B-cell immunoglobulin repertoires with paired heavy and light chain can be determined by means of 10X single-cell V(D)J sequencing. Precise and quick analysis of 10X single-cell immunoglobulin repertoires remains a challenge owing to the high diversity of immunoglobulin repertoires and a lack of specialized software that can analyze such diverse data.In this study, specialized software for 10X single-cell immunoglobulin repertoire analysis was developed. SCIGA  is an easy-to-use pipeline that performs read trimming, immunoglobulin sequence assembly and annotation, heavy and light chain pairing, statistical analysis, visualization, and multiple sample integration analysis, which is all achieved by using a 1-line command. Then SCIGA was used to profile the single-cell immunoglobulin repertoires of 9 patients with coronavirus disease 2019 (COVID-19). Four neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were identified from these repertoires.SCIGA provides a complete and quick analysis for 10X single-cell V(D)J sequencing datasets. It can help researchers to interpret B-cell immunoglobulin repertoires with paired heavy and light chain. The diversity of B-cell immunoglobulin is an important characteristic of the adaptive immune system. It is developed through the rearrangement of variable (V), diversity (D), and joining (J) gene segments, which is referred to as V(D)J; the pairing of heavy and light chains; and somatic hypermutation (SHM) . Exposur10X single-cell V(D)J sequencing is a powerful tool for investigating paired heavy and light chain repertoires of B-cell immunoglobulins . It has Here, we developed SCIGA , a software package for quickly analyzing the data of 10X single-cell immunoglobulin repertoires. SCIGA performs read trimming, immunoglobulin sequence assembly and annotation, and heavy and light chain pairing by means of a 1-line command. It also computes the statistics of repertoires, including gene usage frequency, SHM rate, length of complementarity-determining region 3 (CDR3), and clonality, and further implements visualization. We profiled the immunoglobulin repertoires of peripheral blood mononuclear cells (PBMCs) from 9 patients with COVID-19 using SCIGA. Finally, we identified 4 neutralizing antibodies against SARS-CoV-2 from these repertoires.RRID:SCR_017344) [RRID:SCR_011848) [RRID:SCR_010753) [de novo assembler for short reads. (iv) Gene call: to detect the usage of the V(D)J gene and C gene (isotype), SCIGA aligns the assembled immunoglobulin sequences against the V-, D-, and J- gene reference database using IgBLAST (RRID:SCR_002873) [RRID:SCR_004870) [H, JH, VL, and JL genes and identical H-CDR3 length and exceed a given similarity threshold of H-CDR3 nucleotide sequences. (viii) Statistical analysis and visualization: SCIGA calculates a list of statistics, including gene usage frequency, SHM rate, CDR3 length, Simpson index, Shannon entropy, and others. SCIGA subsequently generates figures to show the features of the repertoires. (ix) Multiple sample integration analysis: after analyzing each sample, SCIGA consolidates all of the outputs into one. It identifies the shared immunoglobulins that are potential public antibodies suitable for use against a specific pathogen. Shared immunoglobulins are defined as immunoglobulins from different samples that can be clustered into the same clonal lineage. Clustering is performed as described in Step 7 with cells of all samples.SCIGA is a software package for the analysis of 10X single-cell immunoglobulin repertoires. It integrates several tools and algorithms into a single workflow. The input data can be raw reads or the output of Cell Ranger (_010753) , which i_002873)  and agai_004870) . The V(D_004870)  and embeAt the time of this study, Cell Ranger is the only existing software for processing raw data generated by 10X single-cell V(D)J sequencing. A test dataset was therefore built to compare SCIGA to Cell Ranger. The PBMCs from 9 patients with COVID-19 (B1–B9) were collected and 10X single-cell V(D)J sequencing was performed Fig. . The rawIGHV4-34 (12.51%), IGHV3-30 (7.95%), and IGHV3-23 (6.30%) for the heavy chain and IGLV3-19 (9.46%), IGKV1-39 (8.26%), and IGKV3-20 (7.90%) for the light chain. IGHV4-34 and IGLV3-19 had elevated usage frequency in the repertoire of Patient B2 and reached 63.98% and 64.39%, respectively. IGHM had the highest usage in the repertoires of most patients, except for Patient B2, who showed the highest usage of IGHG1 J sequencing data of the 9 patients with COVID-19 were analyzed, and features of the immunoglobulin repertoires were determined. A total of 8,358 B cells were detected  was the most used gene in the heavy chain, IGKV1-39 (9 clones) and IGKV3-20 (9 clones) were the most used genes in the light chain, and IGHV4-34: IGLV3-19 (5 clones) was the most used gene pair  against SARS-CoV-2 pseudovirus, whereas B8-C1 (1.47 μg/mL) and B6-C2 (14.89 μg/mL) were moderate and weak neutralizing antibodies  were screened for the following criteria: IgG antibodies in the clone with fraction ≥1% and cell number ≥20 see . Four mAies Fig. . Similaries Fig. .In this study, we developed the SCIGA software for 10X single-cell immunoglobulin repertoire analysis. SCIGA is easy to use and allows researchers to quickly perform advanced analysis on 10X V(D)J sequencing datasets. Cell Ranger has previously been used for 10X single-cell immunoglobulin repertoire analysis. However, this software includes low-quality cells in the output and disregards the effect of SHM when defining clonal lineage. In addition, some important information about repertoires, including, e.g., the level of SHM, is not included in the output of Cell Ranger. SCIGA performs the quality control process for cells and defines the clonal lineage including the effect of SHM. Larger clones can be detected by using SCIGA. Moreover, SCIGA generates the needed statistical outputs and implements visualization. It is therefore a more efficacious tool for researchers.In this study, SCIGA was used to analyze the single-cell immunoglobulin repertoires of the PBMCs of patients with COVID-19. Large-scale clone expansion was not observed in most patients. In Patient B2, however, B cells expanded, which was indicated by a large size of clonal lineage with the IgG isotype. This indicates that Patient B2 likely generated neutralizing antibodies against SARS-CoV-2.Finally, we tried to identify the SARS-CoV-2–responding antibodies. In previous work, immunoglobulins with an SHM rate <2% were excluded in screens for neutralizing antibodies . HoweverGigaScience GigaDB database [The datasets supporting the results of this article are available in the NCBI repository and can be accessed with PRJNA682839. All additional supporting data and materials are available in the database .Project name: SCIGAhttps://github.com/sciensic/SCIGAProject home page: Operating system(s): LinuxProgramming language: PerlOther requirements: IgBlast 1.15.0 or higher, Blast 2.9.0 or higher, R , ggplot2 License: GNU GPL-3.0 LicenseRRID:SCR_021002, Biotools ID: scigaFigure S1:The usage frequency of V-genes in the repertoire of each patient. Only the genes with frequency >1% are shown.Figure S2: Distribution of the SHM rate in the repertoire of each patient. Color denotes the chain, with IGH shown in red, IGK in yellow, and IGL in blue.Figure S3: Distribution of the CDR3 length in the repertoire of each patient. Color denotes the chain, with IGH shown in red, IGK in yellow, and IGL in blue.Figure S4: Fraction of all clones in the repertoire of each patient. The x-axis captures the clone rank and y-axis captures the clone fraction. *Selected antibody candidate.Figure S5: Number of the used V-genes of the top 10 largest clones of all patients. (A) The count of the used V-gene for the heavy chain. (B) The count of the used V-gene for the light chain. (C) The count of the used V-gene pair. The initials of the gene names denote the chain: H: IGH; K: IGK; L: IGL.Figure S6: Two different examples show how to chose the threshold: (A) having and (B) not having a large difference in the read counts between cells containing GEMs and background.Table S1: Information on the 9 patients with COVID-19.Table S2: Information on the shared immunoglobulins. Columns 7–11 denote the number of shared immunoglobulin sequences.Table S3: Information on the 4 antibody candidates.giab050_GIGA-D-20-00341_Original_SubmissionClick here for additional data file.giab050_GIGA-D-20-00341_Revision_1Click here for additional data file.giab050_GIGA-D-20-00341_Revision_2Click here for additional data file.giab050_GIGA-D-20-00341_Revision_3Click here for additional data file.giab050_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giab050_Response_to_Reviewer_Comments_Revision_1Click here for additional data file.giab050_Response_to_Reviewer_Comments_Revision_2Click here for additional data file.giab050_Reviewer_1_Report_Original_SubmissionRachael Bashford-Rogers -- 1/13/2021 ReviewedClick here for additional data file.giab050_Reviewer_1_Report_Revision_1Rachael Bashford-Rogers -- 4/6/2021 ReviewedClick here for additional data file.giab050_Reviewer_2_Report_Original_SubmissionYariv Wine -- 2/20/2021 ReviewedClick here for additional data file.giab050_Supplemental_FilesClick here for additional data file.ACE2: angiotensin-converting enzyme 2; BLAST: Basic Local Alignment Search Tool; CDR3: complementarity determining region 3; COVID-19: coronavirus disease 2019; ECD: extracellular domain; ELISA: enzyme-linked immunosorbent assay; GEMs: Gel Beads-in-emulsion; IC50: half-maximal inhibitory concentration; Ig: immunoglobulin; mAb: monoclonal antibody; NTD: N-terminal domain; PBMC: peripheral blood mononuclear cell; RBD: receptor binding domain; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SHM: somatic hypermutation; UMI: unique molecular identifier.This study was conducted according to the ethical principles of the Declaration of Helsinki. Ethical approval was obtained from the Research Ethics Committee of Shenzhen Third People’s Hospital (2020-207). All participants provided written informed consent for sample collection and subsequent analyses. Raw data has been approved for release by the Ministry of Science and Technology (MOST) of People's Republic of China, with the reference number 2021BAT3238.The authors declare that they have no competing interests.This study was supported by the National Science Fund for Distinguished Young Scholars (82025022), the Sanming Project for Medicine of Shenzhen (SZSM201612053), the National Key Plan for Scientific Research and Development of China , the National Science and Technology Major Project of the Infectious Diseases (2018ZX10301404 to Z.Z. and S.Z.), the Science and Technology Innovation Committee of Shenzhen Municipality , the National Natural Science Foundation of China (82002140), and the Natural Science Foundation of Guangdong Province of China (2019A1515011197).Z.Z. and L.W. designed this study and wrote the manuscript. H.Y. performed this study and wrote the manuscript. L.C. performed the antibody neutralization test and wrote the manuscript. B.J. performed the ELISA test. G.X. performed the 10X single-cell V(D)J sequencing. Y.L. revised the manuscript. S.Z. contributed to the discussion .


