARS-CoV-2 antibodies and the ACE2 receptor. Antibodies are color-coded based on epitope and function. N-terminal domain (NTD), nonAbs IE, infection enhancing non-neutralizing antibody; nAb, neutralizing antibody; nonAb, non-neutralizing antibody. Mean and standard error from 3 independent experiments are shown. c. Cynomolgus macaque challenge study scheme. Blue arrows indicate 748 RBD-NP immunization timepoints. Intranasal/intratracheal SARS-CoV-2 challenge is indicated at week 10. d. Macaque serum IgG binding determined by ELISA to recombinant SARS-CoV-2 stabilized Spike ectodomain (S-2P), RBD, NTD, and Fusion peptide (FP). Binding titer is shown as area752under-the curve of the log10-transformed curve. Arrows indicate immunization timepoints. e. Plasma antibody blocking of SARS-CoV-2 S-2P binding to ACE2-Fc and RBD neutralizing antibody DH1041. Group mean and standard error are shown. f. Dose-dependent serum neutralization of SARS-COV-2 pseudotyped virus infection of ACE2- expressing 293T cells. Serum was collected after two immunizations. The SARS-CoV-2 pseudovirus spike has an aspartic acid to glycine change at position 614 (D614G). Each curve represents a single macaque. g. Heat map of serum neutralization ID50 and ID80 titers for SARS-COV-2 D614G pseudotyped virus after two immunizations. h. SARS-COV-2 D614G pseudotyped virus serum neutralization kinetics. Each curve represents a single macaque. i. Comparison of serum neutralization ID50 titers from cynomolgus macaques immunized with recombinant protein RBD nanoparticles (blue) or nucleoside-modified mRNA-LNP expressing S- 2P (burgundy) (**P<0.01, Two-tailed Exact Wilcoxon test n = 5). j. Comparison of serum neutralization titers obtained from RBD-scNP-vaccinated macaques (blue) and SARS-CoV-2 infected humans (shades of green). Human samples were stratified based on disease severity as asymptomatic (N=34), symptomatic (n=71), and hospitalized (N=60) (**P<0.01, Two-tailed Wilcoxon test n = 5).

Competitive with the Moderna/Pfizer vaccine for neutralizing antibody titer

When tested against the currently dominant D614G strain of SARS-CoV-2, the RBD-conjugated nanoparticle vaccine induced higher neutralizing antibody titers than another vaccine similar to the Moderna and Pfizer lipid-encapsulated nucleoside-modified mRNA (mRNA-LNP) vaccines that are now being used in the vaccination campaigns against COVID-19.

The measure of antibody titer used here showed an eight-fold increase with the former compared to the latter. The antibody response was also higher with the RBD-nanoparticle vaccine than with natural infection of all grades of severity.

Unaffected by emerging variants

It also showed potent neutralizing activity against the new SARS-CoV-2 variant B.1.1.7, which is rapidly spreading worldwide. This is not only more infective but may be resistant to many RBD-targeting antibodies, as well as more virulent.

While changes in binding affinity of anti-RBD antibody DH1041 to the ACE2 receptor and to the spike protein were observed with different mutations, such as those acquired during mink infection, or those found in the South African or Brazil or UK strains, the cross-neutralizing antibody DH1047 showed unchanged binding to the SARS-CoV-2.

“RBD-scNP (RBD sortase A conjugated nanoparticle) and mRNA-LNP-induced RBD binding antibodies were not sensitive to spike mutations present in neutralization-resistant UK, South Africa or Brazil SARS-CoV-2 variants.”

SARS-CoV-2 spike induces cross-neutralizing antibodies to pre-emergent betaCoVs

SARSr-CoVs still pose a danger of future pandemics to human beings. The researchers, therefore, explored the ability of this vaccine to neutralize other viruses. Similar to the LNP-mRNA vaccines based on the prefusion stabilized spike or the RBD, the RBD-scNP also elicited potent cross-neutralizing antibodies against SARS-CoV and SARSr-bat CoVs (batCoV-WIV-1, and batCoV-SHC014).

The neutralization was most potent against SARS-CoV-2, however. The highest neutralizing antibody titers were observed with RBD-scNP and the least with the RBD-expressing LNP-mRNA vaccine. The high titers may indicate that durable immunity is achieved.  

The RBD-scNP vaccine showed cross-neutralizing activity against batCoV-RaTG13 and pangolin CoV GXP4L spike antigens, in addition to SARS-CoV and SARS-CoV-2. Notably, sera obtained following vaccination with this formulation failed to neutralize the seasonal human CoVs or MERS-CoV, probably because of the difference in RBD among these CoVs, which belong to different groups.

The similarity between the RBD-scNP and DH1047 in terms of cross-neutralizing profile shows that not only do antibodies induced by the former bind near the epitope bound by the latter, but they are not specific to SARS-CoV-2 RBD. In fact, they also block batCoV-SHC01.

Notably, only a third of COVID-19 patients produce antibodies that block DH1047, indicating it is a sub-immunodominant epitope. As such, the RBD-scNP vaccine targets this epitope rather than the immunodominant ACE2 blocking epitope.

Protection against productive infection

The RBD-scNP vaccine was also protective for vaccinated monkeys when challenged with the SARS-CoV-2 virus via the respiratory tract. In all but one of the vaccinated macaques, “RBD-scNP-induced immunity prevented virus replication, and likely provided sterilizing immunity, in the upper and lower respiratory tract.”

What are the implications?

The RBD-scNP platform induced the highest cross-neutralizing antibody titer for group 2b CoVs, and as such, may serve as the basis for a reasonably effective initial broadly neutralizing vaccine against this group – both now, and in the future, if the further zoonotic transmission should occur.

