Enhanced Interfacial H-Bond Networks Promote Glycan-Glycan Recognition and Interaction.

H-bond networks at heterogeneous interfaces play crucial roles in bioseparation, biocatalysis, biochip array profiling, and functional nanosystem self-assembly, but their precise modulation and enhancement remain challenging. In this study, we have discovered that interfacial hydrophobic hydration significantly enhances H-bond networks at the interface between a glycan-modified adsorbent and a methanol-water-acetonitrile ternary solution. The enhanced H-bond networks greatly promote the adsorbent-solution heterogeneous glycan-glycan recognition and interaction. This novel hydrophobic hydration-enhanced hydrophilic interaction (HEHI) strategy improves the affinity and efficiency of intact glycopeptide enrichment. Compared with the commonly used hydrophilic-interaction enrichment strategy, 23.5 and 48.5% more intact N- and O-glycopeptides are identified, and the enrichment recoveries of half of the glycopeptides are increased >100%. Further, in-depth profiling of both N- and O-glycosylation occurring on SARS-CoV-2 S1 and hACE2 proteins has been achieved with more glycan types and novel O-glycosylation information involved. Interfacial hydrophobic hydration provides a powerful tool for the modulation of hydrophilic interactions in biological systems.

Interactions of SARS-CoV-2 and MERS-CoV fusion peptides measured using single-molecule force methods.

We address the challenge of understanding how hydrophobic interactions are encoded by fusion peptide (FP) sequences within coronavirus (CoV) spike proteins. Within the FPs of severe acute respiratory syndrome CoV 2 and Middle East respiratory syndrome CoV (MERS-CoV), a largely conserved peptide sequence called FP1 (SFIEDLLFNK and SAIEDLLFDK in SARS-2 and MERS, respectively) has been proposed to play a key role in encoding hydrophobic interactions that drive viral-host cell membrane fusion. Although a non-polar triad (Leu-Leu-Phe (LLF)) is common to both FP1 sequences, and thought to dominate the encoding of hydrophobic interactions, FP1 from SARS-2 and MERS differ in two residues (Phe 2 versus Ala 2 and Asn 9 versus Asp 9, respectively). Here we explore whether single-molecule force measurements can quantify hydrophobic interactions encoded by FP1 sequences, and then ask whether sequence variations between FP1 from SARS-2 and MERS lead to significant differences in hydrophobic interactions. We find that both SARS-2 and MERS wild-type FP1 generate measurable hydrophobic interactions at the single-molecule level, but that SARS-2 FP1 encodes a substantially stronger hydrophobic interaction than its MERS counterpart (1.91 ± 0.03 nN versus 0.68 ± 0.03 nN, respectively). By performing force measurements with FP1 sequences with single amino acid substitutions, we determine that a single-residue mutation (Phe 2 versus Ala 2) causes the almost threefold difference in the hydrophobic interaction strength generated by the FP1 of SARS-2 versus MERS, despite the presence of LLF in both sequences. Infrared spectroscopy and circular dichroism measurements support the proposal that the outsized influence of Phe 2 versus Ala 2 on the hydrophobic interaction arises from variation in the secondary structure adopted by FP1. Overall, these insights reveal how single-residue diversity in viral FPs, including FP1 of SARS-CoV-2 and MERS-CoV, can lead to substantial changes in intermolecular interactions proposed to play a key role in viral fusion, and hint at strategies for regulating hydrophobic interactions of peptides in a range of contexts.

Fat Checking: Emerging Role of Lipids in Metabolism and Disease.

Lipids are hydrophobic molecules involved in a plethora of biological functions; for example, they are employed for the storage of energy, serve as essential constituents of cell membranes and participate in the assembly of bilayer configuration [...].

Cationic Surfactants as Disinfectants against SARS-CoV-2.

The virucidal activity of a series of cationic surfactants differing in the length and number of hydrophobic tails (at the same hydrophilic head) and the structure of the hydrophilic head (at the same length of the hydrophobic n-alkyl tail) was compared. It was shown that an increase in the length and number of hydrophobic tails, as well as the presence of a benzene ring in the surfactant molecule, enhance the virucidal activity of the surfactant against SARS-CoV-2. This may be due to the more pronounced ability of such surfactants to penetrate and destroy the phospholipid membrane of the virus. Among the cationic surfactants studied, didodecyldimethylammonium bromide was shown to be the most efficient as a disinfectant, its 50% effective concentration (EC50) being equal to 0.016 mM. Two surfactants (didodecyldimethylammonium bromide and benzalkonium chloride) can deactivate SARS-CoV-2 in as little as 5 s.

