Zn2+ and Cu2+ Interaction with the Recognition Interface of ACE2 for SARS-CoV-2 Spike Protein.

The spike protein (S) of SARS-CoV-2 is able to bind to the human angiotensin-converting enzyme 2 (ACE2) receptor with a much higher affinity compared to other coronaviruses. The binding interface between the ACE2 receptor and the spike protein plays a critical role in the entry mechanism of the SARS-CoV-2 virus. There are specific amino acids involved in the interaction between the S protein and the ACE2 receptor. This specificity is critical for the virus to establish a systemic infection and cause COVID-19 disease. In the ACE2 receptor, the largest number of amino acids playing a crucial role in the mechanism of interaction and recognition with the S protein is located in the C-terminal part, which represents the main binding region between ACE2 and S. This fragment is abundant in coordination residues such as aspartates, glutamates, and histidine that could be targeted by metal ions. Zn2+ ions bind to the ACE2 receptor in its catalytic site and modulate its activity, but it could also contribute to the structural stability of the entire protein. The ability of the human ACE2 receptor to coordinate metal ions, such as Zn2+, in the same region where it binds to the S protein could have a crucial impact on the mechanism of recognition and interaction of ACE2-S, with consequences on their binding affinity that deserve to be investigated. To test this possibility, this study aims to characterize the coordination ability of Zn2+, and also Cu2+ for comparison, with selected peptide models of the ACE2 binding interface using spectroscopic and potentiometric techniques.

Elucidation of novel compounds and epitope-based peptide vaccine design against C30 endopeptidase regions of SARS-CoV-2 using immunoinformatics approaches.

There has been progressive improvement in immunoinformatics approaches for epitope-based peptide design. Computational-based immune-informatics approaches were applied to identify the epitopes of SARS-CoV-2 to develop vaccines. The accessibility of the SARS-CoV-2 protein surface was analyzed, and hexa-peptide sequences (KTPKYK) were observed having a maximum score of 8.254, located between amino acids 97 and 102, whereas the FSVLAC at amino acids 112 to 117 showed the lowest score of 0.114. The surface flexibility of the target protein ranged from 0.864 to 1.099 having amino acid ranges of 159 to 165 and 118 to 124, respectively, harboring the FCYMHHM and YNGSPSG hepta-peptide sequences. The surface flexibility was predicted, and a 0.864 score was observed from amino acids 159 to 165 with the hepta-peptide (FCYMHHM) sequence. Moreover, the highest score of 1.099 was observed between amino acids 118 and 124 against YNGSPSG. B-cell epitopes and cytotoxic T-lymphocyte (CTL) epitopes were also identified against SARS-CoV-2. In molecular docking analyses, -0.54 to -26.21 kcal/mol global energy was observed against the selected CTL epitopes, exhibiting binding solid energies of -3.33 to -26.36 kcal/mol. Based on optimization, eight epitopes (SEDMLNPNY, GSVGFNIDY, LLEDEFTPF, DYDCVSFCY, GTDLEGNFY, QTFSVLACY, TVNVLAWLY, and TANPKTPKY) showed reliable findings. The study calculated the associated HLA alleles with MHC-I and MHC-II and found that MHC-I epitopes had higher population coverage (0.9019% and 0.5639%) than MHC-II epitopes, which ranged from 58.49% to 34.71% in Italy and China, respectively. The CTL epitopes were docked with antigenic sites and analyzed with MHC-I HLA protein. In addition, virtual screening was conducted using the ZINC database library, which contained 3,447 compounds. The 10 top-ranked scrutinized molecules (ZINC222731806, ZINC077293241, ZINC014880001, ZINC003830427, ZINC030731133, ZINC003932831, ZINC003816514, ZINC004245650, ZINC000057255, and ZINC011592639) exhibited the least binding energy (-8.8 to -7.5 kcal/mol). The molecular dynamics (MD) and immune simulation data suggest that these epitopes could be used to design an effective SARS-CoV-2 vaccine in the form of a peptide-based vaccine. Our identified CTL epitopes have the potential to inhibit SARS-CoV-2 replication.

The Omicron Sub-Variant BA.4 Displays a Remarkable Lack of Clinical Signs in a Golden Syrian Hamster Model of SARS-CoV-2 Infection.