The study also showed that the use of both RBD-scNP and the LNP-spike mRNA vaccines, the latter resembling those which have been recently rolled out, is capable of inducing cross-neutralizing antibodies to the dominant D614G variant and the newer variants of SARS-CoV-2, but at lower titers.

The findings indicate the ability of the SARS-CoV-2 Spike to be included in an RBD-scNP or LNP-mRNA formulation to induce cross-neutralizing antibodies against several SARSr-CoVs. Thus, even the currently used vaccines are likely to prevent future pandemics if immunization is successfully achieved.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Saunders, K. O. et al. (2021). SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys. bioRxiv preprint. doi: https://doi.org/10.1101/2021.02.17.431492. https://www.biorxiv.org/content/10.1101/2021.02.17.431492v1

Posted in: Medical Science News | Medical Research News | Disease/Infection News | Healthcare News

Tags: ACE2, Amino Acid, Angiotensin, Angiotensin-Converting Enzyme 2, Antibodies, Antibody, Aspartic Acid, binding affinity, Cell, Conjugation, Coronavirus, Coronavirus Disease COVID-19, Electron, Electron Microscopy, Enzyme, Glycine, heat, Helicobacter pylori, Immunization, MERS-CoV, Microscopy, Nanoparticle, Nanoparticles, Nucleoside, Pandemic, Pathogen, Protein, Pseudovirus, Receptor, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Vaccine, Virus

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Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.

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Substrain of SARS-CoV-2 variant in UK may resist antibody neutralization

Researchers at the Polish Academy of Sciences in Warsaw have identified a substrain of the recently emerged B.1.1.7 variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that may confer resistance to antibody neutralization.

The SARS-CoV-2 virus is the agent responsible for the coronavirus disease 2019 (COVID-19) pandemic that has now claimed the lives of more than 2.35 million people.

The substrain of the B.1.1.7 variant of concern (VOC) contains mutations that have previously been shown to compromise the binding of neutralizing antibodies.

Tomasz Lipniacki and colleagues say mutations in the receptor-binding domain (RBD) of the viral spike protein are of particular concern, especially those identified in the receptor-binding motif (RBM).

The spike protein is the surface structure the virus uses to bind to and infect cells by attaching to the host cell receptor angiotensin-converting enzyme 2 (ACE2).

The researchers say the mutations could eventually lead to “immune escape” strains that can reinfect convalescent individuals and reduce the efficacy of the vaccines currently being used in mass immunization efforts.

“Such mutants may hinder the efficiency of existing vaccines and expand in response to the increasing after‐infection or vaccine‐induced seroprevalence,” writes the team.

A pre-print version of the research paper is available on the medRxiv* server, while the article undergoes peer review.

Study: L18F substrain of SARS-CoV-2 VOC-202012/01 is rapidly spreading in England. Image Credit: NIAID

The B.1.1.7 variant has spread rapidly since mid-October 2020

The B.1.1.7 variant has rapidly spread since mid-October 2020, and by January 2021, it constituted about 80% of all SARS-CoV-2 genomes sequenced in England.

The high transmissibility of this VOC may be expressed in terms of its replicative advantage – defined as the ratio of the VOC reproduction number to that of non-VOC strains.

To date, a number of studies have estimated the replicative advantage as lying somewhere between 1.47 and 2.24.

As is the case with all viral strains, the B.1.1.7 variant will continue to mutate, and given its significant replicative advantage, any mutations acquired are likely to spread globally.

“As this strain will likely spread globally towards fixation, it is important to monitor its molecular evolution,” say the researchers.

What did the current study involve?

Using the Global Initiative on Sharing Avian Influenza Data (GISAID) database, Lipniacki and colleagues estimated growth rates of the mutations that B.1.1.7 has acquired.

This revealed a substrain with an L18F substitution in the spike protein that is rapidly growing in the UK.

This leucine‐to‐phenylalanine substitution in residue 18 was first reported to have occurred in a VOC strain genome collected on December 4th, 2020.

As of February 5th, 2021, as many as 850 spikes L18F VOC genomes had been reported in England.

Based on data collected between December 7th, 2020 and January 17th, 2021, the researchers showed that the L18F substrain had spread exponentially in England. They estimated a replicative advantage of 1.70 relative to the remaining B.1.1.7 substrains.

RBM mutations are particularly concerning

Lipniacki and colleagues say that mutations in the RBD of the spike protein are particularly concerning, especially substitutions E484K and S494P found in the RBM.

Importantly, the LI8F mutation has expanded in the South African variant 501Y.V2 that contains the spike mutations E484K and N501Y. Studies have suggested that E484K may compromise the binding of class 2 neutralizing antibodies, while the A501V mutation compromises the binding of class 1 antibodies.

Furthermore, in a 2021 study published in Science, the S494P substitution was characterized as an escape mutation, along with six other escape residues in the RBM that included F490.

In the current study, Lipniacki and colleagues also identified F490S as a potential escape mutation.

What do the authors advise?

“These mutations may potentially lead to immune escape mutants, resulting in reinfection of convalescent individuals and lowering efficacy of current vaccines,” warn the researchers.

“Propagation of such mutations is facilitated by high replicative advantage of the VOC strain and potential selection due to the increasing number of convalescent or immunized individuals,” they add.

Correspondingly, a study published in 2021 showed that L18F substitution compromises the binding of neutralizing antibodies, suggesting that the replicative advantage of L18F mutants may be partly associated with the ability to infect seroprevalent individuals (who already have anti-SARS-CoV-2 antibodies).

