Fragments S1 Research Articles

B-085 Sars-cov-2 nt-chip: A Multiplex eia Platform for Detect Humoral Response and Neutralizing Antibodies Against Sars-cov-2

Abstract Background COVID-19 pandemic is a current health crisis that began in late 2019 and is caused by the pathogen SARS-CoV-2. This virus causes respiratory illness and has led to numerous infections and deaths worldwide. SARS-CoV-2 has an essential protein, the spike protein (S) containing a receptor-binding domain (RBD) binding to the angiotensin-converting enzyme 2 (ACE2) receptor on host cells. Studies have shown that mutations in the RBD can affect its ability to bind to the ACE2 receptor, and some of these mutations have been associated with increased transmissibility or disease severity. During infection, the host immune response can generate a variety of antibodies against SARS-CoV-2 proteins, including neutralizing antibodies that can target RBD. Serological tests could detect the presence of these antibodies in blood samples and are being used to determine whether a person has been infected with SARS-CoV-2 in the past or has developed immunity against the virus after vaccination. These tests also can help to better understand the spreading of new VOCs and epidemiological profiles of COVID-19. However, it is essential to note that the presence of antibodies does not guarantee immunity. Methods SARS-CoV-2 VOC Virachip IgG test was designed for the multiplex detection of binding antibodies to multiple SARS-CoV-2 proteins, including nucleocapsid (N), spike fragment S1 (S1), spike fragment S2 (S2), and the RBDs of Wuhan strain (RBD-w), Delta VOC (RBD-d), and Omicron VOC (RBD-o). This test was modified and further developed to also measure the neutralization effectiveness of antibodies binding against different RBDs. The percentage of inhibition of neutralizing antibodies against Wuhan and Omicron RBDs was determined. To achieve this, a purified ACE-2 Alkaline-Phosphatase conjugate was incorporated into the assay, and its ability to bind RBD spots in the microarray wells was measured after preincubation with sera or plasma. The presence of neutralizing antibodies was assessed for its capability of preventing such interaction, hence allowing us to detect neutralizing antibodies indirectly. Furthermore, it was also possible to generally detect binding antibodies against RBDs and the N protein. Assessing past infections in people immunized with mRNA vaccines, and in the same assay, detecting the subset of antibodies that promote functional neutralization against SARS-CoV-2 infection was feasible. Results A cohort containing 50 pre-pandemic and 61 post-infection sera the SARS-CoV-2 NT-CHIP yielded 100% specificity and 96.7% sensitivity regarding neutralization antibodies against RBD-w. In case of RBD-o analysis of 111 serum samples from patients before the emergence of Omicron and 69 serum samples from patients after infection resulted in 99.1% specifictiy and 95.7% sensitivity. These tests were performed in comparison with PRNT, the gold standard test for measuring neutralization. Conclusion This innovative microarray platform allows for quick upgrades to incorporate new RBDs from currently important VOCs. Given the status of the pandemic, the emergence of new variant strains, and vaccine adjustments, the SARS-COV-2 NT-CHIP could be a very valuable tool for assessing the humoral immunity generated against future VOCs. Thus, it contributes to the decision-making processes in the public health care system and is well suited for new vaccine studies.

Novel and sensitive electrochemical/fluorescent dual-mode biosensing platform based on the cascaded cyclic amplification of enzyme-free DDSA and functional nucleic acids

Herein, we present a novel electrochemical (EC)/fluorescent (FL) dual-mode biosensor for sensitive and accurate detection of target nucleic acids, which was based on the functional nucleic acids-involved enzyme-free dynamic DNA self-assembly of catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) for cascaded cyclic amplification. Originally, the CHA reaction of three well-designed hairpin probes were initiated by target sequence, forming abundant Mg2+-dependent three-way DNAzyme junctions (MTWDJ) which could recognize and cleave the methylene blue-labeled substrate hairpin (MB-Hs) to generate the MB-labeled fragments s1 (MB-s1) and the HCR initiator s2. Then, s2 triggered the HCR of four hairpins to produce long DNA nanowires which contained numerous G-quadruplex sequences and the same Mg2+-dependent DNAzyme (MNAzyme) sequences as MTWDJ. Therefore, the HCR copolymer could not only emerge the fluorescent signals through combining thioflavin T with G-quadruplex, but also generate MB-s1 and s2 via MNAzyme cleavage of MB-Hs to continue initiating the HCR. Meanwhile, MB-s1, the cleavage product of MTWDJ and MNAzyme, was captured on the DNA tetrahedron nanostructure modified electrode surface to bring electrochemical signals. Benefiting from integrating the efficient cyclic cleavage of MTWDJ and MNAzyme, the concatenated CHA and HCR amplification circuit, and the dual-mode detection, the sensitivity and accuracy of this biosensor were significantly improved. Under the optimal conditions, the proposed EC/FL dual-mode sensing strategy exhibited a superior analytical performance toward target nucleic acids, showing the promising application in bioanalysis and early disease diagnosis.