The ongoing emergence of SARS-CoV-2 virus variants remains a source of concern because it is accompanied by the potential for increased virulence as well as evasion of immunity. Here we show that, although having an almost identical spike gene sequence as another Omicron variant (BA.5.2.1), a BA.4 isolate lacked all the typical disease characteristics of other isolates seen in the Golden Syrian hamster model despite replicating almost as effectively. Animals infected with BA.4 had similar viral shedding profiles to those seen with BA.5.2.1 (up to day 6 post-infection), but they all failed to lose weight or present with any other significant clinical signs. We hypothesize that this lack of detectable signs of disease during infection with BA.4 was due to a small (nine nucleotide) deletion (∆686-694) in the viral genome (ORF1ab) responsible for the production of non-structural protein 1, which resulted in the loss of three amino acids (aa 141-143).

In vitro selection of macrocyclic peptide inhibitors containing cyclic γ2,4-amino acids targeting the SARS-CoV-2 main protease.

γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cyclic γ2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ1) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ2), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent Mpro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ1 at the fourth position, manifests a 5.2 nM dissociation constant. An Mpro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ1 interacts with the S1' catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and Mpro enabled production of a variant with a 5-fold increase in potency.

Structural Dynamics Predominantly Determine the Adaptability of Proteins to Amino Acid Deletions.

The insertion or deletion (indel) of amino acids has a variety of effects on protein function, ranging from disease-forming changes to gaining new functions. Despite their importance, indels have not been systematically characterized towards protein engineering or modification goals. In the present work, we focus on deletions composed of multiple contiguous amino acids (mAA-dels) and their effects on the protein (mutant) folding ability. Our analysis reveals that the mutant retains the native fold when the mAA-del obeys well-defined structural dynamics properties: localization in intrinsically flexible regions, showing low resistance to mechanical stress, and separation from allosteric signaling paths. Motivated by the possibility of distinguishing the features that underlie the adaptability of proteins to mAA-dels, and by the rapid evaluation of these features using elastic network models, we developed a positive-unlabeled learning-based classifier that can be adopted for protein design purposes. Trained on a consolidated set of features, including those reflecting the intrinsic dynamics of the regions where the mAA-dels occur, the new classifier yields a high recall of 84.3% for identifying mAA-dels that are stably tolerated by the protein. The comparative examination of the relative contribution of different features to the prediction reveals the dominant role of structural dynamics in enabling the adaptation of the mutant to mAA-del without disrupting the native fold.

Consensus insertion/deletions and amino acid variations of all coding and noncoding regions of the SARS-CoV-2 Omicron clades, including the XBB and BQ.1 lineages.

The currently dominant Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has swiftly diverged into clades. To predict the probable impact of these clades, the consensus insertions/deletions (indels) and amino acid substitutions of the whole genome of clades were compared with the original SARS-CoV-2 strain. The evolutionary history of representatives of clades and lineages was inferred using the maximum-likelihood method and tested using the bootstrap method. The indels and polymorphic amino acids were found to be either clade-specific or shared among clades. The 21K clade has unique indels and substitutions, which probably represent reverted indels/substitutions. Three variations that appear to be associated with SARS-CoV-2 attenuation in the Omicron clades included a deletion in the nucleocapsid gene, a deletion in the 3'untranslated region, and a truncation in open reading frame 8. Phylogenetic analysis showed that the Omicron clades and lineages form three separate clusters.

The novel Nsp9-interacting host factor H2BE promotes PEDV replication by inhibiting endoplasmic reticulum stress-mediated apoptosis.