“In turn, propagation of mutations in escape residues (L18, E484, F490S, or S494) on the VOC strain suggests an increasing selection pressure resulting from the growth of the seroprevalent fraction of the population of England,” says Lipniacki and colleagues.

“This trend can be enhanced by the ongoing English vaccination program, in which the relatively large time span between the first and second dose can be a contributing factor,” concludes the team.

*Important Notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Lipniacki T, et al. L18F substrain of SARS-CoV-2 VOC-202012/01 is rapidly spreading in England. medRxiv, 2021. doi: https://doi.org/10.1101/2021.02.07.21251262, https://www.medrxiv.org/content/10.1101/2021.02.07.21251262v1

Posted in: Medical Research News | Disease/Infection News

Tags: ACE2, Angiotensin, Angiotensin-Converting Enzyme 2, Antibodies, Antibody, Avian Influenza, Cell, Coronavirus, Coronavirus Disease COVID-19, Efficacy, Enzyme, Evolution, Genome, Immunization, Influenza, Leucine, Mutation, Pandemic, Phenylalanine, Propagation, Protein, Receptor, Reproduction, Research, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Vaccine, Virus

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Written by

Sally Robertson

Sally has a Bachelor's Degree in Biomedical Sciences (B.Sc.). She is a specialist in reviewing and summarising the latest findings across all areas of medicine covered in major, high-impact, world-leading international medical journals, international press conferences and bulletins from governmental agencies and regulatory bodies. At News-Medical, Sally generates daily news features, life science articles and interview coverage.

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What is high cholesterol?

What is cholesterol?

Cholesterol (ko-LES-ter-ol) is a fatty substance found in the body. (1) It moves around the body in the blood by attaching itself to proteins, creating molecules known as lipoproteins. (2)

Types of cholesterol

There are two types of cholesterol: low-density lipoprotein (LDL) and high-density lipoprotein (HDL).

These types are often labelled as good and bad respectively. (3) LDL is considered to be harmful to the body; whereas HDL is considered to be protective. (2)

This is because too much LDL can lead to a build-up in the arteries; whereas excess HDL is broken down and removed from the body. (3)

Where is cholesterol found?

Cholesterol is produced by the liver, but it is also found in some foods. (3)

Foods that are particularly high in cholesterol include:

  • Kidneys and other offal
  • Eggs
  • Prawns
  • Full fat dairy products
  • Meats (4, 5, 6)

Despite cholesterol being found in some foods; it is important to remember that a lot of the cholesterol found in the body actually comes from foods that are high in saturated fat. This is because this fat is turned into cholesterol by the liver. (6)

What does cholesterol do in the body?

The two types of cholesterol do different things in the body. LDL carries cholesterol from the liver to cells that require it; whereas HDL carries cholesterol in the opposite direction – from the cells to the liver. (3)

Cholesterol is also used in the body to make some hormones, vitamin D and some substances that aid digestion. (1)

Specifically, cholesterol is used to make the stress hormones. These are also known as adrenal corticoid hormones and include cortisol, corticosterone and so forth. Cholesterol is also used to make sex hormones: androgens and estrogens. (16)

Cholesterol is used in the making of bile acids, which aid the digestion of food – particularly the digestion of fats. Cholesterol in the bile acid is reabsorped into the intestinal tract once it has been used to break down the fats. Thus, one way of lowering cholesterol levels is to target this reabsorption. (16)

Overall, it is important that your body has cholesterol as these things are essential for the body to function. For example, hormones carry signals around the body. (6)

High cholesterol

Despite cholesterol being important for the body to function, too much cholesterol can be a bad thing.

Video following the life of a man recently diagnosed with high cholesterol. Source: British Heart Foundation

But how high do cholesterol levels have to be to be considered dangerous?

According to the NHS, the UK government states that healthy cholesterol levels are defined as total cholesterol levels below 5mmol/L and LDL levels below 3mmol/L. (7)

The BBC, however, states that healthy cholesterol levels are controversial. (6)

Both the NHS and the BBC stress that healthy levels for people with (or at high risk of) heart disease, hypertension or diabetes should be lower. Specifically they recommend that these high risk individuals should keep their cholesterol levels below 4mmol/L and their LDL levels below 2mmol/L. (6, 7)

How can you tell if you have high cholesterol?

There are not many symptoms of high cholesterol; consequently it may be hard to tell that you have high cholesterol. (8)

One symptom that you may find is yellow patches, called xanthomas, on your skin. These particularly affect the skin around the eye area. (9)

Xanthomas can, however, be caused by other problems, such as diabetes, primary biliary cirrhosis and some cancers. (10)

The main way to tell whether you have high cholesterol is to have a blood test. This may involve fasting for 10-12 hours before the test, to make sure your food does not influence the test. (7)

The blood may be taken either using a needle or syringe or by pricking your finger. (9)

What causes high cholesterol?

The three main things that affect your cholesterol levels are diet, weight and level of physical activity.

If your diet is high in saturated fat, then your blood cholesterol levels will be higher. Similarly, if you are overweight then you are also at a higher risk of high cholesterol.

Physical activity levels can also contribute to your cholesterol levels. Being active for 30 minutes most days can help lower bad cholesterol and raise good cholesterol. (11)

Despite eating a healthy diet, some people may still have high cholesterol. This may be something that they have inherited. There is a condition called familial hyperlipidaemia which is inherited and causes high cholesterol. (2)

What diseases does high cholesterol cause?