Development of a Clinical MALDI-ToF Mass Spectrometry Assay for SARS-CoV-2: Rational Design and Multi-Disciplinary Team Work.

The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has stretched national testing capacities to breaking points in almost all countries of the world. The need to rapidly screen vast numbers of a country’s population in order to control the spread of the infection is paramount. However, the logistical requirement for reagent supply (and associated cost) of RT-PCR based testing (the current front-line test) have been hugely problematic. Mass spectrometry-based methods using swab and gargle samples have been reported with promise, but have not approached the task from a systematic analysis of the entire diagnostic process. Here, the pipeline from sample processing, the biological characteristics of the pathogen in human biofluid, the downstream bio- and physical-chemistry and the all-important data processing with clinical interpretation and reporting, are carefully compiled into a single high-throughput and reproducible rapid process. Utilizing MALDI-ToF mass spectrometric detection to viral envelope glycoproteins in a systems biology-multidisciplinary team approach, we have achieved a multifaceted clinical MALDI ToF MS screening test, primarily (but not limited to) SARS-CoV-2, with direct application to other future epidemics/pandemics that may arise. The clinical information generated not only includes SARS-CoV-2 coronavirus detection–(Spike protein fragments S1, S2b, S2a peaks), but other respiratory viral infections detected as well as an assessment of generalised oral upper respiratory immune response (elevated total Ig light chain peak) and a measure of the viral immune response (elevated intensity of IgA heavy chain peak). The advantages of the method include; (1) ease of sampling, (2) speed of analysis, and much reduced cost of testing. These features reveal the diagnostic utility of MALDI-ToF mass spectrometry as a powerful and economically attractive global solution

Electrochemical biosensor for telomerase activity assay based on HCR and dual interaction of the poly-adenine DNA with Au electrode and Ce-Ti dioxide nanorods

Abstract A new electrochemical biosensor for sensitive detection of telomerase activity based on the hybridization chain reaction and the dual interaction of the poly-adenine (polyA) DNA with Au electrode and Ce-Ti dioxide nanorods (Ce-Ti NRs) has been constructed. Deoxynucleotide triphosphates (dNTPs) can be incorporated at the 3´-OH of TS primer by telomerase to generate the extension products with telomeric repeats (TTAGGG)n (TEP), which can hybridize with hairpin (HP) DNA and open it to form a duplex DNA with a short poly-adenine DNA single-stranded tail (s1). The duplex DNA can be recognized and selectively cleaved into single-stranded DNA fragment s1 and TEP by exonuclease III (Exo III). The released TEP can then hybridize with the HP again to initiate the s1 recycling process, which will result in the massive release of s1 fragments from HP. The s1 fragments can be immobilized on the Au electrode surface through the interaction between polyA and Au electrode on one hand. On the other hand, Ce-Ti NRs can be adsorbed on the Au electrode surface by the interaction between Ce-Ti NRs and the phosphate groups of s1, resulting in the decrease of the differential pulse voltammetry (DPV) current of the redox probe. The higher activity of telomerase, more HP can be hybridized and cleaved, more poly-adenine and Ce-Ti NRs can be immobilized on electrode surface, and remarkable decrease of the DPV signal can be observed. The biosensor exhibited good sensitivity toward telomerase activity in HeLa cells.