Porcine epidemic diarrhoea (PED) caused by porcine epidemic diarrhoea virus (PEDV) has led to significant economic losses in the swine industry worldwide. Histone Cluster 2, H2BE (HIST2H2BE), the main protein component in chromatin, has been proposed to play a key role in apoptosis. However, the relationship between H2BE and PEDV remains unclear. In this study, H2BE was shown to bind and interact with PEDV nonstructural protein 9 (Nsp9) via immunoprecipitation-mass spectrometry (IP-MS). Next, we verified the interaction of Nsp9 with H2BE by immunoprecipitation and immunofluorescence. H2BE colocalized with Nsp9 in the cytoplasm and nuclei. PEDV Nsp9 upregulated the expression of H2BE by inhibiting the expression of IRX1. We demonstrated that overexpression of H2BE significantly promoted PEDV replication, whereas knockdown of H2BE by small interfering RNA (siRNA) inhibited PEDV replication. Overexpression of H2BE led to significantly inhibited GRP78 expression, phosphorylated PERK (p-PERK), phosphorylated eIF2 (p-eIF2), phosphorylated IRE1 (p-IRE1), and phosphorylated JNK (p-JNK); negatively regulated CHOP and Bax expression and caspase-9 and caspase-3 cleavage; and promoted Bcl-2 production. Knocking down H2BE exerted the opposite effects. Furthermore, we found that after deletion of amino acids 1-28, H2BE did not promote PEDV replication. In conclusion, these studies revealed the mechanism by which H2BE is associated with ER stress-mediated apoptosis to regulate PEDV replication. Nsp9 upregulates H2BE. H2BE plays a role in inhibiting apoptosis and thus facilitating viral replication, which depends on the N-terminal region of H2BE (amino acids 1-28). These findings provide a reference for host-PEDV interactions and offer the possibility for developing strategies for PEDV decontamination and prevention.

Protein nanopore reveals the renin-angiotensin system crosstalk with single-amino-acid resolution.

The discovery of crosstalk effects on the renin-angiotensin system (RAS) is limited by the lack of approaches to quantitatively monitor, in real time, multiple components with subtle differences and short half-lives. Here we report a nanopore framework to quantitatively determine the effect of the hidden crosstalk between angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) on RAS. By developing an engineered aerolysin nanopore capable of single-amino-acid resolution, we show that the ACE can be selectively inhibited by ACE2 to prevent cleavage of angiotensin I, even when the concentration of ACE is more than 30-fold higher than that of ACE2. We also show that the activity of ACE2 for cleaving angiotensin peptides is clearly suppressed by the spike protein of SARS-CoV-2. This leads to the relaxation of ACE and the increased probability of accumulation of the principal effector angiotensin II. The spike protein of the SARS-CoV-2 Delta variant is demonstrated to have a much greater impact on the crosstalk than the wild type.

HIV-1 Tat amino acid residues that influence Tat-TAR binding affinity: a scoping review.

HIV-1 remains a global health concern and to date, nearly 38 million people are living with HIV. The complexity of HIV-1 pathogenesis and its subsequent prevalence is influenced by several factors including the HIV-1 subtype. HIV-1 subtype variation extends to sequence variation in the amino acids of the HIV-1 viral proteins. Of particular interest is the transactivation of transcription (Tat) protein due to its key function in viral transcription. The Tat protein predominantly functions by binding to the transactivation response (TAR) RNA element to activate HIV-1 transcriptional elongation. Subtype-specific Tat protein sequence variation influences Tat-TAR binding affinity. Despite several studies investigating Tat-TAR binding, it is not clear which regions of the Tat protein and/or individual Tat amino acid residues may contribute to TAR binding affinity. We, therefore, conducted a scoping review on studies investigating Tat-TAR binding. We aimed to synthesize the published data to determine (1) the regions of the Tat protein that may be involved in TAR binding, (2) key Tat amino acids involved in TAR binding and (3) if Tat subtype-specific variation influences TAR binding. A total of thirteen studies met our inclusion criteria and the key findings were that (1) both N-terminal and C-terminal amino acids outside the basic domain (47-59) may be important in increasing Tat-TAR binding affinity, (2) substitution of the amino acids Lysine and Arginine (47-59) resulted in a reduction in binding affinity to TAR, and (3) none of the included studies have investigated Tat subtype-specific substitutions and therefore no commentary could be made regarding which subtype may have a higher Tat-TAR binding affinity. Future studies investigating Tat-TAR binding should therefore use full-length Tat proteins and compare subtype-specific variations. Studies of such a nature may help explain why we see differential pathogenesis and prevalence when comparing HIV-1 subtypes.

Probing Ligand Binding Sites on Large Proteins by Nuclear Magnetic Resonance Spectroscopy of Genetically Encoded Non-Canonical Amino Acids.

