The oncogenic circular RNA circ_63706 is a potential therapeutic target in sonic hedgehog-subtype childhood medulloblastomas.

Medulloblastoma (MB) develops through various genetic, epigenetic, and non-coding (nc) RNA-related mechanisms, but the roles played by ncRNAs, particularly circular RNAs (circRNAs), remain poorly defined. CircRNAs are increasingly recognized as stable non-coding RNA therapeutic targets in many cancers, but little is known about their function in MBs. To determine medulloblastoma subgroup-specific circRNAs, publicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify circRNAs that differentiate between MB subgroups. circ_63706 was identified as sonic hedgehog (SHH) group-specific, with its expression confirmed by RNA-FISH analysis in clinical tissue samples. The oncogenic function of circ_63706 was characterized in vitro and in vivo. Further, circ_63706-depleted cells were subjected to RNA-seq and lipid profiling to identify its molecular function. Finally, we mapped the circ_63706 secondary structure using an advanced random forest classification model and modeled a 3D structure to identify its interacting miRNA partner molecules. Circ_63706 regulates independently of the host coding gene pericentrin (PCNT), and its expression is specific to the SHH subgroup. circ_63706-deleted cells implanted into mice produced smaller tumors, and mice lived longer than parental cell implants. At the molecular level, circ_63706-deleted cells elevated total ceramide and oxidized lipids and reduced total triglyceride. Our study implicates a novel oncogenic circular RNA in the SHH medulloblastoma subgroup and establishes its molecular function and potential as a future therapeutic target.

Molecular Modeling and Simulation of the Peptidoglycan Layer of Gram-Positive Bacteria Staphylococcus aureus.

The peptidoglycan (PG) layer is a vital component of the bacterial cell wall that protects the cell from rupturing due to internal pressure. Its ubiquity across the bacterial kingdom but not animals has made it the target of drug discovery efforts. The PG layer composed of cross-linked PG strands is porous enough to allow the diffusion of molecules through the PG mesh and into the cell. The lack of an accurate atomistic model of the PG mesh has limited the computational investigations of drug diffusion in Gram-positive bacteria, which lack the outer membrane but consist of a much thicker PG layer compared to Gram-negative bacteria. In this work, we built an atomistic model of the Staphylococcus aureus PG layer architecture with horizontally aligned PG strands and performed molecular dynamics simulations of the diffusion of curcumin molecules through the PG mesh. An accurate model of the Gram-positive bacterial cell wall may aid in developing novel antibiotics to tackle the threat posed by antibiotic resistance.

Immunoinformatic screening of Marburgvirus epitopes and computational investigations of epitope-allele complexes.

Marburgvirus (MARV), a member of the Filovirus family, causes severe hemorrhagic fever in humans. Currently, there are no approved vaccines or post exposure treatment methods available against MARV. With the aim of identifying vaccine candidates against MARV, we employ different sequence-based computational methods to predict the MHC-I and MHC-II T-cell epitopes as well as B-cell epitopes for the complete MARV genome. We analyzed the variations in the predicted epitopes among four MARV variants, the Lake Victoria, Angola, Musoke, and Ravn. We used a consensus approach to identify several epitopes, including novel epitopes, and narrowed down the selection based on different parameters such as antigenicity and IC50 values. The selected epitopes can be used in various vaccine constructs that give effective antibody responses. The MHC-I epitope-allele complexes for GP and NP with favorably low IC50 values were investigated using molecular dynamics computations to determine the molecular details of the epitope-allele complexes. This study provides information for further experimental validation of the potential epitopes and the design and development of MARV vaccines.

Potential Autoimmunity Resulting from Molecular Mimicry between SARS-CoV-2 Spike and Human Proteins.