High cholesterol makes you more likely to get coronary heart disease. (1)

This is because the LDL can build up inside the coronary arteries, this makes the arteries become narrower. The narrowing of arteries is also known as atherosclerosis. (3)

Atherosclerosis means that blood clots can more easily block the arteries. This can lead to heart attacks. (8)

LDL can also build up in other arteries, such as those that lead to the brain. This can lead to stroke. (1)

High cholesterol can also lead to something that is called a mini-stroke. This is also known as a transient ischaemic attack (TIA). A mini-stroke has similar symptoms to a stroke and is caused by a temporary reduction in the blood supply to the brain. This can be caused by atherosclerosis. (3, 12, 13)

What treatments are there for high cholesterol?

There are several ways that you can lower your cholesterol levels: through a healthy diet; exercising more and taking medications.

Healthy diet

A healthy diet involves lowering your intake of saturated fats, such as:

  • Fatty meats
  • Meat pies
  • Butter
  • Cream
  • Cakes and biscuits (2, 4)

The Food Standards Agency recommends that men and women eat no more than 30g and 20g of saturated fat per day respectively. (14)

It also involves eating more healthy foods like:

  • Oily fish, such as mackerel and salmon
  • High-fibre foods such as beans, pulses and lentils
  • Fruit and vegetables
  • Garlic, cooked or raw
  • Foods that contain antioxidants and vitamins C and E, such as strawberries, broccoli and so forth (2, 4, 11)

Exercise

The NHS recommends that you do 150 minutes of moderate-intensity aerobic activity every week. They define this activity as exercise that makes your heart beat faster and causes you to break into a sweat; yet still allows you to be able to talk whilst working. (4)

Medications to lower cholesterol

Some people may need to take medications to lower their cholesterol. Whether this is deemed necessary depends on your LDL and HDL levels, along with your risk of cardiovascular disease. (2)

Video following a man who decides to take statins to lower his cholesterol levels. Source: British Heart Foundation

There are several different medications available to lower cholesterol; these include statins, aspirin, niacin and so forth. (14)

Statins are perhaps the most well-known medicines used to lower cholesterol. They are in fact a group of medicines that include simvastatin, atorvastatin, fluvastatin, pravastatin and rosuvastatin. (9)

They work by blocking an enzyme in your liver that helps make cholesterol.

They can, however, cause side effects including muscle pain and stomach problems, such as indigestion. (9, 14)

Aspirin may be prescribed to prevent blood clots from forming.

Niacin may also be given to lower cholesterol. This is because in high doses it can lower LDL and increase HDL.

Yet again there are potential side effects with this drug. It can lead to liver damage if taken for long periods of time. Also it may cause flushing, which is where the face turns red.

In order to reduce these side effects, it is recommended that you do not take too many niacin supplements and instead try to get as much niacin as you can though your diet. (14)

Several foods contain niacin, which is also known as vitamin B3. These include:

  • Beef, pork and generally foods that are high in protein
  • Fish
  • Some nuts, including peanuts
  • Whole grains (15)

Sources

  1. http://www.nhlbi.nih.gov/health/health-topics/topics/hbc/
  2. http://www.bhf.org.uk/heart-health/conditions/high-cholesterol.aspx
  3. http://www.nhs.uk/conditions/Cholesterol/Pages/Introduction.aspx
  4. http://www.nhs.uk/Livewell/Healthyhearts/Pages/Cholesterol.aspx
  5. www.betterhealth.vic.gov.au/…/Cholesterol_explained
  6. www.bbc.co.uk/health/physical_health/conditions/cholesterol1.shtml
  7. http://www.nhs.uk/Conditions/Cholesterol/Pages/Diagnosis.aspx
  8. http://www.cdc.gov/cholesterol/faqs.htm
  9. www.bupa.co.uk/…/high-cholesterol
  10. http://www.nlm.nih.gov/medlineplus/ency/article/001447.htm
  11. http://www.indstate.edu/humres/docs/high_cholesterol.pdf
  12. www.nhs.uk/…/Introduction.aspx
  13. www.nhs.uk/Conditions/Transient-ischaemic-attack/Pages/Causes.aspx
  14. http://www.nhs.uk/Conditions/Cholesterol/Pages/Treatment.aspx
  15. http://www.moh.gov.my/images/gallery/rni/8_chat.pdf
  16. http://www.mendedhearts.org/Docs/HB-Fall04-Cholesterol.pdf

Further Reading

  • All Cholesterol Content
  • Cholesterol – What is Cholesterol?
  • Cholesterol Physiology
  • Hypercholesterolemia and Hypocholesterolemia
  • High Cholesterol and Stroke Risk
More…

Last Updated: Apr 19, 2019

Written by

April Cashin-Garbutt

April graduated with a first-class honours degree in Natural Sciences from Pembroke College, University of Cambridge. During her time as Editor-in-Chief, News-Medical (2012-2017), she kickstarted the content production process and helped to grow the website readership to over 60 million visitors per year. Through interviewing global thought leaders in medicine and life sciences, including Nobel laureates, April developed a passion for neuroscience and now works at the Sainsbury Wellcome Centre for Neural Circuits and Behaviour, located within UCL.

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Regulation of Gene Expression

Gene expression can be regulated by various cellular processes with the aim to control the amount and nature of the expressed genes.

Expression of genes can be controlled with the help of regulatory proteins at numerous levels. These regulatory proteins bind to DNA and send signals that indirectly control the rate of gene expression.

The up-regulation of a gene refers to an increase in expression of a gene whilst down-regulation refers to the decrease in expression of a gene.

Gene expression, central dogma of molecular biology – Image Copyright: Alila Medical Media / Shutterstock

The control of gene expression is more complex in eukaryotes than in prokaryotes. This is because of the presence of a nuclear membrane in eukaryotes which separates the genetic material from the translation machinery.