A. thaliana villin 4 (AtVLN4). This procedure significantly reduces the complexity of NMR spectra and enables group identification of signals arising from the labeled IDR fragment, a process we refer to as segmental assignment. The validity of our segmental assignment approach is corroborated by backbone residue-specific assignment of the IDR using a minimal set of standard heteronuclear NMR methods. Using segmental assignment, we further demonstrate that the IDR region adjacent to the headpiece exhibits nonuniform spectral alterations in response to temperature. Subsequent residue-specific characterization revealed two segments within the IDR that responded to temperature in markedly different ways. Overall, this study represents an important step toward the selective labeling and probing of target segments within much larger IDR contexts. Additionally, the approach described offers significant savings in NMR recording time, a valuable advantage for the study of unstable IDRs, their binding interfaces, and functional mechanisms.A significant number of proteins possess sizable intrinsically disordered regions (IDRs). Due to the dynamic nature of IDRs, NMR spectroscopy is often the tool of choice for characterizing these segments. However, the application of NMR to IDRs is often hindered by their instability, spectral overlap and resonance assignment difficulties. Notably, these challenges increase considerably with the size of the IDR. In response to these issues, here we report the use of sortase-mediated ligation (SML) for segmental isotopic labeling of IDR-containing samples. Specifically, we have developed a ligation strategy involving a key segment of the large IDR and adjacent folded headpiece domain comprising the C-terminus of  Intrinsically disordered regions (IDRs), often involving hundreds of residues, are widespread in all kingdoms of life and perform many key cellular roles –5. TheseMethods for the investigation of IDRs commonly include NMR spectroscopy, FRET and others, provided that the appropriate probes can be inserted into the IDR sequence (e.g. fluorophores or NMR-active isotopic labels) –10. Of tGiven the difficulties listed above, a complementary alternative to more sophisticated and time-consuming 3D/4D/5D schemes is desirable and could allow a much broader user base to probe sizable IDRs by NMR. To this end, segmental labeling with NMR-active nuclei has the potential to circumvent many of these challenges by reducing spectral overlap and enabling inherently faster 2D NMR schemes. This could allow for meaningful, segment-specific identification of IDR resonances and their usage as functionally relevant probes. It should be noted that segmental labeling options for protein NMR also offer the advantage of allowing production of large cytotoxic samples from smaller non-toxic components, which is a feature that has been reviewed elsewhere and may be particularly relevant for large IDRs . Despitein vitro under mild reaction conditions, requires modest alterations to the polypeptide target, and relies on easily accessible sortase enzymes . Reaction progress was estimated using LC-ESI-MS. (C) Representative ESI-MS spectrum for ligation reaction between triglycine (GGG) and FH8-EDEED . (D) Representative ESI-MS spectrum for ligation reaction between triglycine (GGG) and FH8-IDR-(G4S)2 2 substrate = 14636 Da, calcd MW ligation product = 13870 Da). ESI-MS spectra in B and C represent the 30 min reaction time point. Calculated MW values are average molecular weight predicted using the BMRB Molecular Mass Calculator. * = MeCN adducts from LC-ESI-MS mobile phase .((PDF)Click here for additional data file.S7 FigA) Characterization data for FH8-IDR-HP63 with selective incorporation of 15N in the HP63 segment . (B) Characterization data for FH8-IDR-HP63 with selective incorporation of 15N in the FH8-IDR segment . (C) Characterization data for IDR-HP63 (FH8 tag removed) with selective incorporation of 15N in the IDR segment . Calculated MW values are average molecular weight predicted using the BMRB Molecular Mass Calculator. * = MeCN adducts from LC-ESI-MS mobile phase . All gels were visualized by Coomassie staining, and molecular weight standards (kDa) are indicated to the left of each gel image. The original, uncropped gel images are also provided in S14 Fig.((PDF)Click here for additional data file.S8 Fig(A) Overlaid 15N-HSQC spectra for isolated, uniformly 15N-labeled GG-HP63 (blue contours) and FH8-IDR-HP63 segmentally labeled with 15N in the HP63 domain (red contours). The two spectra are nearly identical, with the majority of resonances overlapping or being directly adjacent. Only four pairs of signals deviate from this pattern (indicated with arrowed brackets). The largest observed difference (*) corresponds to leucine residue L899 (numbering based on wild type villin 4), which directly follows the N-terminal, non-native diglycine of GG-HP63. Thus, in isolated GG-HP63 L899 can experience significant end effects due to its location just three positions from the free N-terminus. In segmentally labeled FH8-IDR-HP63, L899 is an internal residue far removed from either terminus. (B) atVHP76  [y 30289) .(PDF)Click here for additional data file.S9 FigA. thaliana villin 4 (residues 877–974), and includes the 35-residue IDR segment and C-terminal headpiece domain without the sortase-mediated ligation motif (LPXTG), FH8 domain, or the TEV cleavage site. The only non-native element of IDR-HP(877–974) is the N-terminal His6 tag (see S2 and ). Based on the sequence of IDR-HP(877–974), we expect to observe up to 101 resonances originating from non-proline residues. A conservative analysis of the spectrum acquired gives 88 resonances of variable intensity. We attribute this apparent shortage in part to the presence of the His6 tag on the protein N-terminus. Due to their identical chemical nature, these six contiguous residues would likely have nearly overlapping or closely packed peaks. Additionally, there is a region of poor spectral resolution at ~8.3 ppm (1H dimension) and ~122 ppm (15N dimension) where significant spectral overlap may prevent identification of more individual peaks. Lastly, resonances may be undetectable due to their respective residues experiencing intermediate time-scale dynamics.Data recorded at 25°C on a 500 MHz instrument. This sample represents the native sequence of (PDF)Click here for additional data file.S10 Fig(A)15N-HSQC spectra of the isolated FH8 domain (at 500 MHz 1H frequency) recorded at 25°C. For comparison, the 15N-HSQC spectrum of FH8-IDR-HP63 at 25°C  is shown in panel B.(PDF)Click here for additional data file.S11 Fig15N-HSQC spectra for FH8-IDR-HP63 segmentally labeled with 13C/15N in the FH8-IDR portion  and uniformly 15N-labeled IDR-HP(877–974) . The filled red contours indicate segmentally assigned FH8-IDR-HP63 resonances . An “X” signifies the eight FH8-IDR-HP63 resonances for which there are no overlapping or adjacent resonances in the IDR-HP(877–974) control. Seven out of these eight FH8-IDR-HP63 resonances were assigned to the introduced TEV site (L74Y75F76Q77G78), the single glycine spacer (G114), or the leucine (L115) of the sortase ligation site. Double arrows indicate four segmentally assigned resonances  for which there are nearby matching signals in the native control (open blue contours). The length of every arrow is equal or smaller than 0.05 ppm in the 1H dimension. Primary structure diagrams are shown below the spectra for segmentally labeled FH8-IDR-H63 (sequence position numbering begins with the first residue of the FH8 domain) and uniformly labeled IDR-HP(877–974) (residue numbering corresponds to the sequence of native villin 4). Non-black coloration in the structure diagrams (red or blue) indicates the position of isotopic labels (13C and/or 15N).Superimposed (PDF)Click here for additional data file.S12 Fig15N-HSQC spectra of isolated G5-HP60  at (A) 25°C and (B) 5°C. For comparison, each spectrum of G5-HP60 is overlaid with the 15N-HSQC spectrum of atVHP60  [1H frequency). Circled resonances correspond to amino acid side chains. The sequences of G5-HP60 and atVHP60 are shown at the bottom . Underlined residues correspond to non-native amino acids added to the HP60 domain. In the case of G5-HP60, five glycines were included to ensure accessibility of the N-terminus for potential sortase-mediated ligation. et al.) . The spe(PDF)Click here for additional data file.S13 FigA) 15°C, (B) 25°C, and (C) 45°C. The spectra recorded at 15°C and 45°C were re-referenced to the 1H / H2O chemical shift value of the spectrum recorded at 25°C according to the standard approach [A and C were transferred from the spectrum acquired at 25°C . Peaks for which no residue specific assignments were obtained are marked with “+” or “x”, with “+” indicating unassigned peaks that were observed in the FH8-IDR-HP63 construct (13C/15N-labeled FH8-IDR segment), and “x” indicating unassigned peaks unique to the IDR-HP63 derivative (15N-labeled IDR segment). Two of the peaks marked “x” are presumed to correspond to resonances for residues E79 and E80 (adjacent to G78 at the N-terminus in the IDR-HP63 sample).Spectra recorded at (approach . Residue(PDF)Click here for additional data file.S14 Fig(PDF)Click here for additional data file.S1 TableaCalculated values are average molecular weight predicted using the BMRB Molecular Mass Calculator [15N. bA secondary mass peak of lower intensity was observed at 18158 Da, which is consistent with the presence of a glutathione adduct  [cMass of FH8-IDR-G doubly labeled with 13C and 15N was not determined.lculator . For iso8157 Da) . No evid(PDF)Click here for additional data file.S2 TableaResidue number based on position in the FH8-IDR-HP63 construct. Value in parentheses indicates the corresponding position in wild-type villin 4. The native 35-residue portion of the IDR covers position E79 (E877)–D113 (D911). bEffect of FH8 assessed by comparison of 15N-HSQC spectra for FH8-IDR-HP63  and IDR-HP63 . All spectra recorded at 25°C. cEffect of GLPETGG insertion assessed by comparison of spectra for FH8-IDR-HP63 and IDR-HP(877–974) . All spectra recorded at 25°C. dObserved chemical shift differences attributed to the presence of the His6-tag on the N-terminus of IDR-HP(877–974) as opposed to the non-native, N-terminal G78 residue in the case of IDR-HP63. eNearly no effect indicates chemical shift differences of <0.04 ppm for the 1H dimension and <0.2 ppm for the 15N dimension. fAlignment of relevant sequences (35-residue IDR fragment indicated in bold).(PDF)Click here for additional data file.


