miRNA-211 maintains metabolic homeostasis in medulloblastoma through its target gene long-chain acyl-CoA synthetase 4.

The prognosis of childhood medulloblastoma (MB) is often poor, and it usually requires aggressive therapy that adversely affects quality of life. microRNA-211 (miR-211) was previously identified as an important regulator of cells that descend from neural cells. Since medulloblastomas primarily affect cells with similar ontogeny, we investigated the role and mechanism of miR-211 in MB. Here we showed that miR-211 expression was highly downregulated in cell lines, PDXs, and clinical samples of different MB subgroups (SHH, Group 3, and Group 4) compared to normal cerebellum. miR-211 gene was ectopically expressed in transgenic cells from MB subgroups, and they were subjected to molecular and phenotypic investigations. Monoclonal cells stably expressing miR-211 were injected into the mouse cerebellum. miR-211 forced expression acts as a tumor suppressor in MB both in vitro and in vivo, attenuating growth, promoting apoptosis, and inhibiting invasion. In support of emerging regulatory roles of metabolism in various forms of cancer, we identified the acyl-CoA synthetase long-chain family member (ACSL4) as a direct miR-211 target. Furthermore, lipid nanoparticle-coated, dendrimer-coated, and cerium oxide-coated miR-211 nanoparticles were applied to deliver synthetic miR-211 into MB cell lines and cellular responses were assayed. Synthesizing nanoparticle-miR-211 conjugates can suppress MB cell viability and invasion in vitro. Our findings reveal miR-211 as a tumor suppressor and a potential therapeutic agent in MB. This proof-of-concept paves the way for further pre-clinical and clinical development.

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.

The long non-coding RNA SPRIGHTLY and its binding partner PTBP1 regulate exon 5 skipping of SMYD3 transcripts in group 4 medulloblastomas.

Background: Although some of the regulatory genes, signaling pathways, and gene regulatory networks altered in medulloblastomas (MB) are known, the roles of non-coding RNAs, particularly long non-coding RNAs (lncRNAs), are poorly described. Here we report that the lncRNA SPRIGHTLY (SPRY4-IT1) gene is upregulated in group 4 medulloblastoma (G4 MB). Methods: SPRIGHTLY expression was assessed in MB subgroup patient-derived xenografts, cell lines, and patient samples. The effect of SPRIGHTLY hemizygous deletion on proliferation, invasion, apoptosis, and colony formation were assessed in vitro and on tumor growth in vivo. dChIRP pull-down assays were used to assess SPRIGHTLY-binding partners, confirmed by immunoprecipitation. SMYD3 ΔE5 transcripts were examined in cell lines and publicly available RNA-seq data. Pathway analysis was performed by phospho-kinase profiling and RNA-seq. Results: CRISPR/Cas9 deletion of SPRIGHTLY reduced cell viability and invasion and increased apoptosis in G4 MB cell lines in vitro. SPRIGHTLY hemizygous-deleted G4 MB cells injected into mouse cerebellums produced smaller tumors than those derived from parental cells expressing both copies of SPRIGHTLY. SPRIGHTLY lncRNA bound to the intronic region of the SMYD3 pre-mRNA transcript. SPRIGHTLY also interacted with PTPB1 protein to regulate SMYD3 exon skipping to produce an aberrant protein. SPRIGHTLY-driven SMYD3 regulation enhanced the expression of EGFR pathway genes in G4 MB cell lines and activated cell coagulation/hemostasis-related gene expression, suggesting a novel oncogenic role in G4 MB. Conclusions: These results demonstrate the importance of SPRIGHTLY lncRNA as a promoter of G4 MB and the role of the SPRIGHTLY-SMYD3-PTPB1 axis as an important oncogenic regulator in MB.

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.

Measles and Nipah virus assembly: Specific lipid binding drives matrix polymerization.