A paramagnetic molecular voltmeter

We have developed a general electron paramagnetic resonance (EPR) method to measure electrostatic potential at spin labels on proteins to millivolt accuracy. Electrostatic potential is fundamental to energy-transducing proteins like myosin, because molecular energy storage and retrieval is primarily electrostatic. Quantitative analysis of protein electrostatics demands a site-specific spectroscopic method sensitive to millivolt changes. Previous electrostatic potential studies on macromolecules fell short in sensitivity, accuracy and/or specificity. Our approach uses fast-relaxing charged and neutral paramagnetic relaxation agents (PRAs) to increase nitroxide spin label relaxation rate solely through collisional spin exchange. These PRAs were calibrated in experiments on small nitroxides of known structure and charge to account for differences in their relaxation efficiency. Nitroxide longitudinal ( R 1) and transverse ( R 2) relaxation rates were separated by applying lineshape analysis to progressive saturation spectra. The ratio of measured R 1 increases for each pair of charged and neutral PRAs measures the shift in local PRA concentration due to electrostatic potential. Voltage at the spin label is then calculated using the Boltzmann equation. Measured voltages for two small charged nitroxides agree with Debye–Hückel calculations. Voltage for spin-labeled myosin fragment S1 also agrees with calculation based on the p K shift of the reacted cysteine.

Prothrombin fragments containing kringle domains induce migration and activation of human neutrophils

The cross-talk between inflammatory and coagulation cascades has been demonstrated. Prothrombin processing releases the protease domain (thrombin) along with two catalytically inactive kringle-containing derivatives: prothrombin fragments 1 (F1) and 2 (F2). It is well established that thrombin is able to trigger an inflammatory response but the possible effects of prothrombin fragments on leukocyte functions are still unknown. In this report, we demonstrate for the first time that both F1 and F2 prothrombin fragments, interfere with intracellular functional signaling pathways to modulate human neutrophil migration. In addition, we show that thrombin, fragment 1 and fragment 2 induce human neutrophil chemotaxis. The effect of fragment 2, but not fragment 1, was partially inhibited by pertussis toxin, an inhibitor of G αi-signaling. The pre-treatment of cells with fragment 2 inhibited thrombin-induced chemotaxis, while both fragments impaired neutrophil migration induced by interleukin-8. F1 and F2 increased the expression and activation of G-protein-coupled receptor kinase-2, which has emerged as a key effector in the desensitization of chemokine receptors. In parallel, prothrombin fragments activated extracellular signal-regulated kinase 1/2, stimulating its phosphorylation and nuclear translocation, and induced inhibitor of kappa-B phosphorylation and degradation followed by nuclear factor-kappa B translocation to nucleus. Furthermore, both prothrombin fragments induced interleukin-8 gene expression in human neutrophils. These findings suggest that the interference with neutrophil signaling and function, caused by kringle-containing prothrombin fragments may desensitize these cells to respond to further activation by thrombin and interleukin-8 during inflammatory and coagulation responses.

The immunity induced by recombinant spike proteins of SARS coronavirus in Balb/c mice

SummaryThe immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322 1464 bp) and S2 (2170 2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E. coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and purified by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with complete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-concentration was increased significantly but the concentrations of Il-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.

DNA Binding Selectivity of MeCP2 Due to a Requirement for A/T Sequences Adjacent to Methyl-CpG

DNA methylation is interpreted by a family of methyl-CpG binding domain (MBD) proteins that repress transcription through recruitment of corepressors that modify chromatin. To compare in vivo binding of MeCP2 and MBD2, we analyzed immunoprecipitated chromatin from primary human cells. Genomic sites occupied by the two MBD proteins were mutually exclusive. As MeCP2 was unable to colonize sites vacated by depletion of MBD2, we tested the hypothesis that methyl-CpG alone is insufficient to direct MeCP2 binding. In vitro selection for MeCP2 bound DNA-enriched fragments containing A/T bases ([A/T] > or = 4) adjacent to methyl-CpG. [A/T] > or = 4 was found to be essential for high-affinity binding at selected sites and at known MeCP2 target regions in the Bdnf and Dlx6 genes. MBD2 binding, however, did not require an A/T run. The unexpected restriction of MeCP2 to a defined subset of methyl-CpG sites will facilitate identification of genomic targets that are relevant to Rett Syndrome.

Phylogenomics and bioinformatics of SARS-CoV

Tracing the history of molecular changes in coronaviruses using phylogenetic methods can provide powerful insights into the patterns of modification to sequences that underlie alteration to selective pressure and molecular function in the SARS-CoV (severe acute respiratory syndrome coronavirus) genome. The topology and branch lengths of the phylogenetic relationships among the family Coronaviridae, including SARS-CoV, have been estimated using the replicase polyprotein. The spike protein fragments S1 (involved in receptor-binding) and S2 (involved in membrane fusion) have been found to have different mutation rates. Fragment S1 can be further divided into two regions (S1A, which comprises approximately the first 400 nucleotides, and S1B, comprising the next 280) that also show different rates of mutation. The phylogeny presented on the basis of S1B shows that SARS-CoV is closely related to MHV (murine hepatitis virus), which is known to bind the murine receptor CEACAM1. The predicted structure, accessibility and mutation rate of the S1B region is also presented. Because anti-SARS drugs based on S2 heptads have short half-lives and are difficult to manufacture, our findings suggest that the S1B region might be of interest for anti-SARS drug discovery.

Field desorption mass spectra of flavonoid acylglycosides. I. Natural acetyl derivatives

The field desorption mass spectra (FD spectra) of 17 natural flavonoid acetylglycosides have been studied. In the spectra of each of the 0-monoglycosides the molecular ion (M or M + H) appears as the main peak and it is accompanied by the ions of the aglycone (A or A + H) and of the acylated anhydrosugar (S). The intensity of the latter peak is largely connected with the structure of the substance. In the FD spectra of flavone 0-biosides, fragment S is absent but its mass can be calculated from the difference (M − A). Useful information for establishing the position of the acetyl group is given by the fragments S1 and (M − S1) corresponding to the detachment of the terminal sugar residue. The FD spectra of flavone C-glycosides differ greatly from the spectra of the 0-glycosides: In them the main peak is that of the ion (M), but peaks (A) and (S) are absent and the ions present resemble the fragmentation of the C-glycosides under the action of electron impact.

Actin microfilaments in paramecium: localization and role in intracellular movements.

Using heavy meromyosin (HMM) or the fragment S1 of myosin as probes for actin microfilaments, we studied their organization in Paramecium both by fluorescence and electron microscopy. In interphasic cells, HMM decorates (a) most prominently the periphery of nascent and young food vacuoles and their route during the early phase of their intracellular transit; (b) a thin meshwork radiating from the gullet throughout the cytoplasm; (c) a small area beneath the pore of contractile vacuoles and beneath the cytoproct when open to release food residues. Most of these HMM-decorated structures are in close contact with microtubular arrays. All HMM decoration disappears in dividing cells and in cytochalasin-treated cells. In vivo, the drug immediately blocks food vacuole formation but does not affect cytokinesis, cyclosis, contractile vacuole pulsation, defecation, or nuclear movements. The data show that, as in the cells of other organisms, actin microfilaments form defined arrays that undergo physiologically controlled cycles of assembly/disassembly. These arrays contribute (at least in the phagocytotic process) to diverse types of movement: constriction, membrane fusion, and migration of food vacuoles. However, aside from their massive concentration along the phagocytotic tractus, actin microfilaments are neither major structural components of Paramecium cytoplasm nor the only cytoskeletal components ensuring motility or contractility processes.

Chemical cleavage of bovine rhodopsin at tryptophanyl bonds; characterization of the polypeptidic fragments and the phosphorylated site

Bovine rhodopsin from retinal rod photoreceptors, a protein of 39,000 molecular weight, was cleaved by BNPS-Skatole at the level of tryptophanyl bonds. This hydrolysis yields five fragments S1, S2, S3, S4 and S5 (molecular weights: 35,000, 28,000, 19,500, and 15,500 and 12,000, respectively) and four peptides. Large fragments were purified by polyacrylamide gel electrophoresis in SDS. S2, S3 and S4 contain the glycanes of native rhodopsin and their N-termini are blocked. S5 has the same C-terminal extremity as rhodopsin and contains the phosphorylated site. Phosphate groups are incorporated in this fragment on serines and threonines.

Nucleation of polar actin filament assembly by a positively charged surface.

Polylysine-coated polystyrene beads can nucleate polar assembly of monomeric actin into filamentous form. This nucleation has been demonstrated by a combination of biochemical and structural experiments. The polylysine-coated beads accelerate the rate of actin assembly as detected by two different biochemical assays. Subsequent examination of the beads by electron microscopy reveals numerous actin filaments of similar length radiating from the beads. ATP promotes this bead-induced acceleration of assembly. Decoration of the filaments with the myosin fragment S1 shows that these filaments all have the same polarity, with the arrowhead pattern pointing toward the bead. The relevance of the system to in vitro mechanisms and its usefulness in other studies are discussed.