Rattus norvegicus heterotrimeric G protein α-subunit and human small G protein Ras-related G protein C and analyzed the binding modes of these G proteins with GTP or ATP by performing molecular dynamics simulations. In summary, it will provide new insights into the enormous diversity of biological function of G proteins.Guanine nucleotide-binding (G) proteins, namely, phosphate-binding (P) loop GTPases, play a critical role in life processes among different species. Based on the structural characteristics, G proteins can be divided into heterotrimeric G proteins, small G proteins and multiple unique unconventional G proteins. The highly conserved unconventional G protein YchF is composed of a core G domain, an inserted coiled-coil domain, and a TGS domain from the N-terminus to the C-terminus. In this review, we compared the structural characteristics of the G domain in rice OsYchF1 with those of  Heterotrimeric G proteins and small G proteins belong to Ras-like GTPases, whereas OsYchF1, a rice YchF, belongs to the YchF subfamily in Obg-related GTPases of the TRAFAC superclass of P-loop GTPases.2Guanine nucleotide-binding (G) proteins, so-called phosphate-binding (P) loop GTPases, regulate multiple aspects of signaling pathways and cellular processes in all kingdoms of life.3 OsYchF1 is a kind of unconventional G protein, which comprises three conserved domains, a N-terminal core G domain, flanked by a coiled-coil domain, and a C-terminal TGS  domain  domain . The OsY) domain . The C-t2+) that is crucial for nucleotide binding and hydrolysis. Mg2+ not only connects the β and γ of GTP with walk B motif but also coordinates the conserved threonine (Thr) residue in the G2 motif and moves its backbone carbonyl group into a position with the hydrolytic water to facilitate the nucleophilic assault, resulting in γ-phosphate hydrolysis from GTP.5 The walk B motif overlaps with switch I and switch II regions, where conformational switch occurs accompanying with nucleotide hydrolysis; the G4 motif ((N/T)KxD) is critical for the adenosine or guanosine recognition, determining the specific guanine base signature. The G5 motif ((T/G) (C/S) A) supports the specific binding of guanine base, and the backbone of alanine (Ala/A) in the G5 motif forms a hydrogen bond with the oxygen (O-6) in guanine.6 The OsYchF1 G domain maintained five conserved motifs as other G proteins. G1, G2, G3, and G5 motifs are conserved and invariant with other G proteins, but the G4 motif ((N/T)KxD) in the YchF subfamily exhibits a unique (N/T)(M/L/V)xE amino acid sequence (All G proteins possess a core G domain including five conserved fingerprint motifs that range from G1 to G5 motifs. The G1 motif (GxxxxGK(S/T)), namely, P-loop or walk A motif, is associated with the binding for α- and β-phosphate of guanosine triphosphate (GTP). G2 motif (x (T/S) x) and G3 motif (hhhDxxG), namely, walk B motif, bind to the terminal γ-phosphate of GTP. The conserved aspartic acid (Asp/D) residue in the G3 motif (hhhDxxG) binds to the essential cofactor magnesium  accession code: 1NI3) was the first solved 3-Dimension (D) structure in the YchF subfamily, and another bacterium Haemophilus influenzae YchF was also solved soon (PDB code: 1JAL).4 After that, a crystal structure of human YchF ortholog hOLA1 (human Obg-like ATPase 1) (PDB code: 2OHF) was determined. Interestingly, hOLA1 displayed higher ATP binding affinities and hydrolysis rates than GTP.7Escherichia coli YchF protein also displayed ATPase priority, while the 70S ribosome binds to and enhances the ATPase activity of YchF (~10 fold).8 OsYchF1 crystal structures in the absence or presence of AMPPNP and GMPPNP  are the first solved YchF in plants. Additionally, the OsYchF1 Switch I region includes the G2 motif (xT/Sx), while the G3 motif (DxxG) overlaps with the switch II region in OsYchF1  that anchor across the plasma membrane to perceive the extracellular signals. The heterotrimeric G proteins function mainly in vision, smell, and taste in animals.6 Second, small G proteins are monomeric G proteins with a molecular weight of ~20-40kDa and consists of five families: Ras, Rho, Rab, Ran, and Arf families.Herein, heterotrimeric G protein and small G protein, two categories in the TRAFAC superclass, were chosen to compare with OsYchF1 in the 3-D structure . First, Rattus norvegicus heterotrimeric G protein α-subunit (XP_010846404.1), Oryza sativa heterotrimeric G protein α-subunit (XP_015639183.1), human Ras-related G protein C (NP_071440.1), Oryza sativa Ras-related G protein Rab-2-B (XP_015626284.1), and YchF homolog  were performed by using the Jalview version 2.11.1.3 software   . The phy homolog .Figure R. norvegicus heterotrimeric G protein α-subunit (PDB code: 1SVS) and small human Ras-related G protein C (PDB code: 3LLU) were compared with those of OsYchF1 in plants (PDB code: 5EE0). The G1 motif (phosphate binding loop) in OsYchF1 is highly identical to that of the R. norvegicus heterotrimeric G protein α-subunit and human Ras-related G protein C (KxD) of the R. norvegicus heterotrimeric G protein α-subunit and human Ras-related G protein C pointed inside the nucleotide-binding site and came into contact with ribose of guanosine nucleotide to support the GTP docking (MSE) of OsYchF1, Met, and glutamic acid (Glu/E) were replaced by Lys and Asp, respectively. The OsYchF1 G4 motif mutant became GTP priority again.11 These results indicated that the G4 motif determined the ATP or GTP binding and hydrolysis specificity of OsYchF1.To extrapolate the features to all plants and animals, the G1-G5 motifs of the rotein C . Further docking . Oδ1/2 o docking . In the  docking . The sid docking . TherefoR. norvegicus heterotrimeric G protein α-subunit with GMPPNP (guanylyl-imidodiphosphate) (PDB code: 1SVS), small human Ras-related G protein C with GMPPNP (PDB code: 3LLU), and OsYchF1 in the presence of GMPPNP (PDB code: 5EE9) or AMPPNP (adenylyl-imido-diphosphate) (PDB code: 5EE3), and conducted all-atomic molecular dynamics (MD) simulations. Either GTP or ATP binds to G proteins tightly, and the disassociation of G protein complexes was not observed during MD simulations. Yet, each complex showed specific interactions.To further illuminate the binding modes of G proteins with GTP or ATP, we selected four G protein complexes as examples, including First, for 1SVS, GTP was completely surrounded by the protein. There were on average 5.3 hydrogen bonds between 1SVS and GTP. Two key serine residues of the protein, S16 and S120, formed critical hydrogen bonds with the small molecule .Figure Second, although GTP partially exposed to the solution, more hydrogen bonds (11.1) were observed between 3LLU and GTP. However, many of such hydrogen bonds involved G protein backbone atoms. Notably, the hydroxy groups from S15, S16, and S35 sidechains hydrogen-bonded to GTP .Third, OsYchF1 with GMPPNP (PDB code: 5EE9) and AMPPNP (PDB code: 5EE3) was structurally similar, and therefore, their complexes resembled each other. It was worth noting that the whole ligand molecules were attached to OsYchF1 in crystal structures of both complexes. Interestingly, GTP and ATP exhibited high flexibility in conformation during MD simulations. Only triphosphate groups were anchored upon the unconventional G protein OsYchF1, while either guanine or adenine groups were solvated by water molecules. We speculated that there existed strong electrostatic interactions regarding triphosphate groups. Such a binding mode resulted in less hydrogen bonds . We identified that T26 was essential for GTP and ATP binding modes . In bothOsYchF1 in rice is still not fully understood. Especially, the OsYchF1 loss-of-function mutants and gain-of-function transgenic lines have not been constructed and observed in rice, and the important agronomic traits of OsYchF1 need to be clarified in rice.Although the 3-D structure of OsYchF1 in the absence or presence of AMPPNP and GMPPNP had been determined by X-ray crystallography, the biological function of


However, it is still unknown whether a similar expression mechanism of P3N-PIPO is used in the other genera of the family Potyviridae. Moreover, due to the extremely low expression level of P3N-PIPO in natural virus-infected plants, the peptides spanning the slippery site which provide direct evidence of the slippage at the protein level, have not been identified yet. In this study, a potato virus X (PVX)-based expression vector was utilized to investigate the expression mechanism of P3N-PIPO. A high expression level of the P3N-PIPO(WT) of turnip mosaic virus  was observed based on the PVX expression vector. For the first time, we successfully identified the peptides of P3N-PIPO spanning the slippery site by mass spectrometry. Likewise, the P3N-PIPO(WT) of wheat yellow mosaic virus  was also successfully expressed using the PVX expression vector. Integrated proteome and transcriptome analyses revealed that WYMV P3N-PIPO was expressed at the conserved G2A6 site through transcriptional slippage. Moreover, as revealed by mutagenesis analysis, Hexa-adenosine of the G2A6 site was important for the frameshift expression of P3N-PIPO in WYMV. According to our results, the PVX-based expression vector might be used as an excellent tool to study the expression mechanism of P3N-PIPO in Potyviridae. To the best of our knowledge, this is the first experimental evidence dissecting the expression mechanism of a bymovirus P3N-PIPO in the experimental host Nicotiana benthamiana.P3N-PIPO , the movement protein of potyviruses, is expressed as a translational fusion with the N-terminus of P3 in potyviruses. As reported in previous studies, P3N-PIPO is expressed via transcriptional slippage at a conserved G Most RNA viruses have a relatively small genome size, which seems to be insufficient for RNA viruses to cope with long-term selective pressure. RNA virus has explored various strategies to maximize its coding capacities, such as splicing, transcriptional slippage, ribosomal frameshifting, leaky scanning, and stop-codon read-through ,2.Escherichia. Coli [E. coli operons, pyrBI and codBA, were regulated by transcriptional slippage under special conditions [Potyvirus [Amongst them, transcriptional slippage often takes place on repetitive nucleotides such as poly(A) or poly(T) tracts, resulting in the synthesis of heterogeneous mRNAs that always insert one or more extra nucleotides but seldom delete one or two bases . If thisia. Coli ,4. For enditions ,6. As fonditions ,8,9 , known as PIPO (Pretty Interesting Potyviridae ORF), is discovered within the genome sequences of analysis . PIPO waP3N-PIPO ,14. TherP3N-PIPO ,16,17,18yviruses . It is wyviruses . Owing tyviruses ,21. Bymovirus, was also successfully identified and characterized. In this study, the PVX-based vector system was utilized to investigate the expression mechanism of P3N-PIPO. The frameshift expression of P3N-PIPO(WT)-GFP was successfully identified using the PVX-based vector in plants. In addition, the frameshift of P3N-PIPO(WT) in another plant virus, wheat yellow mosaic virus (WYMV) of the genus Potyviridae, but also provides an excellent tool to study the slippage efficiency of P3N-PIPO.Our research not only sheds more light on the expression mechanism of P3N-PIPO in the family gfp fragment without the start codon was amplified from a GFP binary vector using specific oligonucleotide primers .The PVX vector (pgR106) used in this study was obtained from David Baulcombe’s laboratory. A  primers  that incClaI and SnaBI restrictions sites at the 5′- and 3′-terminus, respectively. Afterwards, this fragment was cloned into the pJET1.2/blunt vector  following the standard molecular clone protocols and was named the pJET-P3N-PIPO (ClaI/SnaBI) vector.Additionally, P3N-PIPO(WT) was amplified from TuMV and WYMV infectious clones, respectively, by the use of specific oligonucleotide primers  that inc2A6 site of WYMV P3N-PIPO(WT) with the QuikChange Lightning Site-Directed Mutagenesis Kit , according to the manufacturer’s instructions (The in-frame control construct WYMV P3N-PIPO(FS-1)-GFP was generated by inserting adenosine into the Gructions . Other mructions .AscI/SnaBI, SalI) and pJET-P3N-PIPO (ClaI/SnaBI) were digested with ClaI and SnaBI, respectively. Then, PVX-GFP (ClaI/SnaBI) and P3N-PIPO (ClaI/SnaBI) were purified by gel extraction  and ligated by T4 DNA ligase  25 °C: 22 °C ± 2 °C, and 16 h of light. Agrobacterium tumefaciens strain GV3101 (carrying the helper plasmid pSoup), as described previously [N. benthamiana with PVX chimeric virus vectors was also implemented, as described previously [All the PVX chimeric virus vectors constructed in this study were transformed into the eviously . Moreoveeviously . Plant leaves expressing recombinant proteins were imaged using a Leica TCS SP2 confocal microscope. Green fluorescent protein (GFP) was excited at 488 nm, and the emitted light was captured at 507 nm. Images were captured digitally and processed using the Leica LCS software.N. benthamiana leaves infected with PVX chimeric viruses using a lysis buffer (100 mM Tris-HCl (pH = 6.8), 20% SDS, and 2% β-mercaptoethanol) and were separated by 12% SDS-PAGE. Proteins were electroblotted onto a preactivated PVDF membrane. Then, the membrane was blocked with block buffer (50 mL 1 × TBS buffer containing 5% skimmed milk powder) and probed with the GFP monoclonal antibody  at room temperature for 2 h. Following three washes of 5 min each in 1 × TBS containing 0.1% Tween 20, proteins were incubated with alkaline phosphatase (AP)-conjugated polyclonal goat anti-mouse IgG  and visualized using the NBT/BCIP® solution .To analyze the frameshift expression of P3N-PIPO, total proteins were extracted from All GFP-fused proteins were purified by the GFP-TRAP-A purification kit  following the manufacturer’s instructions. Thereafter, the GFP-TRAP bound fraction was resolved by SDS-PAGE and stained with the EZBlue™ gel staining reagent .Proteins of interest were excised from the stained SDS-PAGE gels, followed by in-gel digestion with trypsin, and analysis by nano-LC/MS/MS according to standard protocols. Samples were prepared as previously described by Granvogl et al. . Mass spN. benthamiana with viruses. Total RNA was extracted from the collected samples with TRIzol reagent  in line with the manufacturer’s instructions. Sequencing libraries were prepared using NEBNext® UltraTM RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations. Meanwhile, index codes were added to attribute sequences to each sample. The library quality was assessed using the Agilent Bioanalyzer 2100 system. Deep sequencing was performed on the Illumina HiSeq 4000 platform  by Tianjin Novogene Bioinformatic Technology Co., Ltd. .The infiltration patches were harvested eight days after the infiltration of Raw reads of the viral vector from Hiseq 4000 sequencing were quality trimmed. The remaining reads were mapped to P3N-PIPO sequences of TuMV and WYMV using bowtie2 (version 2.4.1) with default parameters. Later, the mapped reads to identify insertion/deletion (indel) were performed by custom Perl scripts and Linux shell bash scripts.AscI/SnaBI; SalI)  and TuMV P3N-PIPO(WT) into the PVX-based vector pgR106, which then named PVX-GFP  . It was N. benthamiana, and the fluorescence of P3N-PIPO-GFP was successfully detected by Laser Scanning Confocal Microscopy (LSCM)  a,b. To further confirm the expression of P3N-PIPO-GFP, we utilized a GFP antibody to detect the frameshift expression of P3N-PIPO. Additionally, an in-frame control, where the predicted shift site GGA_AAA_AAT_C was mutated to GGA_AAA_AAA_T to force the expression of WYMV P3N-PIPO(FS-1)-GFP, was prepared to indicate the approximate size (56 kD) at which the frameshift protein should theoretically migrate in the gels. Using PVX-GFP as a positive control, the frameshift expression of WYMV P3N-PIPO(WT)-GFP and TuMV P3N-PIPO(WT)-GFP was successfully detected c.N. benthamiana infected with PVX-TuMV-P3N-PIPO(WT)-GFP and PVX-WYMV-P3N-PIPO(WT)-GFP. Later, the frameshift proteins were affinity-purified by GFP-TRAP beads and separated through SDS-PAGE. Afterwards, gel slices containing frameshift proteins were excised and digested with trypsin and were analyzed by mass spectrometry.To determine the precise sites and direction of the frameshift expression of P3N-PIPO, total proteins were extracted from 2A6 site in the –1 directions , GFP fluorescence was easily detected from 4 to 5 dpi in the infiltration area  was cloned into the PVX-based expression vector to test the validity of this approach. Our results indicated that the PVX-based expression vector was an excellent tool to study the expression mechanism of P3N-PIPO. In addition, peptides spanning the slippery site of P3N-PIPO were identified by mass spectrometry, which provided the first direct evidence for this frameshift expression. Therefore, the PVX-based expression vector was used in this study to achieve an abundant P3N-PIPO expression for analysis, and TuMV P3N-PIPO of a bymovirus (WYMV) was cloned into the PVX-based expression vector using the same approach. Integrated proteome and transcriptome analyses confirmed that both P3N-PIPOs of TuMV and WYMV were expressed in the G2A6 conserved motif by means of transcriptional slippage.To investigate the differences in the expression mechanism of P3N-PIPO between different genera in the family Bymovirus showed frameshift expression in the G2A6 conserved motif. All in all, the PVX-based system developed in this study contributed to future research on the functional study of P3N-PIPO in Potyviridae.In this study, the PVX-based vector system was successfully developed and proved to be an efficient and convenient tool for exploring the P3N-PIPO slippage efficiency. Furthermore, we provided the first consolidated evidence to support that P3N-PIPO of viruses in the genus


VEGFA) was constructed into a dicistronic reporter vector, psiCHECK2, and the effect of RBM4 on the IRES activity was tested in 293T cells. The results showed that the IRES insertion significantly increased the FLuc expression activity, indicating that this G4-containing IRES was active in 293T cells. When the G4 structure in the IRES was disrupted by base mutation, the IRES activity was significantly decreased. The IRES activity was notably increased when the cells were treated with G4 stabilizer PDS. EMSA results showed that RBM4 specifically bound the G4 structure in the IRES element. The knockdown of RBM4 substantially reduced the IRES activity, whereas over-expressing RBM4 increased the IRES activity. Taking all results together, we demonstrated that RBM4 promoted the mRNA translation of VEGFA gene by binding to the G4 structure in the IRES.In eukaryotes, mRNAs translation is mainly mediated in a cap-dependent or cap-independent manner. The latter is primarily initiated at the internal ribosome entry site (IRES) in the 5′-UTR of mRNAs. It has been reported that the G-quadruplex structure (G4) in the IRES elements could regulate the IRES activity. We previously confirmed RBM4  as a G4-binding protein in human. In this study, to investigate whether RBM4 is involved in the regulation of the IRES activity by binding with the G4 structure within the IRES element, the IRES-A element in the 5′-UTR of vascular endothelial growth factor A ( In protein synthesis, the genetic information in mRNA is translated into an amino acid sequence with the help of ribosomes and transfer RNAs (tRNAs) . The mRNThe mRNA translation can be subjectively separated into three stages: initiation, extension, and termination. The main difference between cap-dependent and cap-independent translation lies in the initiation stage. The initial step of cap-dependent translation initiation involves the binding of ribosomes, together with tRNAMet, to the 7-methylguanosine (m7-GpppG) 5′-cap of an mRNA. However, some viral and cellular mRNAs contain an internal ribosome entry site (IRES) that attracts ribosomes directly to the interior of the RNA to initiate cap-independent translation, which is also known as IRES mediated translation. IRES-mediated translation was first found in the RNAs of the virus ,7. VirusVEGFA is G-rich and harbors two separate IRES sites (IRES-A at 749–1038 nt and IRES-B at 91–554 nt) that facilitate VEGFA translation initiation in a cap-independent manner to respond to various cellular stresses [VEGFA translation at AUG and synthesize a secreted form of VEGFA, which is crucial for the tumor angiogenesis [VEGFA IRES mediated translation [VEGFA was used to investigate the specific mechanism of G4 structures in regulating mRNA translation.The human vascular endothelial growth factor (VEGF) is a homodimeric glyco protein which plays critical roles in vasculogenesis and angiogenesis. The VEGF family consists VEGF-A , VEGF-B, VEGF-C, VEGF-D, VEGF-E , VEGF-F (snake venom VEGF), placenta growth factor (PlGF), and the endocrine gland-derived vascular endothelial growth factor (EG-VEGF). More attention was focused on the VEGFA due to its key roles in tumor growth and metastasis and eye disease . The 5′Ustresses ,21. It hogenesis . Moreovenslation . ConsequBombyx mori, is ubiquitously expressed in human tissues [Human RNA-binding motif protein 4 (RBM4), a homologue of LARK in  tissues . We prev tissues . Studies tissues ,27. RBM4 tissues ,26. HoweVEGFA as a target gene to explore whether RBM4 can promote the IRES-mediated mRNA translation by binding the G4 structure in the IRES element. The results revealed that RBM4 favors IRES-mediated translation as a G4 binding protein.This study takes IRES-A of VEGFA 5′-UTR mediates mRNA translation, the dual luciferase reporter psiCHECK2 was applied to detect its function in mRNA translation. The HSV-TK promoter upstream of the firefly luciferase (FLuc) marker gene was replaced by the VEGFA IRES-A sequence to generate a pR-IRES-F vector. The control pRF was the empty vector without the HSV-TK promoter (VEGFA IRES-A activity (FLuc/RLuc) was significantly increased, as compared to the control plasmid pRF  and one zinc finger domain. RBM4, a homolog of silkworm LARK, was first identified in eclosion . We preveclosion . Human Rosophila , implyinKIT gene . In thisthe IRES . Our stuVEGFA could be affected, tumor growth and metastasis could be inhibited to some extent. Here, we demonstrated that the G4 structure at VEGFA IRES-A facilitated its cap-independent translation through binding with RBM4, elaborating the mechanism whereby the G4 structure in the IRES element affects the IRES-mediated translation. Compounds which could specifically target the G4 structure in VEGFA IRES, affecting its folding or unfolding, and then affecting VEGFA expression, would be novel potential therapeutic applications. As a matter of fact, a quinazoline-based compound that targets G4 structures has been revealed to inhibit DNA replication, DNA damage checkpoint activation, and promote apoptosis [VEGFA is a key mediator of angiogenesis in cancer. VEGFA is overexpressed in tumor cells, and VEGFA protein secreted by tumor cells and surrounding stroma induces the formation of new blood vessels, leading to tumor outgrowth, metastasis, and prognosis ,39. Givepoptosis . MoreoveVEGFA (Gene ID: 7422) IRES-A sequence (with Xho I and Mlu I restriction enzyme sites at 5′ and 3′ respectively) obtained from IRESbase, available online: http://reprod.njmu.edu.cn/cgi-bin/iresbase/index.php website (accessed on 5 January 2022) was synthesized and inserted into pMD18T vector . The dual luciferase reporter vector psiCHECK2 was used to study the IRES activity. The HSV-TK promoter region was excised with unique Xho I and Mlu I restriction sites, and replaced with the VEGFA IRES-A fragment, generating a pR-IRES-F vector. The control vector pRF was obtained by self-ligation of the blunted liner psiCHECK2 vector digested by Xho I and Mlu I. To exclude ribosomal read-through, a 60 bp oligonucleotides encoding a stable hairpin was cloned into the unique Nhe I site upstream of the Renilla luciferase, resulting in a p-hp-R-IRES-F vector. The sequence of the hairpin was as follows: 5′-AGCCATGGTGCTAGAGAGATCTGGTACCGAGCTCCCCGGGCTGCAGGATATCCTGCAGCCCGGGGAGCTCGGTACCAGATCTAGCCTATAGTGAG-3′.The 306 bp To construct the RBM4 eukaryotic expression vector, the open reading frame (ORF) sequence of RBM4 was cloned with cDNAs of 293T cells as template. Specific primers for cloning RBM4 ORF were as follows: Forward primer: 5′-GGTACCGCCACCATGGTGAAGCTGTTCATCGGAA-3′; Reverse primer: 5′-GGATCCAAAGGCTGAGTACCGCGC-3′. The RBM4 ORF fragment was cloned into the EGFP-N1 vector with Kpn I and BamH I, generating a RBM4-GFP vector.2 with DEME medium supplemented with 10% fetal bovine serum (FBS). The cells were passaged every 2–3 days.The 293T cell line was purchased from ATCC. The cells were cultured at 37 °C in an incubator containing 5% COThe 293T cells were inoculated in 12-well cell culture plates prior to transfection. Transfection was conducted when the cells grew to 80–90% density the next day. A 50 μL mixture containing 1 μg plasmid DNA (pRF or PR-IRES-F), 3 μL Fugene liposome, and Opti-MEM medium was added to the medium after 15 min incubation at room temperature. Then, 24 h later, the cells were collected, and the enzyme activity was determined. For the over-expression of RBM4, 1 μg PR-IRES-F vector DNA and 1 μg RBM4-GFP over-expression vector DNA (control EGFP-N1 vector) were transfected into the cells. For RNA interference (RNAi), 1 μg PR-IRES-F vector DNA and 40 pmol RBM4 siRNA (non-specific siRNA as control) were co-transfected into the cells. The cells were collected at 24 h post transfection and the enzyme activity was measured.Luciferase activity was measured using the Dual Luciferase Kit . The treated cells were washed once with PBS and then lysed with 300 μL lysis buffer at 4 °C for 5 min. The supernatant was collected to determine the enzyme activity after centrifugation at 14,000 rpm for 1 min at 4 °C.+, pH 7.5) at a final concentration of 5 μM, heated at 95 °C for 10 min, and slowly cooled to room temperature. CD was detected with a J-815 CD spectrometer . The wavelength range of the spectrum collected was 220~350 nm, the step length of the light wave was 1 nm, and the response time was 1 s.IRES RNA oligonucleotides were dissolved in 50 mM Tris buffer  on ice for 30 min, and centrifuged for 10 min at 4 °C. The supernatant was collected to conduct Western blot analysis. The protein concentration was determined with a BCA Kit . Hence, 30 μg protein was separated by SDS-PAGE and transferred to the PVDF membrane. Incubation took place with anti-RBM4 antibody  or anti-GAPDH antibody (1:5000) and the horseradish peroxidase (HRP) labeled secondary antibodies . Enhanced chemiluminescence (ECL) kit  was used to observe and record the experimental results.Escherichia coli BL21 cells for protein expression. One mM IPTG  was used to induce the protein expression. The cells were collected after 4 h induction and re-suspended in 10 mL pre-cooled 20 mmol/L Tris-HCl buffer . After sonication and centrifugation, the supernatant was passed through a Ni-NTA nickel column  for purification. The nonspecific binding proteins were eluted with a gradient solution of imidazole prepared with buffer solution. The gradients of imidazole solution were 0, 20, 50, and 400 mmol/L, respectively. The elution of the target recombinant protein was validated by SDS-PAGE.RBM4 ORF was constructed into a pET-28a vector between the Nco I and Xho I restriction sites. The recombinant plasmid DNA was transformed into ® Chemiluminescent EMSA Kit  according to the instruction. RNA probes  were annealed to allow the formation of G4 structures . The purified recombinant RBM4 protein (1 μg) was incubated with the annealed RNA G4 probes at room temperature for 20 min, and the reaction mixture was separated by 4% native PAGE electrophoresis. After electrophoresis, a gel imaging system was used to record the results.EMSA was conducted using LightShiftp < 0.05, ** p < 0.01, *** p < 0.001 indicate statistically significant differences. All experiments were conducted for three repeats.All data were statistically analyzed by GraphPad Prism8, shown as mean ± standard deviation, and pairwise comparison was performed with T test, where *


B-Myb is an important transcription factor that plays a critical role in gene expression regulation and tumorigenesis. However, its functional implication in colorectal cancer remains elusive. In this study, we found that B-Myb was significantly upregulated at both mRNA and protein levels in colorectal cancer samples compared to non-tumor counterparts. B-Myb overexpression accelerated cell proliferation, cell cycle progression and cell motility in colorectal cancer cells, and promoted tumor growth in orthotopic nude mouse models in vivo. In contrast, B-Myb depletion inhibited these malignant phenotypes. Mechanistic investigations revealed that E2F2 was a novel transcriptional target of B-Myb and is essential to B-Myb-induced malignant phenotypes. Notably, B-Myb and E2F2 exhibited positive expression correlation, and interacted with each other in colorectal cancer cells. In addition to their autoregulatory mechanisms, B-Myb and E2F2 can also directly transactivate each other, thus constituting consolidated reciprocal feed-forward transactivation loops. Moreover, both B-Myb and E2F2 are required for the activation of ERK and AKT signaling pathways in colorectal cancer cells. Taken together, our data clarified a critical role for B-Myb in colorectal cancer and unraveled an exquisite mutual collaboration and reciprocal cross regulation between B-Myb and E2F2 that contribute to the malignant progression of human colorectal cancer. The colorectal cancer (CRC) is one of the most common cancers and the main cause of cancer-related deaths worldwide . AlthougThe myb gene family consists of three members, encoding three corresponding transcription factors of MYBL1 (A-Myb), MYBL2 (B-Myb), and MYB (c-Myb) with distinct biological functions –7. A-MybIn this study, we demonstrated that the expression of B-Myb is frequently upregulated in CRC. We further discovered that E2F2 is a novel target gene of B-Myb, and B-Myb plays a tumor-promoting role in CRC via mutual collaboration and forming reciprocal feed-forward loops with E2F2, which is essential for the activation of ERK and AKT signaling pathways in CRC.https://portal.gdc.cancer.gov/). The heatmap data showed that the expression of B-Myb mRNA increased in both COAD and READ tissues  confirmed the significant upregulation of B-Myb mRNA in CRC relative to normal colorectal tissues , and of note, the expression level of B-Myb mRNA was positively correlated with pathologic grade  and rectum adenocarcinoma (READ) from the Cancer Genome Atlas  staining showed that B-Myb co-localized with E2F2 in cell nuclei Fig. . Then, wWe next sought to determine the role of E2F2 in the B-Myb-induced malignant phenotypes in CRC cells. Intriguingly, in the stable B-Myb-overexpressing HCT116 or RKO cells, silencing of either B-Myb or E2F2 significantly repressed the expression of both B-Myb and E2F2 mRNAs Fig. . ConversOur GSEA analysis yielded several remarkable growth-related and oncogene-related gene sets, such as CSR_LATE_UP.V1_UP, E2F1_UP.V1_UP, and MYC_UP.V1_UP Figs. . TherefoPrevious studies have shown that B-Myb, as a key transcription factor, plays an important role in regulating cell cycle progression and tumorigenesis. Nearly 30 years ago, Sala et al. firstly reported that constitutive expression of human B-Myb induced a transformed phenotype accompanied by activation of cyclin D1 and cdc2 expression . HoweverCurrently, B-Myb has been proposed to exert its biological functions through two main mechanisms , 6. FirsIn this study, our mechanistic investigation revealed an exquisite mutual collaboration and cross regulation between B-Myb and E2F2. Feed-forward loop regulation is an effective and widespread strategy of transcriptional control program in both prokaryotes and metazoans , 23. FeePrevious studies reported that E2Fs including E2F2 and B-Myb have been shown to be coordinated by the DREAM complex, and E2Fs mainly regulate gene expression in G1/S and B-Myb regulates gene expression in S and G2/M phases during cell cycle –7. HowevE2F2 belongs to the E2F family of transcription factors which generally divided into the two groups, i.e, “activators” (E2F1-3) and “repressors” (E2F4-8) , 25. AloIn conclusion, our study revealed that B-Myb is overexpressed in CRC, and transactivates and interacts with E2F2 to promote human CRC cell proliferation, cell cycle progression, motility, at least in part, through activation of ERK and AKT pathways. We additionally demonstrated that B-Myb and E2F2 forms reciprocal feed-forward loops that function prominently to maintain the malignant phenotype in CRCs cells Fig. . Taken t7 U/L), and streptomycin (10 mg/L) in a humidified incubator containing 5% CO2 at 37 °C. Cell lines were tested by short tandem repeat analysis and tested for mycoplasma, and the last time of authentication for these cells was April 2019 from the supplementary file of our previous study  to generate the 3 × E2F reporter. A series of luciferase reporter mutants harboring mutations in E2F and/or Myb-binding sites have been constructed by the site-directed mutagenesis kit  based on the three parental constructs of E2F2-P1314, B-Myb-P1064 and 3 × E2F reporter as described previously . The pCD/+148 werThe details of other materials and methods are provided in the Supplementary materials and methods.Supplementary Materials and methodsSupplementary TablesSupplementary Figure LegendsSupplementary Figure 1-4


An emphasis is placed on the discussion of the mechanisms and limitations of these reactions. Finally, my personal perspective on the future development of this rapidly emerging field was provided.The creation of new bonds via C-F bond cleavage of readily available per- or oligofluorinated compounds has received growing interest. Using such a strategy, a myriad of valuable partially fluorinated products can be prepared, which otherwise are difficult to make by the conventional C-F bond formation methods. Visible light photoredox catalysis has been proven as an important and powerful tool for defluorinative reactions due to its mild, easy to handle, and environmentally benign characteristics. Compared to the classical C-F activation that proceeds via two-electron processes, radicals are the key intermediates using visible light photoredox catalysis, providing new modes for the cleavage of C-F bonds. In this review, a summary of the visible light-promoted C-F bond cleavage since 2018 was presented. The contents were classified by the fluorosubstrates, including polyfluorinated arenes,  The C-F bond is the strongest covalent single bond that carbon can form. The strength of the C-F is among the reasons why organofluorine compounds have found wide applications in diverse fields ,2,3,4. TTwo main synthetic strategies have been developed for the C-F bond cleavage: one is the direct C-F bond functionalization, and the other is based on β-F elimination. The direct C-F bond functionalization can be achieved by (1) forming a carbon-metal bond via the addition of the C-F bond to an electron-rich metal center ; (2) gen−. Single-electron transfer (SET) between PC− and fluorosubstrate generates highly reactive radical anion, which eliminates fluoride to give a carbon radical. In the oxidative quenching cycle, the excited photocatalyst PC* is proposed to donate an electron to the fluorosubstrates. Since the reducing ability of PC* is lower than PC−, the oxidative quenching cycle is less common in photocatalytic C-F bond cleavage reactions. Generally, the presence of electron-withdrawing groups and π-systems in the fluorosubstrates are favorable for accepting an electron from the photocatalyst. This activation mode is suitable for the C-F bond cleavage of polyfluorinated arenes, gem-difluoroalkenes, and trifluoromethyl arenes, producing oligofluorinated aryl radicals, monofluoroalkenyl radicals, and α,α-difluororobenzylic radicals, respectively. Another key feature of photoredox catalysis is its potential to crossover from a radical to a polar pathway during the overall catalytic process . Th. Th38]. 2(dtbpy)PF6 as the photocatalyst, Xie and Hashmi disclosed the photocatalyzed C(sp3)–H multifluoroarylation of tertiary amines with perfluoroarenes via the cross coupling of α-amino alkyl radicals and perfluoroaryl radicals in 2017 [6H5NHAc (E1/2 = −1.60 V vs. SCE) and [Cu(bcp)(XP)]PF6 3 may lower the redox potentials of C6H5NHAc and/or tertiary amines by coordination. However, the yields only diminished slightly in the absence of Gd(OTf)3. The authors found that an 18 W pulsed blue LED  was an ideal light source, which provided higher yields than the reactions irradiated by a conventional 24 W blue LED.Except for H-atom abstraction and addition to π systems, radical-radical cross coupling is also a possible pathway to trap the perfluoroaryl radicals. Using Ir(ppy) in 2017 . Subsequ in 2017 . ElectroA. α-Alkoxyl alkyl radical B was generated by the HAT between radical cation A and ether, while perfluoroaryl radical anion was formed from perfluoroarene by accepting an electron from Ir(II). Finally, radical–radical cross-coupling of α-alkoxyl radical B and perfluoroaryl radical anion C with the extrusion of a fluoride ion afforded the products. For polyfluoroarenes bearing a strong electron-withdrawing group, the C-F bond cleavage occurred selectively at the para position. However, the regioselectivities were poor when polyfluoroarenes bearing an electron-rich group were used as the substrates. For example, para-, meta-, and ortho- C-F bond functionalization of PhO-substituted polyfluoroarene were observed in the ratio of 7:1:3.Although the direct SET oxidation of tertiary amines followed by deprotonation is effective to produce α-amino alkyl radicals, this method is not suitable for the generation of α-alkoxyl alkyl radicals from ethers due to their high oxidation potentials. Using quinuclidine as the H–atom transfer (HAT) catalyst, Xia and Yang reported a visible light-induced multifluoroarylation of ethers with perfluoroarenes  41]. Th. Th41]. 3 as the boryl source . Therefore, NHC-boryl radical A can be generated through a HAT process mediated by thiyl radical B. Single electron oxidation of thiols by the excited photocatalyst followed by the deprotonation in the presence of bases is an efficient method to generate thiyl radicals, while the reduced Ir(II) can activate polyfluoroarenes to give polyfluoroaryl radical anions C. This photocatalytic defluoroborylation provides a mild and operationally simple protocol to fluorinated organoboranes, which are versatile synthetic precursors for the preparation of valuable fluorinated organic compounds.Wu disclosed the defluoroborylation of polyfluoroarenes via the radical–radical cross-coupling pathway with NHC-BHl source  42]. Th. Th3 as 2 and electron-withdrawing CF3 group, were compatible with the present conditions with good reaction efficiencies. Interestingly, bis(perfluorophenyl)dimethylsilane underwent a desilylation process during the borylation. In this work, NHC-boryl radical was proposed to be generated via the direct SET between photoexcited Ir(III)* and NHC-BH3. Because the redox potential of perfluoropyridine (E(IrII/III) = −1.51 V vs. SCE), the authors speculated that the reduction of perfluoropyridine to its radical ion by Ir(II) is not favorable. However, the addition of boryl radical to perfluoropyridine would afford the radical intermediate C, whose calculated redox potential is −0.24 V, making it an ideal oxidant to accept one electron from Ir(II). Therefore, a mechanism involving radical addition and fluoride elimination was proposed, which was further supported by the detection of radical intermediate C by HRMS and the DFT calculations.Soon after Wu’s report, Yang and co-workers reported the same photocatalytic C–F bond borylation of polyfluoroarenes without a HAT catalyst  43]. Va. Va43]. E(IrII/III) = −1.32 V vs. SCE and Ered(C6F6) = −2.85 V vs. SCE); (2) when tBu-acetylene, norbornene, and 1,3,5-trimethoxybenzene were used as scavengers, no perfluorophenyl radical adduct was observed; and (3) exclusive fluorine substitution was observed using chloro-fluoro arenes. As a result, the formation of polyfluoroaryl radical anion is less likely. The reaction tolerates a wide range of aliphatic carboxylic acids, as exemplified by the late-stage functionalization of substrates derived from the nature product glycyrrhetic acid as well as drug molecules bezafibrate and ciprofibrate. Because the nucleophilicity of primary radicals is lower than secondary and tertiary carbon radicals, carboxylic acids that result in the formation of primary radicals are poor substrates in this reaction, which is consistent with the radical addition mechanism.Recently, Ritter reported the decarboxylative polyfluoroarylation of alkylcarboxylic acids  44]. Th. Th44]. 2O can reduce the number of side products and avoid the darkening of the reaction mixture. Replacing H2O by D2O led to the partial deuteration products, which suggested there were other potential proton sources in the reaction. Therefore, as with previous photocatalytic arylation of perfluoroarenes, the authors proposed a mechanism involving the formation of doubly allylic radical B via the addition of fluoroaryl radical A to arenes. SET reduction of B and protonation might be the major pathway to form 1,4-cyclohexadienes. Alternatively, the direct HAT of B from amine radical cation D or disproportionation of B is also the possible route.As shown in 2-hybridized C-F bond of gem-difluoroalkenes by visible light photocatalysis might occur via radical–radical cross-coupling or the addition-elimination pathway. The first visible light-induced defluorinative monofluoroalkenylation of tertiary amines with gem-difluoroalkenes was developed by the Hashmi group in 2016 2(dtbbpy)PF6 as the photocatalyst and K2CO3 as the base. Despite the base-mediated SNV reaction of thiols and gem-difluoroalkenes through nucleophilic addition-elimination being a possible pathway . Th. Thgem-d pathway , the con M−1 s−1 , indicat3 as the reductant, which was applied to the synthesis of α-fluoro-β-arylalkenyl sulfides by the reactions with gem-difluoroalkenes using organic dye Na2(Eosin Y) as the photosensitizer (2(Eosin Y)]* to produce an oxygen-centered sulfinic radical A. The reaction of A with PPh3 afforded a phosphoranyl radical B, which underwent β-scission to form sulfoxide radical C and phosphine oxide. Radical C was converted to thiyl radical via a second deoxygenation mediated by PPh3. The SET reduction of thiyl radical by reduced [Na2(Eosin Y)]− gave thiyl anion and regenerated the ground state [Na2(Eosin Y)]. The key carbon ion intermediate G for β-fluoride elimination was formed via the direct addition of ArS− to gem-difluoroalkenes or the addition of ArS• to gem-difluoroalkenes followed by SET reduction. To minimize the electronic repulsion between the fluorine atom and aryl group, Z-isomer was obtained as the major product via favorable conformation II. Interestingly, the E/Z selectivity of the products can be improved via Z → E isomerization under green light irradiation in the absence of a photocatalyst.Shi reported the generation of thiyl radicals by the deoxygenation of S–O bonds of sodium sulfinates with PPhnsitizer  55]. In. In3 as 3 is an ideal protocol to prepare medicinally interesting difluoroalkylated arenes (ArCF2–FG). König . St. St3 is tBu2PhP)2Cl2 as the catalyst and Xantphos as the ligand. However, a new palladium complex Pd(Xantphos)2 was demonstrated as the active species for promoting the reaction, whose oxidation potential was calculated to be +/Fc in THF. Therefore, the SET reduction of trifluoromethylarenes by an excited-state [Pd(0)Ln]* generates α,α-difluororobenzylic radical B and [F-Pd(I)Ln] species through C–F bond cleavage. The recombination of B and [F-Pd(I)Ln] forms [ArCF2-Pd(II)FLn] complex C. Transmetalation of C with arylboronic acid produces [ArCF2-Pd(II)Ln-Ar] D, which undergoes reductive elimination to deliver defluoroarylation product and regenerates Pd(0) catalyst. The proposed mechanism gave a reasonable explanation for the formation of side products, such as the ArCF2H generated by the hydrogen abstraction of α,α-difluororobenzylic radical B from solvent THF and over-defluorination for the formation of dimerization products F.A visible light-induced palladium-catalyzed selective defluoroarylation of trifluoromethylarenes with arylboronic acids was recently disclosed by the Zhang group via the Pd(0)-Pd(I)-Pd(II) catalytic cycle  59]. Th. Th59]. − addition and back electron transfer (BET). To overcome these problems, a highly reactive allylic stannane was used as the coupling partner to prevent the retro-reaction, which generates Bu3SnF in situ to trap F− by forming thermodynamically stable Bu3SnF2−. The Stern–Volmer luminescence quenching experiments indicated that iPr2Net and allylic stannane exhibited less efficient quenching effects for the excited state of Ir(ppy)3 in comparison with perfluoroalkylarenes. Therefore, a mechanism involving the oxidative quenching of excited Ir(ppy)3 by perfluoroalkylarenes was proposed. Various perfluoroalkyl groups, such as linear Ar–nC4F9 and Ar–nC6F13, as well as heptafluoroisopropylarenes (Ar–CF(CF3)2) underwent site-selective defluoroallylation regardless of the large steric hindrance.Nishimoto and Yasuda reported the site-selective C-F bond activation of perfluoroalkylarenes at the benzylic position through perfluoroalkyl radicals generated by photoredox catalysis  60]. Pr. Pr60]. 3 group or auxiliary CN, ester, and –SO2NH2 groups are typical substrates used in the above-mentioned reactions. Jui reported the C-F functionalization of trifluoromethylarenes substituted by electron-rich groups at an elevated temperature using a phenoxazine-based photocatalyst  must be reductively quenched by a radical precursor to give PC− and radical R•. The addition of R• to α-CF3 alkenes provide α-CF3 alkyl radical A, which is SET reduced by PC−, affording anionic intermediate B concomitant with the regeneration of ground-state photocatalyst (PC). Finally, β-fluoride elimination from B leads to gem-difluoroalkene products silicates were applied as the precursors of alkyl radicals in the C-F cleavage of trifluoromethyl alkenes by Molander a 64]. D. D64]. Dus media b 65]..−, base 3SO2Na (−) to switch the subsequent α-CF3 carbon radicals to carbon ions.The robustness of this visible-light-mediated radical-polar crossover addition–elimination protocol for the C-F bond cleavage of α-trifluoromethyl alkenes was further demonstrated by the use of alkylboronic acids a 66], C, C66], C3SO2Na b 67], a, a3SO2Na3SO2Na c 68] as as3SO2Na2. These compounds can also be prepared using readily available aryl carboxylic acids as the acyl radical precursors through the direct deoxygenation of acids in the presence of PPh3  species. The reaction of radical cation with carboxylates generated phosphoryl radical, which underwent β-C–O bond cleavage to form triphenylphosphine oxide and acyl radicals.As mentioned in  of PPh3  69]. In. In2. Th2)-H bond in the aldehydes, the activation of C(sp3)–H adjacent to a heteroatom, such as N, O, and S, is more effective, offering a convenient method to convert amides, ethers, and thioethers into the corresponding γ,γ-difluoroallylic compounds in good yields via the combination of HAT and photoredox catalysis.The HAT of aldehydes is also a useful method to generate acyl radicals. Wang developed a photocatalytic C–H difluoroallylation of alkyl aldehydes using quinuclidine as the HAT catalyst  70]. Th. Th70]. p-toluenesulfonic acid (PTSA) gave enamine A, which was single-electron oxidized by Ir(III)* to form radical cation B and Ir(II). The acidic β-C–H of radical cation B was deprotonated by DABCO, producing β-enaminyl radical C for the subsequent defluorinative coupling with α-trifluoromethyl alkenes. The hydrolysis of difluoroallylated enamine F regenerated organocatalyst Cy2NH and uncovered the carbonyl group.Our group reported the visible-light-mediated β-C–H difluoroallylation of aldehydes through C–F bond cleavage of α-trifluoromethyl alkenes . In cont3)–H bond in amides was developed via an intramolecular 1,5-HAT process (3ppy)2(dtbbpy)PF6 as the photocatalyst. In the presence of base, the deprotonation of trifluoroacetamides followed by single-electron oxidation generated an amidyl radical for the 1,5-HAT. The electron-withdrawing CF3 group in the amides is important for this reaction, which not only acidified the N–H bond but also stabilized the N-centered radicals. Pan independently reported the same reaction using 4CzIPN as the photocatalyst [Regioselective difluoroallylation of remote inactivated C(sp process  72]. Ma. Ma3)–H catalyst .2·diglyme and 5,5′-dimethyl 2,2′-bipyridine were added (A by binding Ni(II) precatalyst to the oxygen site of amide was the reason for the activation of the α-C–H bond. Aryl- or alkyl-substituted secondary amides were more effective substrates for this transformation.Interestingly, Martin found that the site selectivity of difluoroallylation switched from the δ-C–H bond of amides to the α-position when catalytic amounts of NiBrre added  72]. Th. Th2·dig5-exo-trig radical cyclization is a useful method to convert N-centered radicals to carbon radicals . N. NEred <reported ,64, this reagent b 82]..Ered < 23SiOH) as the XAT reagent  than 4CzIPN (pE = +1.35 V vs. SCE) due to the introduction of two electron-withdrawing chloro groups to the 3,6-positions of carbazole motifs.Molander reported the first defluorinative arylation of trifluoromethyl alkenes, in which aryl radicals were generated from aryl halides with tris(trimethylsilyl)silanol , and fluoride elimination provided gem-difluoroalkene products. The use of 0.25 equivalent of chlorotrimethylsilane (TMSCl) was essential for reproducibility, although its precise role is not clear. These phosphine-mediated deoxygenative or desulfurative reactions with trifluoromethyl alkenes significantly expand the scope of radical precursors for photocatalytic reductive cross-coupling.As described above, phosphinyl radical cations have been employed for the deoxygenative activation of sodium sulfinates , carboxystyrenes  84]. Th. Th3 stygem-difluoroallylborane products, their mechanisms are a little different. Wu proposed a HAT process for the generation of NHC-boryl radical (see 3 by excited Ir(III) species followed by deprotonation in Yang’s work  in the β-position of α-CF3 styrenes, these substrates are easier to be SET reduced by Ir(III)* (B and Ir(IV). Then, NHC-BH3 (E1/2 = + 0.76 V vs. SCE) was oxidized by Ir(IV) 3, a strongly reducing photocatalyst in its excited state, are crucial for the formation of radical anion B. The cross-coupling of NHC-boryl radical A and radical anion B occurred at sterically less hindered α-carbon, which has lower activation Gibbs energy than the reaction at the β-carbon according to the DFT calculations. Such radical–radical combination provides the α-CF3 carbon ion C, which undergoes β-fluoride elimination to form the borylation products.Boron-centered radicals are important reactive intermediates in organic synthesis. Wu , Yang 8, and Wanical see , while twork see . The sub3 group attached to other conjugated π-systems. Our group reported the first photoredox defluorinative alkylation of 1-trifluoromethyl 1,3-butadienes with 1,4-dihydropyridines as the alkylation reagents  annulation of trifluoromethyl alkenes with tertiary amines  annulation of o-hydroxyphenylacetic acids and trifluoromethyl alkenes (gem-difluoroalkenes, which were readily converted into fluorinated dihydrobenzoxepines by cleaving a second C-F bond in the presence of base.The cleavage of one C-F bond of trifluoromethyl alkenes via the radical-polar cross over strategy allows the facile synthesis of various y amines , or dihyy amines  via dual alkenes  90]. Th. Thgem-dgem-difluoroalkenes, trifluoromethyl arenes, and trifluoromethyl alkenes enabled by visible light photoredox catalysis from 2018 to the present. The level of interest in this area is growing rapidly, because the reactions in this review trebled those in our previous book chapter with the same focus. If electrochemical methods and transition metal-catalyzed reductive coupling reactions were counted, the progress on radical-involved C-F bond cleavage is even greater. Visible-light-promoted C-F bond cleavage, just like other easily handling reactions by photoredox catalysis, obtained a significant fillip because visible light is mild, green, and infinitely available. Photocatalysts in their excited states can be either single-electron oxidized or single-electron reduced. Due to their unique redox property and moderate redox potentials, visible light photocatalysts are expected to find more applications in selective cleavage of the C-F bond.Selective C-F bond cleavage of readily available per- or poly-fluorinated compounds has emerged as a powerful tool for effective synthesis of fluoroorganic compounds. In this review, we summarized the advances in C-F bond cleavage of polyfluorinated arenes, gem-difluoroalkenes, α-CF3 styrenes are the most common substrates for the defluorinative coupling with various radical precursors while examples of α-CF3 alkenes bearing one or two substituents on the β-positions are rare. Except for the C-F bond attached to the C-C double and aromatic rings, readily available poly-fluorinated reagents bearing other π-systems, such as trifluoroacetyl derivates and trifluoromethyl acetylenes, have not achieved by visible light photocatalysis [Since this field is currently shaping up, there is a lot more that needs to be explored by chemists in the future especially in terms of the broad scope of poly-fluorinated substrates. For example, an additional electron-withdrawing group on the benzene ring is usually needed for the C-F bond cleavage of poly-fluorinated arenes and trifluoromethyl arenes; at least one aryl group is necessary for the efficient C-F bond cleavage of atalysis . The C-F


Understanding the course of the antibody response directed to individual epitopes of SARS-CoV-2 proteins is crucial for serological assays and establishment of vaccines. Twenty-one synthetic peptides were synthesized that have ten amino acids overlap and cover the complete membrane (M) protein. Plasma samples from 32 patients having acute disease and 30 patients from the convalescent phase were studied. Only peptide M01 (aa 1–20) and to a lesser extent peptide M21 (aa 201–222) showed specific reactivity as compared to historical control plasma samples. Peptide M01 was recognized by IgM- (71.9%) and IgG-specific antibodies (43.8%) during the acute phase as early as day 8 PIO. In a longitudinal analysis, a higher reactivity was observed for the IgM response directed to peptide M01 following day 20 PIO as compared to earlier time points of the acute phase. In the convalescent phase, antibody reactivity to the two M-specific peptides was significantly lower (<30% seropositivity). A fusion protein encoding major parts of RBD also showed higher rates of recognition during acute (50.0%) and lower rates in the convalescent phase (23.3%). Taken together, our results suggest that during the acute phase of COVID-19 antibodies are raised to two linear epitopes of the SARS-CoV-2 M protein, located at the very N- and C-termini, showing almost similar levels of reactivity as immunodominant linear epitopes derived from the spike and nucleocapsid protein. Anti-M is also present in the convalescent phase of COVID-19 patients, however at lower levels, with the N-terminus of the M protein as a preferred target. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the pandemic coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a member of the betacoronavirus genus and belongs to the class of enveloped viruses having a diameter of ~80–120 nm containing a positive single-stranded RNA genome of about 30 kb , whereasThe spike protein, proteolytically processed by host proteases into two independent domains, named S1 and S2, mediates host cell entry , 11. TheCOVID‐19 is typically accompanied by fever, cough, fatigue, and difficulty of breathing; however mild or asymptomatic courses and other symptoms, like nasal congestion, loss of taste and smell, and diarrhea have been reported . Given tIn silico prediction of B cell epitopes was employed for the structural proteins, including S and M proteins . Participants were informed regarding the research project and written informed consent was obtained from all participants before enrollment. COVID-19 patients were enrolled between April and November 2020. Diagnosis followed the clinical description released by the World Health Organization . COVID‐1SARS-CoV-2 sequences spanning the membrane (M) gene were obtained from NCBI GenBank accession number MN908947.3. Approximately 21 N-terminal biotinylated 20-mer peptides having overlapping sequences of 10 residues were synthesized . Peptide S and peptide N were synthesized accordingly and contained sequences as reported previously  were incubated with peptides at a final concentration of 1 µg/ml in PBS for 2 h at room temperature (RT). Patient plasma was diluted at 1:250 in PowerBlock . Plasma samples were incubated overnight at RT. Horseradish peroxidase (HRP)-conjugated polyclonal goat antiserum was used that is directed to the constant region (Fc) of all four human immunoglobulin (IgG) subclasses . HRP-conjugated polyclonal goat antiserum was used for detection of the μ-chain of human IgM . Antibody incubation was performed at a dilution of 1:10,000 (IgG) and 1:5,000 (IgM) for 2 h. All washing steps (five times) were performed using PBS/0.05% Tween-20 . Tetramethylbenzidine (TMB) substrate  was incubated for 20 min and reaction was stopped by the addition of 2N sulfuric acid . Absorbance was measured at 450 nm using an Infinite M200 plate reader . OD values from a control well without peptide were used for each plasma sample and obtained values were subtracted from background OD. Repeated measurements of identical probes resulted in a low variance (mean 0.08 ± 0.01). A cut-off for positivity was set as the mean value plus two standard deviations (2SD) obtained with control sera.Complementary DNA was generated with 1 µg of RNA from a COVID-19 patient using SuperScript III and hexamers with random sequences according to the protocol of the manufacturer (Invitrogen). Cloning of amino acids S407-579 of the SARS-CoV-2 spike protein was performed by using Platinum SuperFI II Tag-polymerase (Invitrogen) and primersGCCATGTCAGACAAATCGCTCCAGGG-3’/5’-GGGGATCCTCAGTGGTGGTGGTGGTGGTGTGGATCACGGACAGCATCAGTAG-3’  . The resulting 0.5 kb PCR fragment was digested with NcoI and BamHI restriction enzymes (New England Biolabs) and cloned into T7 polymerase regulated E. coli expression vector OGWA (E. coli BL21 (DE3) . Induction was initiated at an OD600 of 0.6–0.8 by addition of 1 mM Isopropyl-β-D-thiogalactopyranosid  at 37°C. Protein purification after lysozyme (Thermo Fischer Scientific) digested bacterial lysates was performed using NI-NTA agarose according to the protocol of the manufacturer (CubeBiotec).5’-GCtor OGWA . ExpressThe purified protein was separated on a 12.5% SDS polyacrylamide gel and blotted onto a PVDF membrane (Bio-Rad Laboratories). The membrane was blocked for 2 h in Tris-buffered saline (TBS) containing 0.05% Tween 20  and 5% non-fat dry milk (Carl Roth). The membrane was dissected into individual stripes which were incubated with patient plasma (1:250 in 1 ml) overnight at RT. Polyclonal horseradish peroxidase (HRP)-linked secondary antibody (BioRad goat anti-human IgG (Fc):HRP, Cat. No. AHP1323P at 1:10,000 dilution) was incubated for 1 h. As control, an antibody directed against the histidine tag  was used at 1:1,000 dilution for every blotting experiment. Amersham ECL Western Blotting Detection Reagent  was added for staining. Images were obtained using a FUSION SOLO 6S EVOLUTION-CAPT . Determination of antibody binding was investigated by visual inspection of the blots and by ImageJ  analysis. Each experiment contained strips of a positive (anti-histidin antibody) and a negative control (negative control plasma or no plasma). Identical rectangle boxes were identified by anti-histidin positives. The number of pixels within boxes was referenced to the number of pixels in the negative control for each blot (pixel number investigated/pixel number of negative control). A fold value >1.5 was arbitrarily chosen to indicate positives. All positives identified by ImageJ analysis were in agreement with positives identified by visual inspection. Repeated measurements of identical plasma samples did not result in variant results (data not