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of the SARS-CoV-2 Spike Receptor-Binding Domain.

SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor-binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein's structure and function, and thus, comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid chromatography coupled to mass spectrometry has been widely used to characterize post-translational modifications in proteins, including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogeneous glycans at the intact protein level. Herein, we evaluated several online separation techniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2) capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS). Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facile comparison of biosimilars in quality control applications.

Superhydrophobic polypropylene sorbent derived from discarded face masks: A highly efficient adsorbent for oil spill sorbent.

Globally, an estimated 130 billion face masks are used and disposed of every month. Thus, recycling or upcycling discarded face masks has attracted significant attention due to economic benefits and environmental concerns. To reduce the amount of used face masks going to waste, this study features a superhydrophobic face mask prepared by simple chemical modification with environmentally preferable alkane solvents (n-hexane, n-heptane, and n-decane), that is effective as a sorbent for oil spill cleanup. All alkanes examined increased the surface roughness of the face masks and improved face mask hydrophobicity. The heptane treated face mask (at 90 °C for 1 h), can adsorbed Arabian light crude oil up to 21 times of their weight on the water surface. In addition, chloroform, toluene, gasoline, and diesel were adsorbed 18, 13, 8 and 16 times, respectively. More importantly, heptane has a high recycling efficiency as a treatment solvent and is reusable for at least 10 cycles of mask surface treatment. Consequently, this inexpensive and easily fabricated material is a promising development in waste face mask (WFM) upcycling.

New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition.

The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinones as their electron acceptors. We have designed this study to understand the interaction of an N-terminally truncated human DHODH (HsΔ29DHODH) and the DHODH from Escherichia coli (EcDHODH) with ubiquinone (Q10) in supported lipid membranes using neutron reflectometry (NR). NR has allowed us to determine in situ, under solution conditions, how the enzymes bind to lipid membranes and to unambiguously resolve the location of Q10. Q10 is exclusively located at the center of all of the lipid bilayers investigated, and upon binding, both of the DHODHs penetrate into the hydrophobic region of the outer lipid leaflet towards the Q10. We therefore show that the interaction between the soluble enzymes and the membrane-embedded Q10 is mediated by enzyme penetration. We can also show that EcDHODH binds more efficiently to the surface of simple bilayers consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine, and tetraoleoyl cardiolipin than HsΔ29DHODH, but does not penetrate into the lipids to the same degree. Our results also highlight the importance of Q10, as well as lipid composition, on enzyme binding.

Omicron and Delta variant of SARS-CoV-2: A comparative computational study of spike protein.

Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially those of concern, may have an impact on the virus's transmissibility and pathogenicity, as well as diagnostic equipment performance and vaccine effectiveness. Even though the SARS-CoV-2 Delta variant (B.1.617.2) emerged during India's second wave of infections, Delta variants have grown dominant internationally and are still evolving. On November 26, 2021, World Health Organization identified the variant B.1.1.529 as a variant of concern, naming it Omicron, based on evidence that Omicron contains numerous mutations that may influence its behavior. However, the mode of transmission and severity of the Omicron variant remains unknown. We used computational studies to examine the Delta and Omicron variants in this study and found that the Omicron variant had a higher affinity for human angiotensin-converting enzyme 2 (ACE2) than the Delta variant due to a significant number of mutations in the SARS-CoV-2 receptor-binding domain (RBD), indicating a higher potential for transmission. Based on docking studies, the Q493R, N501Y, S371L, S373P, S375F, Q498R, and T478K mutations contribute significantly to high binding affinity with human ACE2. In comparison to the Delta variant, both the entire spike protein and the RBD in Omicron include a high proportion of hydrophobic amino acids such as leucine and phenylalanine. These amino acids are located within the protein's core and are required for structural stability. We observed a disorder-order transition in the Omicron variant between spike protein RBD regions 468-473, and it may be significant in the influence of disordered residues/regions on spike protein stability and binding to ACE2. A future study might investigate the epidemiological and biological consequences of the Omicron variant.

Structural polymorphism of coiled-coils from the stalk domain of SARS-CoV-2 spike protein.

Spike trimer plays a key role in SARS-CoV-2 infection and vaccine development. It consists of a globular head and a flexible stalk domain that anchors the protein into the viral membrane. While the head domain has been extensively studied, the properties of the adjoining stalk are poorly understood. Here, we characterize the coiled-coil formation and thermodynamic stability of the stalk domain and its segments. We find that the N-terminal segment of the stalk does not form coiled-coils and remains disordered in solution. The C-terminal stalk segment forms a trimeric coiled-coil in solution, which becomes significantly stabilized in the context of the full-length stalk. Its crystal structure reveals a novel antiparallel tetramer coiled-coil with an unusual combination of a-d and e-a-d hydrophobic core packing. Structural analysis shows that a subset of hydrophobic residues stabilizes different coiled-coil structures: trimer, tetramer, and heterohexamer, underscoring a highly polymorphic nature of the SARS-CoV-2 stalk sequence.

The spectrum between substrates and inhibitors: Pinpointing the binding mode of dengue protease ligands with modulated basicity and hydrophobicity.

Peptides can be inhibitors and substrates of proteases. The present study describes the inhibitor- vs. substrate-like properties of peptidic ligands of dengue protease which were designed to provide insight into their binding modes. Of particular interest was the localization of the cleavable peptide bond and the placement of hydrophobic elements in the binding site. The findings provide clues for the design of covalent inhibitors in which electrophilic functional groups bind to the catalytic serine, and in addition for the development of inhibitors that are less basic than the natural substrate and therefore have an improved pharmacokinetic profile. We observed a tendency of basic elements to favor a substrate-like binding mode, whereas hydrophobic elements decrease or eliminate enzymatic cleavage. This indicates a necessity to include basic elements which closely mimic the natural substrates into covalent inhibitors, posing a challenge from the chemical and pharmacokinetic perspective. However, hydrophobic elements may offer opportunities to develop non-covalent inhibitors with a favorable ADME profile and potentially improved target-binding kinetics.

Underlying selection for the diversity of spike protein sequences of SARS-CoV-2.

The global spread of SARS-CoV-2 is fast moving and has caused a worldwide public health crisis. In the present article, we analyzed spike protein sequences of SARS-CoV-2 genomes to assess the impact of mutational diversity. We observed from amino acid usage patterns that spike proteins are associated with a diversity of mutational changes and most important underlying cause of variation of amino acid usage is the changes in hydrophobicity of spike proteins. The changing patterns of hydrophobicity of spike proteins over time and its influence on the receptor binding affinity provides crucial information on the SARS-CoV-2 interaction with human receptor. Our results also show that spike proteins have evolved to prefer more hydrophobic residues over time. The present study provides a comprehensive analysis of molecular sequence data to consider that mutational variants might play a crucial role in modulating the virulence and spread of the virus and has immediate implications for therapeutic strategies.

In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI-MS spectrum.

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs.

Nanometer-Thick Superhydrophobic Coating Renders Cloth Mask Potentially Effective against Aerosol-Driven Infections.

The advent of COVID-19 pandemic has made it necessary to wear masks across populations. While the N95 mask offers great performance against airborne infections, its multilayered sealed design makes it difficult to breathe for a longer duration of use. The option of using highly breathable cloth or silk masks especially for a large populace is fraught with the danger of infection. As a normal cloth or silk mask absorbs airborne liquid, it can be a source of plausible infection. We demonstrate the chemical modification of one such mask, Eri silk, to make it hydrophobic (contact angle of water is 143.7°), which reduces the liquid absorption capacity without reducing the breathability of the mask significantly. The breathability reduces only 22% for hydrophobic Eri silk compared to the pristine Eri silk, whereas N95 shows a 59% reduction of breathability. The modified hydrophobic silk can repel the incoming aqueous liquid droplets without wetting the surface. The results indicate that a multilayered modified silk mask to make it hydrophobic can be an affordable and breathable alternative to the N95 mask.

Phospholipids dock SARS-CoV-2 spike protein via hydrophobic interactions: a minimal in-silico study of lecithin nasal spray therapy.

Understanding the physical and chemical properties of viral infections at molecular scales is a major challenge for the scientific community more so with the outbreak of global pandemics. There is currently a lot of effort being placed in identifying molecules that could act as putative drugs or blockers of viral molecules. In this work, we computationally explore the importance in antiviral activity of a less studied class of molecules, namely surfactants. We employ all-atoms molecular dynamics simulations to study the interaction between the receptor-binding domain of the SARS-CoV-2 spike protein and the phospholipid lecithin (POPC), in water. Our microsecond simulations show a preferential binding of lecithin to the receptor-binding motif of SARS-CoV-2 with binding free energies significantly larger than [Formula: see text]. Furthermore, hydrophobic interactions involving lecithin non-polar tails dominate these binding events, which are also accompanied by dewetting of the receptor binding motif. Through an analysis of fluctuations in the radius of gyration of the receptor-binding domain, its contact maps with lecithin molecules, and distributions of water molecules near the binding region, we elucidate molecular interactions that may play an important role in interactions involving surfactant-type molecules and viruses. We discuss our minimal computational model in the context of lecithin-based liposomal nasal sprays as putative mitigating therapies for COVID-19.

Toward nanotechnology-enabled face masks against SARS-CoV-2 and pandemic respiratory diseases.

Wearing a face mask has become a necessity following the outbreak of the coronavirus (COVID-19) disease, where its effectiveness in containing the pandemic has been confirmed. Nevertheless, the pandemic has revealed major deficiencies in the ability to manufacture and ramp up worldwide production of efficient surgical-grade face masks. As a result, many researchers have focused their efforts on the development of low cost, smart and effective face covers. In this article, following a short introduction concerning face mask requirements, the different nanotechnology-enabled techniques for achieving better protection against the SARS-CoV-2 virus are reviewed, including the development of nanoporous and nanofibrous membranes in addition to triboelectric nanogenerators based masks, which can filter the virus using various mechanisms such as straining, electrostatic attraction and electrocution. The development of nanomaterials-based mask coatings to achieve virus repellent and sterilizing capabilities, including antiviral, hydrophobic and photothermal features are also discussed. Finally, the usability of nanotechnology-enabled face masks is discussed and compared with that of current commercial-grade N95 masks. To conclude, we highlight the challenges associated with the quick transfer of nanomaterials-enabled face masks and provide an overall outlook of the importance of nanotechnology in counteracting the COVID-19 and future pandemics.

Targeted Delivery of mRNA with One-Component Ionizable Amphiphilic Janus Dendrimers.

Targeted and efficient delivery of nucleic acids with viral and synthetic vectors is the key step of genetic nanomedicine. The four-component lipid nanoparticle synthetic delivery systems consisting of ionizable lipids, phospholipids, cholesterol, and a PEG-conjugated lipid, assembled by microfluidic or T-tube technology, have been extraordinarily successful for delivery of mRNA to provide Covid-19 vaccines. Recently, we reported a one-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) synthetic delivery system for mRNA relying on amphiphilic Janus dendrimers and glycodendrimers developed in our laboratory. Amphiphilic Janus dendrimers consist of functional hydrophilic dendrons conjugated to hydrophobic dendrons. Co-assembly of IAJDs with mRNA into dendrimersome nanoparticles (DNPs) occurs by simple injection in acetate buffer, rather than by microfluidic devices, and provides a very efficient system for delivery of mRNA to lung. Here we report the replacement of most of the hydrophilic fragment of the dendron from IAJDs, maintaining only its ionizable amine, while changing its interconnecting group to the hydrophobic dendron from amide to ester. The resulting IAJDs demonstrated that protonated ionizable amines play dual roles of hydrophilic fragment and binding ligand for mRNA, changing delivery from lung to spleen and/or liver. Replacing the interconnecting ester with the amide switched the delivery back to lung. Delivery predominantly to liver is favored by pairs of odd and even alkyl groups in the hydrophobic dendron. This simple structural change transformed the targeted delivery of mRNA mediated with IAJDs, from lung to liver and spleen, and expands the utility of DNPs from therapeutics to vaccines.

Diverse Mechanisms of Antimicrobial Activities of Lactoferrins, Lactoferricins, and Other Lactoferrin-Derived Peptides.

Lactoferrins are an iron-binding glycoprotein that have important protective roles in the mammalian body through their numerous functions, which include antimicrobial, antitumor, anti-inflammatory, immunomodulatory, and antioxidant activities. Among these, their antimicrobial activity has been the most studied, although the mechanism behind antimicrobial activities remains to be elucidated. Thirty years ago, the first lactoferrin-derived peptide was isolated and showed higher antimicrobial activity than the native lactoferrin lactoferricin. Since then, numerous studies have investigated the antimicrobial potencies of lactoferrins, lactoferricins, and other lactoferrin-derived peptides to better understand their antimicrobial activities at the molecular level. This review defines the current antibacterial, antiviral, antifungal, and antiparasitic activities of lactoferrins, lactoferricins, and lactoferrin-derived peptides. The primary focus is on their different mechanisms of activity against bacteria, viruses, fungi, and parasites. The role of their structure, amino-acid composition, conformation, charge, hydrophobicity, and other factors that affect their mechanisms of antimicrobial activity are also reviewed.

BCEPS: A Web Server to Predict Linear B Cell Epitopes with Enhanced Immunogenicity and Cross-Reactivity.

Prediction of linear B cell epitopes is of interest for the production of antigen-specific antibodies and the design of peptide-based vaccines. Here, we present BCEPS, a web server for predicting linear B cell epitopes tailored to select epitopes that are immunogenic and capable of inducing cross-reactive antibodies with native antigens. BCEPS implements various machine learning models trained on a dataset including 555 linearized conformational B cell epitopes that were mined from antibody-antigen protein structures. The best performing model, based on a support vector machine, reached an accuracy of 75.38% ± 5.02. In an independent dataset consisting of B cell epitopes retrieved from the Immune Epitope Database (IEDB), this model achieved an accuracy of 67.05%. In BCEPS, predicted epitopes can be ranked according to properties such as flexibility, accessibility and hydrophilicity, and with regard to immunogenicity, as judged by their predicted presentation by MHC II molecules. BCEPS also detects if predicted epitopes are located in ectodomains of membrane proteins and if they possess N-glycosylation sites hindering antibody recognition. Finally, we exemplified the use of BCEPS in the SARS-CoV-2 Spike protein, showing that it can identify B cell epitopes targeted by neutralizing antibodies.

Microscopic interactions between ivermectin and key human and viral proteins involved in SARS-CoV-2 infection.

The identification of chemical compounds able to bind specific sites of the human/viral proteins involved in the SARS-CoV-2 infection cycle is a prerequisite to design effective antiviral drugs. Here we conduct a molecular dynamics study with the aim to assess the interactions of ivermectin, an antiparasitic drug with broad-spectrum antiviral activity, with the human Angiotensin-Converting Enzyme 2 (ACE2), the viral 3CLpro and PLpro proteases, and the viral SARS Unique Domain (SUD). The drug/target interactions have been characterized in silico by describing the nature of the non-covalent interactions found and by measuring the extent of their time duration along the MD simulation. Results reveal that the ACE2 protein and the ACE2/RBD aggregates form the most persistent interactions with ivermectin, while the binding with the remaining viral proteins is more limited and unspecific.

Interaction between SARS-CoV-2 spike glycoprotein and human skin models: a molecular dynamics study.

The possibility of contamination of human skin by infectious virions plays an important role in indirect transmission of respiratory viruses but little is known about the fundamental physico-chemical aspects of the virus-skin interactions. In the case of coronaviruses, the interaction with surfaces (including the skin surface) is mediated by their large glycoprotein spikes that protrude from (and cover) the viral envelope. Here, we perform all atomic simulations between the SARS-CoV-2 spike glycoprotein and human skin models. We consider an "oily" skin covered by sebum and a "clean" skin exposing the stratum corneum. The simulations show that the spike tries to maximize the contacts with stratum corneum lipids, particularly ceramides, with substantial hydrogen bonding. In the case of "oily" skin, the spike is able to retain its structure, orientation and hydration over sebum with little interaction with sebum components. Comparison of these results with our previous simulations of the interaction of SARS-CoV-2 spike with hydrophilic and hydrophobic solid surfaces, suggests that the "soft" or "hard" nature of the surface plays an essential role in the interaction of the spike protein with materials.