This necessitates some additional steps such as messenger RNA (mRNA) transport and resultant eukaryotic gene regulation at many different points. In contrast, prokaryotes lack a clearly defined nucleus hence the key point at which their gene regulation occurs is during transcriptional initiation.  

Regulation of Gene Expression in Eukaryotes

In eukaryotes, the expression of biologically active proteins can be modulated at several points as follows:

Chromatin Structure

Eukaryotic DNA is compacted into chromatin structures which can be altered by histone modifications. Such modifications can result in the up- or down-regulation of a gene.

Initiation of Transcription

This is a key point of regulation of eukaryotic gene expression. Here, several factors such as promoters and enhancers alter the ability of RNA polymerase to transcribe the mRNA, thus modulating the expression of the gene.

Post-Transcriptional Processing

Modifications such as polyadenylation, splicing, and capping of the pre-mRNA transcript in eukaryotes can lead to different levels and patterns of gene expression. For example, different splicing patterns for the same gene will generate biologically different proteins following translation.

RNA Transport

After post-transcriptional processing, the mature mRNA must be transported from the nucleus to the cytosol so that it can be translated into a protein. This step is a key point of regulation of gene expression in eukaryotes.

Stability of mRNAs

Eukaryotic mRNAs differ in their stability and some unstable transcripts usually have sequences that bind to microRNAs and reduce the stability of mRNAs, resulting in down-regulation of the corresponding proteins.

Initiation of Translation

At this stage, the ability of ribosomes in recognizing the start codon can be modulated, thus affecting the expression of the gene. Several examples of translation initiation at non-AUG codons in eukaryotes are available.

Post-Translational Processing

Common modifications in polypeptide chains include glycosylation, fatty acylation, and acetylation – these can help in regulating expression of the gene and offering vast functional diversity.

Protein Transport and Stability

Following translation and processing, proteins must be carried to their site of action in order to be biologically active. Also, by controlling the stability of proteins, the gene expression can be controlled. Stability varies greatly depending on specific amino acid sequences present in the proteins.

Regulation of Gene Expression in Prokaryotes

Prokaryotic genes are clustered into operons, each of which code for a corresponding protein.

In prokaryotes, transcription initiation is the main point of control of gene expression. It is chiefly controlled by 2 DNA sequence elements of size 35 bases and 10 bases, respectively. These elements are called promoter sequences as they help RNA polymerase recognize the start sites of transcription. RNA polymerase recognizes and binds to these promoter sequences. The interaction of RNA polymerase with promoter sequences is in turn controlled by regulatory proteins called activators or repressors based on whether they positively or negatively affect the recognition of promoter sequence by RNA pol.

There are 2 major modes of transcriptional control in E. coli to modulate gene expression. Both of these control mechanisms involve repressor proteins.

Catabolite-Regulated

In this system, control is exerted upon operons that produce genes necessary for the energy utilization. The lac operon is an example of this in E. coli.

In E. coli, glucose has a positive effect on the expression of genes that encode enzymes involved in the catabolism of alternative sources of carbon and energy such as lactose. Due to the preference for glucose, in its presence enzymes involved in the catabolism of other energy sources are not expressed. In this way, glucose represses the lac operon even if an inducer (lactose) is present.

Transcriptional Attenuation

This modulates operons necessary for biomolecule synthesis. This is called attenuated operon as the operons are attenuated by specific sequences present in the transcribed RNA – gene expression is therefore dependent on the ability of RNA Polymerase to continue elongation past specific sequences. An example of an attenuated operon is the trp operon which encodes five enzymes necessary for tryptophan biosynthesis in E.coli.  These genes are expressed only when tryptophan synthesis is necessary i.e. when tryptophan is not environmentally present. This is partly controlled when a repressor binds to tryptophan and prevents transcription for unnecessary tryptophan biosynthesis.

References

  • http://www.nature.com/scitable/topic/gene-expression-and-regulation-15
  • http://www2.le.ac.uk/departments/genetics/vgec/schoolscolleges/topics/geneexpression-regulation
  • http://www.ncbi.nlm.nih.gov/pubmed/20827583
  • http://themedicalbiochemistrypage.org/gene-regulation.php
  • http://www.ncbi.nlm.nih.gov/books/NBK9850/

Further Reading

  • All Gene Expression Content
  • A Guide to Understanding Gene Expression
  • Regulatory Mechanisms Involved in Gene Expression
  • Gene Expression Mechanism
  • Gene Expression Measurement
More…

Last Updated: Aug 23, 2018

Written by

Susha Cheriyedath

Susha has a Bachelor of Science (B.Sc.) degree in Chemistry and Master of Science (M.Sc) degree in Biochemistry from the University of Calicut, India. She always had a keen interest in medical and health science. As part of her masters degree, she specialized in Biochemistry, with an emphasis on Microbiology, Physiology, Biotechnology, and Nutrition. In her spare time, she loves to cook up a storm in the kitchen with her super-messy baking experiments.

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Plants depend on LEAFY protein that enables cells to change their fate

Cells don't express all the genes they contain all the time. The portion of our genome that encodes eye color, for example, doesn't need to be turned on in liver cells. In plants, genes encoding the structure of a flower can be turned off in cells that will form a leaf.

These unneeded genes are kept from becoming active by being stowed in dense chromatin, a tightly packed bundle of genetic material laced with proteins.

In a new study in the journal Nature Communications, biologists from the University of Pennsylvania identified a protein that enables plant cells to reach these otherwise inaccessible genes in order to switch between different identities.

Called a "pioneer transcription factor," the LEAFY protein gets a foothold in particular portions of the chromatin bundle, loosening the structure and recruiting other proteins that eventually allow genes to first be transcribed into RNA and then translated into proteins.

The programs that are not needed in a given cell or tissue or condition are effectively shut off by various chromatin modifications that make them very inaccessible. The question has always been, How do you go from shut to open? We found that LEAFY, this protein that we already knew was important in reprogramming plant cells, is one of these pioneer transcription factors that get a foot in the door, as it were, to alter the program of cells."

Doris Wagner, Study Senior Author and Biologist, School of Arts & Sciences, University of Pennsylvania

Pioneer transcription factors were first characterized by Penn faculty member Kenneth Zaret of the Perelman School of Medicine, whose own work has examined these regulatory proteins in animals, such as in the context of liver development.

Early in her time at Penn, Wagner heard Zaret give a talk about his work in this area and grew curious about looking for similar factors in plants, given that flexible gene expression is so critical to their survival.

Indeed, plants must switch between expressing whole sets of different genes all the time. In rich soils, they may grow more branches to get bigger, while in a drought they may express more genes associated with developing flowers, so they can set seed and reproduce before they succumb.

How plant cells determine their identity and fate has been a focus of Wagner's work since the start of her career, and so has LEAFY. During her postdoc days, Wagner showed that LEAFY could reprogram root cells to produce flowers. "That gave us a good clue that LEAFY might have this 'pioneer' activity, but we had to look more closely to prove it," she says.

To do so, Wagner and colleagues first used isolated protein and strands of genetic material to show that LEAFY, though not other transcription factors, bound to nucleosomes, subunits of chromatin where DNA spools on a cluster of proteins called histones. Specifically, the binding occurred at the gene AP1, which is known to be activated by LEAFY to prompt plants to make flowers.

To confirm that this connection was true in a living organism, the researchers took plant roots and applied a compound that causes them to flower spontaneously. When flowering, they found that not only did LEAFY bind strongly to AP1 but that the binding site was also occupied by a histone. "This tells us that the histones and LEAFY are really occupying the same portion of DNA," Wagner says.

Furthermore, they showed that chromatin structure began to open up at the AP1 region when LEAFY was activated, a key facet of what pioneer transcription factors do.

This opening was limited, and full loosening of chromatin took days. What did happen quickly, the researchers found, was that LEAFY displaced a linker histone protein, creating a small local opening that also allowed other transcription factors to nose their way into the DNA.

Though pioneer transcription factors had been proposed to exist in plants, the new work provides the first concrete support backing this conception for LEAFY. And Wagner believes there are others. "If necessary, plants can alter their entire body plan or generate an entire plant from a little piece of leaf," she says.

"We predict setting this in motion will require pioneer transcription factors. So plants may actually have more of these factors than animals."

In upcoming work, she and her team hope to delve more deeply into the processes that precede and follow this "pioneering" activity of LEAFY: Does anything restrict its activity, and how do the other factors that it recruits fully unpack the hidden-away genes? "It would be great to find out both sides of this equation," Wagner says.

The findings have significance in agriculture and breeding, where LEAFY is already manipulated to encourage earlier flowering, for example. And as more is understood about pioneer transcription factors in plants, Wagner can envision a fine-tuning of other aspects of plant growth and activity, which could be leveraged to help crops adapt to new environmental conditions, such as those being ushered in by climate change.

Source:

University of Pennsylvania

Journal reference:

Jin, R., et al. (2021) LEAFY is a pioneer transcription factor and licenses cell reprogramming to floral fate. Nature Communications. doi.org/10.1038/s41467-020-20883-w.

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Influenza A Structure

Influenza virus is a common cause of human respiratory infection with a high rate of morbidity and mortality, particularly in the elderly and in infants.

Credit: Katryna Kon/Shutterstock.com

Influenza A belongs to the Orthomyxoviridae family. It has a negative sense RNA genome encoding 11 viral genes, contained within a viral envelope. The viral genes are as follows:

  • hemagglutinin (HA)
  • neuraminidase (NP)
  • matrix 1 (M1)
  • matrix 2 (M2)
  • nucleoprotein (NP)
  • non-structural protein (NSP1)
  • non-structural protein 2/nuclear export protein (NS2/NEP)
  • polymerase acidic protein (PA)
  • polymerase basic protein 1 (PB1)
  • polymerase basic protein 2 (PB2)
  • polymerase basic protein 1-F2 (PB1-F2)

Outer virus particle

The influenza particle, or virion, is typically spherical, but sometimes filamentous. It has an outer lipid membrane layer called an envelope derived from the host cell that it replicated in. The envelope is covered with glycoproteins HA and NA which form structures like spikes. The ratio of HA to NA molecules is about four to one.

The matrix ion channel M2 penetrates the envelope. The matrix protein M1 is found beneath the lipid membrane. It forms a shell, providing strength and rigidity to the virion. The spherical form influenza A viruses are typically about 100 nm in diameter and filamentous forms can be longer than 300 nm.

Entry into the host cell begins when spikes formed by HA on the surface of the virion bind to sialic acid on the surface of the host cell. The binding of HA to sialic acid is highly species specific. Humans have a different specificity from horses and birds. However, swine viruses recognize both, which makes swine an effective mixing vessel for avian, equine, and human influenza A virus.

Interior of the virus

Inside the viral particle are the 8 viral RNAs that comprise the genome. Each RNA segment is is joined with proteins B1, PB2, PA, and NP. The protein NEP is also found in the interior of the virion. The ribonucleoprotein (RNP) complex within the virion consists of viral RNA segments coated with NP and heterotrimeric RNA-dependent RNA polymerase, which has subunits PB1, PB2 and PA.

Genome structure

The influenza A genome is comprised of eight negative sense, single-stranded viral RNA segments. They are numbered in order of decreasing length. Segments 1, 3, 4, and 5 encode a single protein per segment. Those are PB2, PA, HA, and NP, respectively. Segment 2 contains PB1.

Some strains of influenza A may also code for PB1-F2, a small protein with pro-apoptotic activity, in an alternate reading frame. Segment 6 of influenza A encodes NA protein and in an alternate reading frame, NB matrix protein. Segment 7 codes for M1 matrix protein and through RNA splicing, M2 ion channel. Segment 8 encodes the NS1 protein, and, through mRNA splicing, NS2/NEP.

Each vRNA segment forms a helical hairpin in shape, and is bound by the heterotrimeric RNA polymerase complex. The rest of it is coated with NP. Each segment has non-coding regions at each end of varying lengths. However, the very end of all segments is a highly conserved sequence which serves as a promoter for vRNA replication and transcription. The non-coding regions also contain the mRNA polyadenylation signal and some signals for virus assembly.

The segmentation of the influenza A genome allows the phenomenon of antigenic shift, wherein the virus acquires an HA segment from another influenza subtype. This occurs in cells infected with different viruses, and the resulting virus may encode a wholly novel protein to which humans have no pre-existing immunity.

Sources:

  • Influenza virus intracellular replication dynamics, release kinetics, and particle morphology during propagation in MDCK cells
  • Structure of the influenza virus
  • The Influenza (Flu) Virus
  • Native morphology of influenza virions

Further Reading

  • All Virology Content
  • Virology – What is Virology?
  • Viral Diseases
  • Virology History
  • Virology Molecular Biology & Viral Therapy
More…

Last Updated: Aug 23, 2018

Written by

Dr. Catherine Shaffer

Catherine Shaffer is a freelance science and health writer from Michigan. She has written for a wide variety of trade and consumer publications on life sciences topics, particularly in the area of drug discovery and development. She holds a Ph.D. in Biological Chemistry and began her career as a laboratory researcher before transitioning to science writing. She also writes and publishes fiction, and in her free time enjoys yoga, biking, and taking care of her pets.

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Dangers of Vitamin Deficiency During Pregnancy

Skip to:

  • What vitamins and nutrients are essential during pregnancy?
  • Vitamin B12
  • Vitamin C
  • Vitamin D
  • Folate
  • Iron
  • Calcium
  • What are the dangers of vitamin deficiency during pregnancy?
  • Vitamin B12 deficiency
  • Vitamin D deficiency
  • Vitamin C deficiency

During pregnancy, the body prepares for the fetus’ development and the breastfeeding process.  The body goes through many physical and hormonal changes. The mother needs to consume more foods rich in vitamins and nutrients to support the baby.

Proper weight gain and eating healthily reduces the risk of complications. Subsequently, the woman’s nutritional condition affects the infant later in life, particularly in his cognitive development, heart health, and the tendency to become overweight or obese.

The food pregnant mothers eat is the major source of nourishment of the body, so it’s vital to consume foods that are rich in vitamins and nutrients.

What vitamins and nutrients are essential during pregnancy?

Vitamin B12

Vitamin B12 or Cobalamin is an essential vitamin that plays a pivotal role in the production of red blood cells. It’s also important for neurological function and DNA synthesis. Vitamin B12, which is bound to protein in food, is released by the activity of gastric protease and hydrochloric acid in the stomach.

When combined with folic acid during pregnancy, vitamin B12 can help prevent spina bifida and other spinal or central nervous system birth defects. Mothers deficient in B12 are more likely to give birth to infants affected by spina bifida.

The major sources of vitamin B12 are beef, ham, pork, fish, dairy products, eggs, chicken, nutritional yeast products, and lamb.

Sources of Vitamin B12 (Cobalamin). Image Credit: Bitt24 / Shutterstock

Vitamin C

Vitamin C, also known as ascorbic acid, is a vitamin that occurs naturally in some foods. It can also be available as a dietary supplement. Vitamin C is an antioxidant that’s important for the skin, bones, and connective tissues. Aside from this, it helps the body absorb iron and boost the immune system.

Vitamin C during pregnancy may help reduce complications such as maternal anemia, intrauterine growth restriction, and pre-eclampsia. The most common sources of vitamin C include broccoli, greens, tomatoes, citrus fruits, and red or green peppers.

Foods High in vitamin C. Image Credit: bitt24 / Shutterstock

Vitamin D

Vitamin D helps the body absorb calcium, which is needed for strong bones. People can get vitamin D by exposing bare skin to sunlight. You can also get vitamin D through supplements and diet.

Vitamin D is very important for mothers and their developing babies. It plays an important role in bone metabolism by regulating the calcium and phosphate balance. It also helps reduce the risk of low birth weight, pre-eclampsia, and preterm birth.

Foods rich in vitamin D include fatty fish such as salmon, mackerel, and tuna, beef liver, egg yolks, and cheese.

Foods rich in vitamin D. Image Credit: bitt24 / Shutterstock

Folate

Folate is a B-vitamin that helps produce DNA and other genetic materials. Folate is important in red blood cell production and to reduce the risk of neural tube defects, such as spina bifida.

Foods rich in folate include legumes, asparagus, leafy green vegetables, beets, eggs, citrus fruits, broccoli, and brussels sprouts. Both dietary consumption and supplementation are recommended. To be effective in preventing neural tube defects during development, women wishing to become pregnant should initiate folate supplementation prior to conception.

Iron

Iron is an important mineral needed by the body for various functions. For instance, iron is a part of hemoglobin, a protein that carries oxygen from the lungs to the different cells of the body. Plus, it aids in the storage and use of oxygen in the muscles.

During pregnancy, the body’s blood volume rises to compensate for the increased demand for nutrients and oxygen. The demand for iron goes up to cope with the increased blood supply. The needed amount of iron should be doubled to about 27 mg per day. The most common sources of iron include green leafy vegetables, beans and lentils, tofu, cashews, fortified breakfast cereals, baked potatoes, and whole grains, to name a few.

Calcium

Calcium, a mineral important for life, helps build bones. It also enables the blood to clot, aids in muscle contraction, and helps the heart to beat. But, about 99 percent of all calcium stores in the body is in the bones and teeth.

During pregnancy, the body needs calcium from food or supplements. The recommended dosage is about 1,000 mg of calcium each day. Foods rich in calcium include cheese, milk, and yogurt.

What are the dangers of vitamin deficiency during pregnancy?

Vitamin deficiency during pregnancy may cause a wide array of maternal and fetal complications.

Vitamin B12 deficiency

Low levels of vitamin B12 during pregnancy may increase the likelihood of neural tube defects. In a study, the researchers found that mothers of children affected by neural tube defects had significantly lower vitamin B12 status.

Vitamin D deficiency

Vitamin D deficiency has been associated with an increased prevalence of pre-eclampsia, which is a common cause of mortality among pregnant women and their infants. According to a study, adverse health outcomes such as low birth weight, pre-eclampsia, neonatal hypocalcemia, bone fragility, heightened risk of developing of autoimmune diseases, and poor postnatal growth have been linked to low vitamin D levels during pregnancy.

Vitamin C deficiency

Vitamin C deficiency among pregnant women may lead to serious health effects on the fetus’ brain. In a study, the researchers said that even marginal vitamin C deficiency in the mother prevents the baby’s hippocampus, an important part of the brain responsible for memory, from developing by about 10 to 15 percent.

The best way to ensure that a woman’s body has appropriate vitamin levels to support a healthy pregnancy is to establish healthful eating habits prior to becoming pregnant. A prenatal care visit is highly recommended to address any potential nutritional deficits that should be addressed prior to trying to conceive. Folate supplementation is also recommended pre-conception and during pregnancy to prevent neural tube defects. The usefulness of vitamin supplements during pregnancy for vitamin B12 and vitamin C is less clear and is currently under study. Healthcare providers may recommend that pregnant women take calcium and iron supplements during pregnancy to ensure that the mother has appropriate levels to maintain her own bone and blood health.

Sources

  • American Pregnancy Association. (2019). https://americanpregnancy.org/pregnancy-health/vitamin-b-pregnancy/
  • National Institute of Child Health and Human Development. (2017). www.nichd.nih.gov/health/topics/pregnancy/conditioninfo/prenatal-care
  • Pannia, E., Cho, C.E., Kubant, R., Sanchez-Hernandez, D., Huot, P.S.,  Anderson, H. (2016). Role of maternal vitamins in programming health and chronic disease. Nutrition Reviews. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4892288/
  • Rumbold, A, Nagata, O., Shahrook, S., and Crowther, C.A. (2015). Vitamin C supplementation in pregnancy. Cochrane Database of Systematic Reviews. https://www.ncbi.nlm.nih.gov/pubmed/26415762
  • Chidambaram, Balasubramaniam. (2012). Folate in pregnancy. Journal of Pediatric Neurosciences. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3519088/
  • Molloy, A., Kirke, P.,Troendle, J., Burke, H., Sutton, M., Brody, L., Scott, J., and Mills, J. (2009). Maternal Vitamin B12 Status and Risk of Neural Tube Defects in a Population With High Neural Tube Defect Prevalence and No Folic Acid Fortification. Pediatrics. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161975/
  • Finkelstein, J., Layden, A., Stover, P. (2015). Vitamin B-12 and Perinatal Health. Advances in Nutrition. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561829/
  • McCullough, M. (2007). Vitamin D Deficiency in Pregnancy: Bringing the Issues to Light. The Journal of Nutrition.  https://academic.oup.com/jn/article/137/2/305/4664522
  • Mulligan, M., Felton, S., Riek, A., and Bernal-Mizrachi, C. (2009). Implications of vitamin D deficiency in pregnancy and lactation. American Journal of Obstetrics and Gynecology. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3540805/
  • Nyborg, P., Vogt, L., Schjoldager, J., Jeannet, N., Hasselholt, S., Paidi, M., Christen, S., and Lykkesfeldt, J. (2012). Maternal Vitamin C Deficiency during Pregnancy Persistently Impairs Hippocampal Neurogenesis in Offspring of Guinea Pigs. Plos One. journals.plos.org/plosone/article?id=10.1371/journal.pone.0048488

Further Reading

  • All Pregnancy Content
  • Early Signs of Pregnancy
  • Is it Safe to Exercise During Pregnancy?
  • Pregnancy: 0-8 weeks
  • Pregnancy: 9 – 12 weeks
More…

Last Updated: Aug 13, 2019

Written by

Angela Betsaida B. Laguipo

Angela is a nurse by profession and a writer by heart. She graduated with honors (Cum Laude) for her Bachelor of Nursing degree at the University of Baguio, Philippines. She is currently completing her Master's Degree where she specialized in Maternal and Child Nursing and worked as a clinical instructor and educator in the School of Nursing at the University of Baguio.

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