shown).p value ≤0.05 was chosen to indicate significance. Data are given as mean ± standard deviation (SD).Statistical analyses were performed by Kruskal–Wallis 1-way ANOVA and Chi Square tests using SPSS 27 software. Pearson’s correlation coefficient (r), receiver operating characteristic (ROC) analyses, and T test were calculated using GraphPad Prism 6. A Linear B-cell epitopes of SARS-CoV-2 membrane protein were studied during the acute and convalescent phase of COVID-19. Plasma samples were derived from patients following positive SARS-CoV-2 RT-PCR analysis. Approximately 32 plasma samples (S01 to S32) were derived during acute disease while being hospitalized. Approximately 30 plasma samples (R01 to R30) were from COVID-19 patients in the convalescent phase. Major characteristics of COVID-19 patients are presented in Having established that peptides M01 and M21 revealed high IgG-specific reactivity, the IgM-specific response was also investigated  between OD value and day PIO was observed for peptide M01  and convalescent phase (63.3%) had IgG-specific antibodies that recognized either one of the four peptides  and/or S407-579his fusion protein, individually or mixed (Serological assays are important to determine the exposure to SARS-CoV-2 in the general population and to gain knowledge about the temporal recognition of epitopes during COVID-19. The immune response directed to the virus spike protein has raised much interest, since this protein directs major arms of the immune system, e.g. by induction of neutralizing antibodies , 16. Howin silico prediction of B cell epitopes also suggested various epitopes, including the N- and C-terminus of M protein (Previously, using a broad epitope mapping of SARS-CoV-2 proteins including the M protein, it was revealed that the primary antibody response of COVID-19 patients is directed to the S and N protein , 26, 27. protein , 34. The protein , 35. The protein . In addiAnti-SARS-CoV-2 antibodies were reported to be detected as early as 3–4 days PIO . In our It was concluded that the level of antibodies to linear epitopes of SARS-CoV-2 proteins, including peptides S and N, correlates to the severity of the disease . Our stuin silico analyses, M protein of SARS-CoV-2 was also suggested to represent a candidate of vaccines (Antibodies to several B cell epitopes of the spike protein were identified to induce neutralizing antibodies –39. RBD vaccines . Whethervaccines –26. As pThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by ethics committee of the University Hospital Münster. The patients/participants provided their written informed consent to participate in this study.AZ and HS contributed to conception, design and supervision of the study. P-RT, RV, IS, KO, and CF organized the database. PJ, PS, and MW performed experiments. PJ, PS, MW, and AZ analyzed data. PJ, PS, and AZ wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The recent identification and rise to dominance of the P.1 and B.1.351 SARS-CoV-2 variants have brought international concern because they may confer fitness advantages. The same three positions in the receptor-binding domain (RBD) are affected in both variants, but where the 417 substitution differs, the E484K/N501Y have co-evolved by convergent evolution. Here we characterize the functional and immune evasive consequences of the P.1 and B.1.351 RBD mutations. E484K and N501Y result in gain-of-function with two different outcomes: The N501Y confers a ten-fold affinity increase towards ACE-2, but a modest antibody evasion potential of plasma from convalescent or vaccinated individuals, whereas the E484K displays a significant antibody evasion capacity without a major impact on affinity. On the other hand, the two different 417 substitutions severely impair the RBD/ACE-2 affinity, but in the combined P.1 and B.1.351 RBD variants, this effect is partly counterbalanced by the effect of the E484K and N501Y. Our results suggest that the combination of these three mutations is a two-step forward and one step back in terms of viral fitness. The continuous genetic drift resulting in immune adaptation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has received international attention since the first identification of new emerging variants in the spring of 2020 . A specihttps://www.gisaid.org, https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/, accessed 7th September 2021). Interestingly, only relatively few positions in the RBD seem to be subjected to selective genetic drift. Some of the key residues involved directly in ACE-2 binding have evolved independently at different continents by convergent evolution. The consequences of these residue changes are being studied intensively and the growing VOC group, which includes to date the strains B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta), has rapidly replaced the parent SARS-CoV-2 strain in the regions where they have been introduced. At the time of writing, new VOC/VOI are being reported almost every week . Nevertheless, a deeper molecular understanding of the interaction of the different strains with the host lags. All three VOC B.1.1.7, B.1.351, and P.1 contain the residue substitution N501Y due to an asparagine to tyrosine exchange in position 501 in the RBD domain. The N501Y substitution impacts transmissibility and disease severity   and 5 variants of interest (VOI)  (severity –15. The n = 150) previously infected with the parent Wuhan strain, sera from individuals after the 1st and 2nd dose with the BNT162b2 vaccine (n = 149), and a group of high-affinity monoclonal antibodies (n = 18) mapping to different epitopes on RBD.Here we present biophysical data of the impact of all the individual and combined residue changes in the RBD of P.1 and B.1.351 and show how these changes influence antibody neutralization of the ACE-2 interaction. We challenged the different constructs with sera from convalescent individuals (417 to either an asparagine (K417N) or a threonine (K417T) results in a significant reduction in RBD/ACE-2 affinity, while the N501Y confers a ten-fold affinity increase towards ACE-2. Neither of these substitutions seems to have a major effect on the antibody neutralization from convalescent or vaccinated individuals. In contrast, the E484K displays a significant antibody evasion capacity without significantly impacting the affinity. Combined in the B.1.351 and P.1 RBD variants, the two different K417 substitutions also lower the overall affinity, but the effect is in part counterbalanced by the E484K and N501Y gain-of-function changes. Our results suggest that the combination of these three mutations is a two-step forward and one step back in terms of viral fitness related to ACE-2 affinity and antibody evasion capacity.The results show that the three residue substitutions in positions 417, 484, and 501 seem to have very different functional impacts on the RBD. The substitution of lysineR319–S593) was optimized regarding the codon adaptation index, 5’ mRNA folding energy, cryptic splice sites, polyadenylation signals, and tandem repeats as described elsewhere  using the default 30°C/min thermal ramp. The RBD mutants were diluted to a final concentration of 0.5 mg/ml in PBS and analyzed in triplicates. Protein unfolding was monitored using the intrinsic fluorescence at 350 and 330 nm, and their ratio was used to determine the inflection temperatures (Ti).Binding kinetics measurements were performed on an Octet RED383 system  using the 16-channel mode with anti-human Fc capture (AHC) sensors . A description of the experimental setup can be found elsewhere .50.The antibody-mediated inhibition potency of sera and mouse monoclonal antibodies (mAbs) was assessed using a previously reported ELISA-based ACE-2/RBD antibody inhibition test . The evρ and p-value p) and linear regressions (reported as goodness-of-fit 2R). IC50 values for the panel of mAbs were interpolated from 6-point 4-fold serial dilutions using the equation [inhibitor] vs normalized response with variable slope. Non-inhibitory antibodies were normalized to 100.Statistical analyses were performed with GraphPad Prism 9 . Friedman test with Dunn’s multiple comparisons correction was used to analyze differences between the inhibition potency of convalescent and vaccinated sera by comparing the mean rank of each RBD mutant with the wt. Differences in the inhibition potency of convalescent sera and mAbs towards the RBD mutant were analyzed using two-tailed Spearman rank correlations , B.1.351 strain  , and P.1K417N = 77 nM, KDK417T = 56.7 nM, KDWT = 23.9 nM) . The E484K variant provides a slight gain in affinity (KD = 15.6 vs 23.9 nM), association rates (4.33x105vs 3.15x105 M−1s−1), and dissociation rates (6.77x10−3vs 7.55x10−3 s−1) compared to the wt . These combined and opposed effects are evidenced when analyzing the response curves of the B.1.351 RBD (N_K_Y) and P.1 RBD (T_K_Y)  . When considered in combination, the B.1.351 RBD (N_K_T) resulted in a 2.4-fold reduction in the median inhibition potency, and the P.1 (T_K_Y) in an even more dramatic 3.2-fold reduction (p < 0.0001 for both). The inhibition potencies towards the wt RBD and the mutations were significantly correlated   of sera or monoclonal antibodies of the ACE-2/RBD interaction . First, n = 150) Figure 350 values of the mutant RBDs were pairwise compared with the wt using linear regression and Spearman correlation analyses . The K417N  , ranging from a 1.5-fold decrease for the N501Y alone, to a 2.39-fold for the combined T_K_Y . The inhibition capacity increased dramatically after the second dose (p < 0.0001 for both). The effect of the N_K_Y and T_K_Y ranged from 1.6- to 1.8-fold inhibition potency decrease (p < 0.0001 for both), comparable to that of E484K alone.Finally, we assessed the immune evasion potential of the variants in sera from individuals immunized with the BNT162b2 vaccine. We collected blood samples from 149 healthy individuals before vaccination, around 3 weeks after the first dose, and 5 weeks after the second dose. The antibody-mediated RBD/ACE-2 inhibition was determined in serial dilutions of serum to account for the marked inhibition differences of naïve and vaccinated sera  Figure 5The relationship between established immunity from either recovered COVID-19 disease or vaccination and new emerging genetic SARS-CoV-2 strains is being heavily studied and debated.https://www.worldometers.info/coronavirus) (The confirmed worldwide COVID-19 cases are relentlessly reaching more than 220 million cases at the time of writing (navirus) . Since tnavirus) . This miin vitro evolution experiments aimed at increasing ACE-2 affinity have shown that the E484K and N501Y were among the first mutations to be selected and fixed  belonging to different epitope clusters, we did not observe a dramatic difference in the neutralization capacity between the variants tested here. Combined, the results thus suggest that the virus neutralization is not just a matter of changes of key residues in immunodominant B-cell epitopes but could also be a balance between the B-cell affinity maturation state and the biophysical affinity of the receptor affinity adapted strains. Interestingly, the same combined E484K/N501Y is found in the VOI B.1.621, originally identified in Colombia . The patients/participants provided their written informed consent to participate in this study.RB-O, PG, and MOS performed conceptualization (idea conception and study design); RB-O, CH, FM and JRB performed investigation ; LBJ performed investigation (RBD/ACE-2 binding kinetics characterization by BLI); RB-O and IJ performed investigation (ELISA-based experiments); CBH, SDN, KKI, SRO, HB, and RF-S performed resources (provision and registration of blood samples); RB-O performed formal analysis; RB-O, MOS, and PG performed writing – original draft. All authors contributed to the article and approved the submitted version.This work was financially supported by grants from the Carlsberg Foundation (CF20-0045) and the Novo Nordisk Foundation (NFF205A0063505 and NNF20SA0064201).LBJ, CH, JRB and FM were employed by Novo Nordisk A/S.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.