N6-(((trimethylsilyl)-methoxy)carbonyl)-l-lysine (TMSK) and N6-trifluoroacetyl-l-lysine (TFAK) are non-canonical amino acids, which can be installed in proteins by genetic encoding. In addition, we describe a new aminoacyl-tRNA synthetase specific for N6-(((trimethylsilyl)methyl)-carbamoyl)-l-lysine (TMSNK), which is chemically more stable than TMSK. Using the dimeric SARS-CoV-2 main protease (Mpro) as a model system with three different ligands, we show that the 1H and 19F nuclei of the solvent-exposed trimethylsilyl and CF3 groups produce intense signals in the nuclear magnetic resonance (NMR) spectrum. Their response to active-site ligands differed significantly when positioned near rather than far from the active site. Conversely, the NMR probes failed to confirm the previously reported binding site of the ligand pelitinib, which was found to enhance the activity of Mpro by promoting the formation of the enzymatically active dimer. In summary, the amino acids TMSK, TMSNK, and TFAK open an attractive path for site-specific NMR analysis of ligand binding to large proteins of limited stability and at low concentrations.

PSAC-PDB: Analysis and classification of protein structures.

This paper presents a novel framework, called PSAC-PDB, for analyzing and classifying protein structures from the Protein Data Bank (PDB). PSAC-PDB first finds, analyze and identifies protein structures in PDB that are similar to a protein structure of interest using a protein structure comparison tool. Second, the amino acids (AA) sequences of identified protein structures (obtained from PDB), their aligned amino acids (AAA) and aligned secondary structure elements (ASSE) (obtained by structural alignment), and frequent AA (FAA) patterns (discovered by sequential pattern mining), are used for the reliable detection/classification of protein structures. Eleven classifiers are used and their performance is compared using six evaluation metrics. Results show that three classifiers perform well on overall, and that FAA patterns can be used to efficiently classify protein structures in place of providing the whole AA sequences, AAA or ASSE. Furthermore, better classification results are obtained using AAA of protein structures rather than AA sequences. PSAC-PDB also performed better than state-of-the-art approaches for SARS-CoV-2 genome sequences classification.

Trinucleotide Substitutions at Two Locations in the SARS-CoV-2 Nucleocapsid (N) Gene.

The genomes of sarbecoviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), incorporate mutations with short sequence exchanges based on unknown processes. Currently, the presence of such short-sequence exchanges among the genomes of different SARS-CoV-2 lineages remains uncertain. In the present study, multiple SARS-CoV-2 genome sequences from different clades or sublineages were collected from an international mass sequence database and compared to identify the presence of short sequence exchanges. Initial screening with multiple sequence alignments identified two locations with trinucleotide substitutions, both in the nucleocapsid (N) gene. The first exchange from 5'-GAT-3' to 5'-CTA-3' at nucleotide positions 28,280-28,282 resulted in a change in the amino acid from aspartic acid (D) to leucine (L), which was predominant in clade GRY (Alpha). The second exchange from 5'-GGG-3' to 5'-AAC-3' at nucleotide positions 28,881-28,883 resulted in an amino acid change from arginine and glycine (RG) to lysine and arginine (KR), which was predominant in GR (Gamma), GRY (Alpha), and GRA (Omicron). Both trinucleotide substitutions occurred before June 2020. The sequence identity rate between these lineages suggests that coincidental succession of single-nucleotide substitutions is unlikely. Basic local alignment search tool sequence search revealed the absence of intermediating mutations based on single-base substitutions or overlapping indels before the emergence of these trinucleotide substitutions. These findings suggest that trinucleotide substitutions could have developed via an en bloc exchange. In summary, trinucleotide substitutions at two locations in the SARS-CoV-2 N gene were identified. This mutation may provide insights into the evolution of SARS-CoV-2.

Inhibition of major histocompatibility complex-I antigen presentation by sarbecovirus ORF7a proteins.

Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8+ cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately fivefold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I down-regulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I down-regulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8+ T cells. Specifically, ORF7a prevented the assembly of the MHC-I peptide loading complex and caused retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccine-elicited immunity.

Genomic Perspectives on the Emerging SARS-CoV-2 Omicron Variant.

A new variant of concern for SARS-CoV-2, Omicron (B.1.1.529), was designated by the World Health Organization on November 26, 2021. This study analyzed the viral genome sequencing data of 108 samples collected from patients infected with Omicron. First, we found that the enrichment efficiency of viral nucleic acids was reduced due to mutations in the region where the primers anneal to. Second, the Omicron variant possesses an excessive number of mutations compared to other variants circulating at the same time (median: 62 vs. 45), especially in the Spike gene. Mutations in the Spike gene confer alterations in 32 amino acid residues, more than those observed in other SARS-CoV-2 variants. Moreover, a large number of nonsynonymous mutations occur in the codons for the amino acid residues located on the surface of the Spike protein, which could potentially affect the replication, infectivity, and antigenicity of SARS-CoV-2. Third, there are 53 mutations between the Omicron variant and its closest sequences available in public databases. Many of these mutations were rarely observed in public databases and had a low mutation rate. In addition, the linkage disequilibrium between these mutations was low, with a limited number of mutations concurrently observed in the same genome, suggesting that the Omicron variant would be in a different evolutionary branch from the currently prevalent variants. To improve our ability to detect and track the source of new variants rapidly, it is imperative to further strengthen genomic surveillance and data sharing globally in a timely manner.

Potential antiviral peptides targeting the SARS-CoV-2 spike protein.

BACKGROUND: The coronavirus disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection became an international pandemic and created a public health crisis. The binding of the viral Spike glycoprotein to the human cell receptor angiotensin-converting enzyme 2 (ACE2) initiates viral infection. The development of efficient treatments to combat coronavirus disease is considered essential. METHODS: An in silico approach was employed to design amino acid peptide inhibitor against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The designed inhibitor (SARS-CoV-2 PEP 49) consists of amino acids with the α1 helix and the ß4 - ß5 sheets of ACE2. The PEP-FOLD3 web tool was used to create the 3D structures of the peptide amino acids. Analyzing the interaction between ACE2 and the RBD of the Spike protein for three protein data bank entries (6M0J, 7C8D, and 7A95) indicated that the interacting amino acids were contained inside two regions of ACE2: the α1 helical protease domain (PD) and the ß4 - ß5 sheets. RESULTS: Molecular docking analysis of the designed inhibitor demonstrated that SARS-CoV-2 PEP 49 attaches directly to the ACE2 binding site of the Spike protein with a binding affinity greater than the ACE2, implying that the SARS-CoV-2 PEP 49 model may be useful as a potential RBD binding blocker.

Analysis of Bacteriophage Behavior of a Human RNA Virus, SARS-CoV-2, through the Integrated Approach of Immunofluorescence Microscopy, Proteomics and D-Amino Acid Quantification.

SARS-CoV-2, one of the human RNA viruses, is widely studied around the world. Significant efforts have been made to understand its molecular mechanisms of action and how it interacts with epithelial cells and the human microbiome since it has also been observed in gut microbiome bacteria. Many studies emphasize the importance of surface immunity and also that the mucosal system is critical in the interaction of the pathogen with the cells of the oral, nasal, pharyngeal, and intestinal epithelium. Recent studies have shown how bacteria in the human gut microbiome produce toxins capable of altering the classical mechanisms of interaction of viruses with surface cells. This paper presents a simple approach to highlight the initial behavior of a novel pathogen, SARS-CoV-2, on the human microbiome. The immunofluorescence microscopy technique can be combined with spectral counting performed at mass spectrometry of viral peptides in bacterial cultures, along with identification of the presence of D-amino acids within viral peptides in bacterial cultures and in patients' blood. This approach makes it possible to establish the possible expression or increase of viral RNA viruses in general and SARS-CoV-2, as discussed in this study, and to determine whether or not the microbiome is involved in the pathogenetic mechanisms of the viruses. This novel combined approach can provide information more rapidly, avoiding the biases of virological diagnosis and identifying whether a virus can interact with, bind to, and infect bacteria and epithelial cells. Understanding whether some viruses have bacteriophagic behavior allows vaccine therapies to be focused either toward certain toxins produced by bacteria in the microbiome or toward finding inert or symbiotic viral mutations with the human microbiome. This new knowledge opens a scenario on a possible future vaccine: the probiotics vaccine, engineered with the right resistance to viruses that attach to both the epithelium human surface and gut microbiome bacteria.

Coordinated evolution at amino acid sites of SARS-CoV-2 spike.

SARS-CoV-2 has adapted in a stepwise manner, with multiple beneficial mutations accumulating in a rapid succession at origins of VOCs, and the reasons for this are unclear. Here, we searched for coordinated evolution of amino acid sites in the spike protein of SARS-CoV-2. Specifically, we searched for concordantly evolving site pairs (CSPs) for which changes at one site were rapidly followed by changes at the other site in the same lineage. We detected 46 sites which formed 45 CSP. Sites in CSP were closer to each other in the protein structure than random pairs, indicating that concordant evolution has a functional basis. Notably, site pairs carrying lineage defining mutations of the four VOCs that circulated before May 2021 are enriched in CSPs. For the Alpha VOC, the enrichment is detected even if Alpha sequences are removed from analysis, indicating that VOC origin could have been facilitated by positive epistasis. Additionally, we detected nine discordantly evolving pairs of sites where mutations at one site unexpectedly rarely occurred on the background of a specific allele at another site, for example on the background of wild-type D at site 614 (four pairs) or derived Y at site 501 (three pairs). Our findings hint that positive epistasis between accumulating mutations could have delayed the assembly of advantageous combinations of mutations comprising at least some of the VOCs.

Origin and Reversion of Omicron Core Mutations in the Evolution of SARS-CoV-2 Genomes.

Genetic analyses showed nearly 30 amino acid mutations occurred in the spike protein of the Omicron variant of SARS-CoV-2. However, how these mutations occurred and changed during the generation and development of Omicron remains unclear. In this study, 6.7 million (all publicly available data from 2020/04/01 to 2022/04/01) SARS-CoV-2 genomes were analyzed to track the origin and evolution of Omicron variants and to reveal the genetic pathways of the generation of core mutations in Omicron. The haplotype network visualized the pre-Omicron, intact-Omicron, and post-Omicron variants and revealed their evolutionary direction. The correlation analysis showed the correlation feature of the core mutations in Omicron. Moreover, we found some core mutations, such as 142D, 417N, 440K, and 764K, reversed to ancestral residues (142G, 417K, 440N, and 764N) in the post-Omicron variant, suggesting the reverse mutations provided sources for the emergence of new variants. In summary, our analysis probed the origin and further evolution of Omicron sub-variants, which may add to our understanding of new variants and facilitate the control of the pandemic.

Nutrition Guidelines for Improved Clinical Care.

Given the importance of poor nutrition as a cause for human chronic disease, it is surprising that nutrition receives so little attention during medical school training and in clinical practice. Specific vitamins, minerals, fatty acids, amino acids and water in the diet are essential for health, and deficiencies lead or contribute to many diseases. Proper use of the dietary guidelines and nutrition facts labeling can improve nutritional status and lead to the consumption of a healthy diet. COVID-19 has altered access to nutritious foods for millions and increased awareness of the importance of diet and immune function. An improved appreciation for nutrition will improve the outcomes of clinical care.

Quantitative profiling of N-glycosylation of SARS-CoV-2 spike protein variants.

With the global spread of the corona virus disease-2019 pandemic, new spike variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continuously emerge due to increased possibility of virus adaptive amino acid mutations. However, the N-glycosylation profiles of different spike variants are yet to be explored extensively, although the spike protein is heavily glycosylated and surface glycans are well-established to play key roles in viral infection and immune response. Here, we investigated quantitatively the N-glycosylation profiles of seven major emerging spike variants including Original, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.671.1), Delta (B.1.671.2), and Omicron (B.1.1.529). The aim was to understand the changing pattern of N-glycan profiles in SARS-CoV-2 evolution in addition to the widely studied amino acid mutations. Different spike variants exhibit substantial variations in the relative abundance of different glycan peaks and subclasses, although no specific glycan species are exclusively present in or absent from any specific variant. Cluster analysis shows that the N-glycosylation profiles may hold the potential for SARS-CoV-2 spike variants classification. Alpha and Beta variants exhibit the closest similarity to the Original, and the Delta variant displays substantial similarity to Gamma and Kappa variants, while the Omicron variant is significantly different from its counterparts. We demonstrated that there is a quantifiable difference in N-glycosylation profiles among different spike variants. The current study and observations herein provide a valuable framework for quantitative N-glycosylation profiling of new emerging viral variants and give us a more comprehensive picture of COVID-19 evolution.