Measles virus, Nipah virus, and multiple other paramyxoviruses cause disease outbreaks in humans and animals worldwide. The paramyxovirus matrix (M) protein mediates virion assembly and budding from host cell membranes. M is thus a key target for antivirals, but few high-resolution structures of paramyxovirus M are available, and we lack the clear understanding of how viral M proteins interact with membrane lipids to mediate viral assembly and egress that is needed to guide antiviral design. Here, we reveal that M proteins associate with phosphatidylserine and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane. Using x-ray crystallography, electron microscopy, and molecular dynamics, we demonstrate that PI(4,5)P2 binding induces conformational and electrostatic changes in the M protein surface that trigger membrane deformation, matrix layer polymerization, and virion assembly.

Medulloblastoma cerebrospinal fluid reveals metabolites and lipids indicative of hypoxia and cancer-specific RNAs.

Medulloblastoma (MB) is the most common malignant brain tumor in children. There remains an unmet need for diagnostics to sensitively detect the disease, particularly recurrences. Cerebrospinal fluid (CSF) provides a window into the central nervous system, and liquid biopsy of CSF could provide a relatively non-invasive means for disease diagnosis. There has yet to be an integrated analysis of the transcriptomic, metabolomic, and lipidomic changes occurring in the CSF of children with MB. CSF samples from patients with (n = 40) or without (n = 11; no cancer) MB were subjected to RNA-sequencing and high-resolution mass spectrometry to identify RNA, metabolite, and lipid profiles. Differentially expressed transcripts, metabolites, and lipids were identified and their biological significance assessed by pathway analysis. The DIABLO multivariate analysis package (R package mixOmics) was used to integrate the molecular changes characterizing the CSF of MB patients. Differentially expressed transcripts, metabolites, and lipids in CSF were discriminatory for the presence of MB but not the exact molecular subtype. One hundred and ten genes and ten circular RNAs were differentially expressed in MB CSF compared with normal, representing TGF-ß signaling, TNF-α signaling via NF-kB, and adipogenesis pathways. Tricarboxylic acid cycle and other metabolites (malate, fumarate, succinate, α-ketoglutarate, hydroxypyruvate, N-acetyl-aspartate) and total triacylglycerols were significantly upregulated in MB CSF compared with normal CSF. Although separating MBs into subgroups using transcriptomic, metabolomic, and lipid signatures in CSF was challenging, we were able to identify a group of omics signatures that could separate cancer from normal CSF. Metabolic and lipidomic profiles both contained indicators of tumor hypoxia. Our approach provides several candidate signatures that deserve further validation, including the novel circular RNA circ_463, and insights into the impact of MB on the CSF microenvironment.

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.

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.

A Conserved Tryptophan in the Ebola Virus Matrix Protein C-Terminal Domain Is Required for Efficient Virus-Like Particle Formation.

The Ebola virus (EBOV) harbors seven genes, one of which is the matrix protein eVP40, a peripheral protein that is sufficient to induce the formation of virus-like particles from the host cell plasma membrane. eVP40 can form different structures to fulfil different functions during the viral life cycle, although the structural dynamics of eVP40 that warrant dimer, hexamer, and octamer formation are still poorly understood. eVP40 has two conserved Trp residues at positions 95 and 191. The role of Trp95 has been characterized in depth as it serves as an important residue in eVP40 oligomer formation. To gain insight into the functional role of Trp191 in eVP40, we prepared mutations of Trp191 (W191A or W191F) to determine the effects of mutation on eVP40 plasma membrane localization and budding as well as eVP40 oligomerization. These in vitro and cellular experiments were complemented by molecular dynamics simulations of the wild-type (WT) eVP40 structure versus that of W191A. Taken together, Trp is shown to be a critical amino acid at position 191 as mutation to Ala reduces the ability of VP40 to localize to the plasma membrane inner leaflet and form new virus-like particles. Further, mutation of Trp191 to Ala or Phe shifted the in vitro equilibrium to the octamer form by destabilizing Trp191 interactions with nearby residues. This study has shed new light on the importance of interdomain interactions in stability of the eVP40 structure and the critical nature of timing of eVP40 oligomerization for plasma membrane localization and viral budding.

Potential RNA-dependent RNA polymerase inhibitors as prospective therapeutics against SARS-CoV-2.