Molecular mimicry between viral antigens and host proteins can produce cross-reacting antibodies leading to autoimmunity. The coronavirus SARS-CoV-2 causes COVID-19, a disease curiously resulting in varied symptoms and outcomes, ranging from asymptomatic to fatal. Autoimmunity due to cross-reacting antibodies resulting from molecular mimicry between viral antigens and host proteins may provide an explanation. Thus, we computationally investigated molecular mimicry between SARS-CoV-2 Spike and known epitopes. We discovered molecular mimicry hotspots in Spike and highlight two examples with tentative high autoimmune potential and implications for understanding COVID-19 complications. We show that a TQLPP motif in Spike and thrombopoietin shares similar antibody binding properties. Antibodies cross-reacting with thrombopoietin may induce thrombocytopenia, a condition observed in COVID-19 patients. Another motif, ELDKY, is shared in multiple human proteins, such as PRKG1 involved in platelet activation and calcium regulation, and tropomyosin, which is linked to cardiac disease. Antibodies cross-reacting with PRKG1 and tropomyosin may cause known COVID-19 complications such as blood-clotting disorders and cardiac disease, respectively. Our findings illuminate COVID-19 pathogenesis and highlight the importance of considering autoimmune potential when developing therapeutic interventions to reduce adverse reactions.

Significance of the RBD mutations in the SARS-CoV-2 omicron: from spike opening to antibody escape and cell attachment.

We computationally investigated the role of the omicron RBD mutations on its structure and interactions with the surrounding domains in the spike trimer as well as with ACE2. Our results suggest that, compared to WT and delta, the mutations in the omicron RBD facilitate a more efficient RBD "down" to "up" conformation as well as ACE2 attachment. These effects, combined with antibody evasion, may have contributed to its dominance over delta.

Lipid II Binding and Transmembrane Properties of Various Antimicrobial Lanthipeptides.

There has been an alarming rise in antibacterial resistant infections in recent years due to the widespread use of antibiotics, and there is a dire need for the development of new antibiotics utilizing novel modes of action. Lantibiotics are promising candidates to engage in the fight against resistant strains of bacteria due to their unique modes of action, including interference with cell wall synthesis by binding to lipid II and creating pores in bacterial membranes. In this study, we use atomic-scale molecular dynamics computational studies to compare both the lipid II binding ability and the membrane interactions of five lanthipeptides that are commonly used in antimicrobial research: nisin, Mutacin 1140 (MU1140), gallidermin, NVB302, and NAI107. Among the five peptides investigated, nisin is found to be the most efficient at forming water channels through a membrane, whereas gallidermin and MU1140 are found to be better at binding the lipid II molecules. Nisin's effectiveness in facilitating water transport across the membrane is due to the creation of several different water trajectories along with no significant water delay points along the paths. The shorter peptide deoxyactagardine B (NVB302) was found to not form a water channel. These detailed observations provide insights into the dual mechanisms of the action of lantibiotic peptides and can facilitate the design and development of novel lanthipeptides by strategic placement of different residues.

Ebola virus protein VP40 binding to Sec24c for transport to the plasma membrane.

Outbreaks of the Ebola virus (EBOV) continue to occur and while a vaccine and treatment are now available, there remains a dearth of options for those who become sick with EBOV disease. An understanding at the atomic and molecular level of the various steps in the EBOV replication cycle can provide molecular targets for disrupting the virus. An important step in the EBOV replication cycle is the transport of EBOV structural matrix VP40 protein molecules to the plasma membrane inner leaflet, which involves VP40 binding to the host cell's Sec24c protein. Though some VP40 residues involved in the binding are known, the molecular details of VP40-Sec24c binding are not known. We use various molecular computational techniques to investigate the molecular details of how EBOV VP40 binds with the Sec24c complex of the ESCRT-I pathway. We employed different docking programs to identify the VP40-binding site on Sec24c and then performed molecular dynamics simulations to determine the atomic details and binding interactions of the complex. We also investigated how the inter-protein interactions of the complex are affected upon mutations of VP40 amino acids in the Sec24c-binding region. Our results provide a molecular basis for understanding previous coimmunoprecipitation experimental studies. In addition, we found that VP40 can bind to a site on Sec24c that can also bind Sec23 and suggests that VP40 may use the COPII transport mechanism in a manner that may not need the Sec23 protein in order for VP40 to be transported to the plasma membrane.

Mutation-induced changes in the receptor-binding interface of the SARS-CoV-2 Delta variant B.1.617.2 and implications for immune evasion.

Following the initial surges of the Alpha (B.1.1.7) and the Beta (B.1.351) variants, a more infectious Delta variant (B.1.617.2) is now surging, further deepening the health crises caused by the pandemic. The sharp rise in cases attributed to the Delta variant has made it especially disturbing and is a variant of concern. Fortunately, current vaccines offer protection against known variants of concern, including the Delta variant. However, the Delta variant has exhibited some ability to dodge the immune system as it is found that neutralizing antibodies from prior infections or vaccines are less receptive to binding with the Delta spike protein. Here, we investigated the structural changes caused by the mutations in the Delta variant's receptor-binding interface and explored the effects on binding with the ACE2 receptor as well as with neutralizing antibodies. We find that the receptor-binding ß-loop-ß motif adopts an altered but stable conformation causing separation in some of the antibody binding epitopes. Our study shows reduced binding of neutralizing antibodies and provides a possible mechanism for the immune evasion exhibited by the Delta variant.

Structural and Dynamical Differences in the Spike Protein RBD in the SARS-CoV-2 Variants B.1.1.7 and B.1.351.

The novel coronavirus (SARS-CoV-2) pandemic that started in late 2019 is responsible for hundreds of millions of cases worldwide and millions of fatalities. Though vaccines are available, the virus is mutating to form new strains among which are the variants B.1.1.7 and B.1.351 that demonstrate increased transmissivity and infectivity. In this study, we performed molecular dynamics simulations to explore the role of the mutations in the interaction of the virus spike protein receptor binding domain (RBD) with the host receptor ACE2. We find that the hydrogen bond networks are rearranged in the variants and also that new hydrogen bonds are established between the RBD and ACE2 as a result of mutations. We investigated three variants: the wild-type (WT), B.1.1.7, and B.1.351. We find that the B.1.351 variant (also known as 501Y.V2) shows larger flexibility in the RBD loop segment involving residue K484, yet the RBD-ACE2 complex shows higher stability. Mutations that allow a more flexible interface that can result in a more stable complex may be a factor contributing to the increased infectivity of the mutated variants.

Structural insights into the repair mechanism of AGT for methyl-induced DNA damage.

Methylation induced DNA base-pairing damage is one of the major causes of cancer. O6-alkylguanine-DNA alkyltransferase (AGT) is considered a demethylation agent of the methylated DNA. Structural investigations with thermodynamic properties of the AGT-DNA complex are still lacking. In this report, we modeled two catalytic states of AGT-DNA interactions and an AGT-DNA covalent complex and explored structural features using molecular dynamics (MD) simulations. We utilized the umbrella sampling method to investigate the changes in the free energy of the interactions in two different AGT-DNA catalytic states, one with methylated GUA in DNA and the other with methylated CYS145 in AGT. These non-covalent complexes represent the pre- and post-repair complexes. Therefore, our study encompasses the process of recognition, complex formation, and separation of the AGT and the damaged (methylated) DNA base. We believe that the use of parameters for the amino acid and nucleotide modifications and for the protein-DNA covalent bond will allow investigations of the DNA repair mechanism as well as the exploration of cancer therapeutics targeting the AGT-DNA complexes at various functional states as well as explorations via stabilization of the complex.

In-silico identification of the vaccine candidate epitopes against the Lassa virus hemorrhagic fever.

Lassa virus (LASV), a member of the Arenaviridae, is an ambisense RNA virus that causes severe hemorrhagic fever with a high fatality rate in humans in West and Central Africa. Currently, no FDA approved drugs or vaccines are available for the treatment of LASV fever. The LASV glycoprotein complex (GP) is a promising target for vaccine or drug development. It is situated on the virion envelope and plays key roles in LASV growth, cell tropism, host range, and pathogenicity. In an effort to discover new LASV vaccines, we employ several sequence-based computational prediction tools to identify LASV GP major histocompatibility complex (MHC) class I and II T-cell epitopes. In addition, many sequence- and structure-based computational prediction tools were used to identify LASV GP B-cell epitopes. The predicted T- and B-cell epitopes were further filtered based on the consensus approach that resulted in the identification of thirty new epitopes that have not been previously tested experimentally. Epitope-allele complexes were obtained for selected strongly binding alleles to the MHC-I T-cell epitopes using molecular docking and the complexes were relaxed with molecular dynamics simulations to investigate the interaction and dynamics of the epitope-allele complexes. These predictions provide guidance to the experimental investigations and validation of the epitopes with the potential for stimulating T-cell responses and B-cell antibodies against LASV and allow the design and development of LASV vaccines.

Conformational Flexibility of the Protein-Protein Interfaces of the Ebola Virus VP40 Structural Matrix Filament.

The Ebola virus (EBOV) is a virulent pathogen that causes severe hemorrhagic fever with a high fatality rate in humans. The EBOV transformer protein VP40 plays crucial roles in viral assembly and budding at the plasma membrane of infected cells. One of VP40's roles is to form the long, flexible, pleomorphic filamentous structural matrix for the virus. Each filament contains three unique interfaces: monomer NTD-NTD to form a dimer, dimer-to-dimer NTD-NTD oligomerization to form a hexamer, and end-to-end hexamer CTD-CTD to build the filament. However, the atomic-level details of conformational flexibility of the VP40 filament are still elusive. In this study, we have performed explicit-solvent, all-atom molecular dynamic simulations to explore the conformational flexibility of the three different interface structures of the filament. Using dynamic network analysis and other calculational methods, we find that the CTD-CTD hexamer interface with weak interdomain amino acid communities is the most flexible, and the NTD-NTD oligomer interface with strong interdomain communities is the least flexible. Our study suggests that the high flexibility of the CTD-CTD interface may be essential for the supple bending of the Ebola filovirus, and such flexibility may present a target for molecular interventions to disrupt the Ebola virus functioning.

Molecular mechanisms of pore formation and membrane disruption by the antimicrobial lantibiotic peptide Mutacin 1140.

The emergence of antibiotic-resistance is a major concern to global human health and identification of novel antibiotics is critical to mitigate the threat. Mutacin 1140 (MU1140) is a promising antimicrobial lanthipeptide and is effective against Gram-positive bacteria. Like nisin, MU1140 targets and sequesters lipid II and interferes with its function, which results in the inhibition of bacterial cell wall synthesis, and leads to bacteria cell lysis. MU1140 contains a structurally similar thioether cage for binding the lipid II pyrophosphate as for nisin. In addition to lipid II binding, nisin is known to form membrane pores. Membrane pore formation and membrane disruption is a common mode of action for many antimicrobial peptides, including gallidermin, a lantibiotic peptide with similar structural features as MU1140. However, whether and how MU1140 and its variants can form permeable membrane pores remains to be demonstrated. In this work, we explored the potential mechanisms of membrane pore formation by performing molecular simulations of the MU1140-lipid II complex in the bacterial membrane. Our results suggest that MU1140-lipid II complexes are able to form water permeating membrane pores. We find that a single chain of MU1140 complexed with lipid II in the transmembrane region can permeate water molecules across the membrane via a single-file water transport mechanism. The ordering of the water molecules in the single-file chain region as well as the diffusion behavior is similar to those observed in other biological water channels. Multiple complexes of MU1140-lipid II in the membrane showed enhanced permeability for the water molecules, as well as a noticeable membrane distortion and lipid relocation, suggesting that a higher concentration of MU1140 assembly in the membrane can cause significant disruption of the bacterial membrane. These investigations provide an atomistic level insight into a novel mode of action for MU1140 that can be exploited to develop optimized peptide variants with improved antimicrobial properties.