Introduction. The emergence of SARS-CoV-2 has taken humanity off guard. Following an outbreak of SARS-CoV in 2002, and MERS-CoV about 10 years later, SARS-CoV-2 is the third coronavirus in less than 20 years to cross the species barrier and start spreading by human-to-human transmission. It is the most infectious of the three, currently causing the COVID-19 pandemic. No treatment has been approved for COVID-19. We previously proposed targets that can serve as binding sites for antiviral drugs for multiple coronaviruses, and here we set out to find current drugs that can be repurposed as COVID-19 therapeutics.Aim. To identify drugs against COVID-19, we performed an in silico virtual screen with the US Food and Drug Administration (FDA)-approved drugs targeting the RNA-dependent RNA polymerase (RdRP), a critical enzyme for coronavirus replication.Methodology. Initially, no RdRP structure of SARS-CoV-2 was available. We performed basic sequence and structural analysis to determine if RdRP from SARS-CoV was a suitable replacement. We performed molecular dynamics simulations to generate multiple starting conformations that were used for the in silico virtual screen. During this work, a structure of RdRP from SARS-CoV-2 became available and was also included in the in silico virtual screen.Results. The virtual screen identified several drugs predicted to bind in the conserved RNA tunnel of RdRP, where many of the proposed targets were located. Among these candidates, quinupristin is particularly interesting because it is expected to bind across the RNA tunnel, blocking access from both sides and suggesting that it has the potential to arrest viral replication by preventing viral RNA synthesis. Quinupristin is an antibiotic that has been in clinical use for two decades and is known to cause relatively minor side effects.Conclusion. Quinupristin represents a potential anti-SARS-CoV-2 therapeutic. At present, we have no evidence that this drug is effective against SARS-CoV-2 but expect that the biomedical community will expeditiously follow up on our in silico findings.

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.

Membrane pore formation and ion selectivity of the Ebola virus delta peptide.

The Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causes severe hemorrhagic fever with a high fatality rate in humans. The EBOV encodes a glycoprotein that when cleaved, produces the delta peptide. Experimental evidence suggests that the delta peptide functions as a viroporin that enhances virus particle release through the host cell membrane. However, the viroporin forming mechanism of the delta peptide is still not well understood. Guided by experimental information, we have computationally investigated the pore formation by different oligomers of the delta peptide. We have performed all-atom molecular dynamics (MD) simulations in an explicit membrane environment to investigate the pore-forming mechanism and stability of the pores. Our results suggest that the delta peptide forms stable pentameric pores. In addition, the pore is selective with respect to chloride ions, and the disulfide bond formed between Cys-29 and Cys-38 in the C-terminal of the peptide is essential for the pore stabilization and ion permeation. Our study provides helpful information on the pore-forming mechanism of filovirus delta peptides and such structural information can be important in designing and developing molecular modulators that target the delta peptide pore and disrupt the pathology of the Ebola virus.

In Silico Investigations of Calcium Phosphate Mineralization in Extracellular Vesicles.

Calcification in bone, cartilage, and cardiovascular tissues involves the release of specialized extracellular vesicles (EVs) that promote mineral nucleation. The small size of the EVs, however, makes molecular level studies difficult, and consequently uncertainty exists on the role and function of these structures in directing mineralization. The lack of mechanistic understanding associated with the initiators of ectopic mineral deposition has severely hindered the development of potential therapeutic options. Here, we used multiscale molecular dynamics simulations to investigate the calcification within the EVs. Results show that Ca2+-HPO42- and phosphatidylserine complexes facilitate the early nucleation. Use of coarse-grained simulations allows investigations of Ca2+-PO43- nucleation and crystallization in the EVs. Systematic variation in the ion-to-water ratio shows that the crystallization and growth strongly depend on the enrichment of the ions and dehydration inside the EVs. Our investigations provide insights into the role of EVs on calcium phosphate mineral nucleation and growth in both physiological and pathological mineralization.

Graphene-VP40 interactions and potential disruption of the Ebola virus matrix filaments.

Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic.