Ziyu Kuai

A novel “turn-on” thiooxofluorescein-based colorimetric and fluorescent sensor for Hg2+ and its application in living cells

A novel water-soluble fluorescent probe FLS2 based on the thiooxofluorescein derivative has been firstly designed and synthesized. UV-vis absorption and fluorescence spectra studies showed that the FLS2 as a colorimetric and ratiometric fluorescent probe exhibited high selectivity and sensitivity towards Hg2+, which was mainly attributed to the special binding with the receptor unit accompanied with the spirolactam ring-opening progress. In addition, the probe FLS2 could be used as a naked-eye indicator for Hg2+ with reversible response. It displayed approximate 37-fold fluorescent enhancement at 529nm in the presence of only 2.0 equiv. Hg2+ and the detection limit was calculated at about 39nM. Whats more, cellular imaging experiment revealed that the sensor had excellent biocompatibility and low cytotoxicity that could be utilized for monitoring Hg2+ in living cells.

Evaluation of the immunogenicity and protective effects of a trivalent chimeric norovirus P particle immunogen displaying influenza HA2 from subtypes H1, H3 and B

The ectodomain of the influenza A virus (IAV) hemagglutinin (HA) stem is highly conserved across strains and has shown promise as a universal influenza vaccine in a mouse model. In this study, potential B-cell epitopes were found through sequence alignment and epitope prediction in a stem fragment, HA2:90-105, which is highly conserved among virus subtypes H1, H3 and B. A norovirus (NoV) P particle platform was used to express the HA2:90-105 sequences from subtypes H1, H3 and B in loops 1, 2 and 3 of the protrusion (P) domain, respectively. Through mouse immunization and microneutralization assays, the immunogenicity and protective efficacy of the chimeric NoV P particle (trivalent HA2-PP) were tested against infection with three subtypes (H1N1, H3N2 and B) of IAV in Madin–Darby canine kidney cells. The protective efficacy of the trivalent HA2-PP was also evaluated preliminarily in vivo by virus challenge in the mouse model. The trivalent HA2-PP immunogen induced significant IgG antibody responses, which could be enhanced by a virus booster vaccination. Moreover, the trivalent HA2-PP immunogen also demonstrated in vitro neutralization of the H3 and B viruses, and in vivo protection against the H3 virus. Our results support the notion that a broadly protective vaccine approach using an HA2-based NoV P particle platform can provide cross-protection against challenge viruses of different IAV subtypes. The efficacy of the immunogen should be further enhanced for practicality, and a better understanding of the protective immune mechanism will be critical for the development of HA2-based multivalent vaccines.

Conserved stem fragment from H3 influenza hemagglutinin elicits cross-clade neutralizing antibodies through stalk-targeted blocking of conformational change during membrane fusion

Currently available influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies due to the mutability of virus sequences and conformational changes during protective immunity, thereby limiting their efficacy. This problem needs to be addressed by further understanding the mechanisms of neutralization and finding the desired neutralizing site during membrane fusion. This study specifically focused on viruses of the H3N2 subtype, which have persisted as a principal source of influenza-related morbidity and mortality in humans since the 1968 influenza pandemic. Through sequence alignment and epitope prediction, a series of highly conserved stem fragments (spanning 47 years) were found and coupled to the Keyhole Limpet Hemocyanin (KLH) protein. By application of a combinatorial display library and crystal structure modeling, a stem fragment immunogen, located at the turning point of the HA neck undergoing conformational change during membrane fusion with both B- and T-cell epitopes, was identified. After synthesis of the optimal stem fragment using a multiple antigen peptide (MAP) system, strong humoral immune responses and cross-clade neutralizing activities against strains from the H3 subtype of group 2 influenza viruses after animal immunizations were observed. By detection of nuclear protein immunofluorescence with acid bypass treatment, antisera raised against MAP4 immunogens of the stem fragment showed the potential to inhibit the conformational change of HA in stem-targeted virus neutralization. The identification of this conserved stem fragment provides great potential for exploitation of this site of vulnerability in therapeutic and vaccine design.

Elicitation of HIV-1 neutralizing antibodies by presentation of 4E10 and 10E8 epitopes on Norovirus P particles.

Eliciting efficient broadly neutralizing antibodies (BnAbs) is a major goal in vaccine development against human immunodeficiency virus type 1 (HIV-1). Conserved epitopes in the membrane-proximal external region (MPER) of HIV-1 are a significant target. In this study, Norovirus P particles (NoV PPs) were used as carriers to display conformational 4E10 and 10E8 epitopes in different patterns with an appropriate linker. Immune responses to the recombinant NoV PPs were characterized in guinea pigs and Balb/c mice and could induce high levels of MPER-binding antibodies. Modest neutralizing activities could be detected in sera of guinea pigs but not of Balb/c mice. The 4E10 or 10E8 epitopes dispersed on three loops on the outermost surface of NoV PPs (4E10-loop123 PP or 10E8-loop123 PP) elicited higher neutralizing activities than the equivalent number of epitopes presented on loop 2 only (4E10-3loop2 PP or 10E8-3loop2 PP). The epitopes on different loops of the PP were well-exposed and likely formed an appropriate conformation to induce neutralizing antibodies. Although sera of immunized guinea pigs could neutralize several HIV envelope-pseudoviruses, a vaccine candidate for efficiently inducing HIV-1 BnAbs remains to be developed.

Exploration of binding and inhibition mechanism of a small molecule inhibitor of influenza virus H1N1 hemagglutinin by molecular dynamics simulation

Influenza viruses are a major public health threat worldwide. The influenza hemagglutinin (HA) plays an essential role in the virus life cycle. Due to the high conservation of the HA stem region, it has become an especially attractive target for inhibitors for therapeutics. In this study, molecular simulation was applied to study the mechanism of a small molecule inhibitor (MBX2329) of influenza HA. Behaviors of the small molecule under neutral and acidic conditions were investigated, and an interesting dynamic binding mechanism was found. The results suggested that the binding of the inhibitor with HA under neutral conditions facilitates only its intake, while it interacts with HA under acidic conditions using a different mechanism at a new binding site. After a series of experiments, we believe that binding of the inhibitor can prevent the release of HA1 from HA2, further maintaining the rigidity of the HA2 loop and stabilizing the distance between the long helix and short helices. The investigated residues in the new binding site show high conservation, implying that the new binding pocket has the potential to be an effective drug target. The results of this study will provide a theoretical basis for the mechanism of new influenza virus inhibitors.

A simple colorimetric and fluorescent probe with high selectivity towards cysteine over homocysteine and glutathione

A novel fluorescent probe (AQDA) based on quinizarin is designed and synthesized. Owing to a nucleophilic addition and a specific intramolecular cyclization reaction, the probe displays high selectivity towards cysteine (Cys) relative to other natural amino acids. The maximum fluorescent intensity is 30-fold that of the initial value in the presence of 5.0 equiv. Cys, and its detection limit is 0.158 μM. The recognition mechanism is further confirmed through mass spectroscopy and proton nuclear magnetic resonance titration. Simultaneously, the fluorescence enhancement mechanism is characterized by theoretical calculations, and experimental data are consistent with the theoretical results. Finally, the cellular imaging experiment verifies that AQDA possesses the capacity to detect endogenous Cys in living cells.

Potential of a novel peptide P16-D from the membrane-proximal external region of human immunodeficiency virus type 1 to enhance retrovirus infection

The peptide P13 (Ac-671NWFDITNWLWYIK683-NH2), derived from the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) transmembrane protein and its derivative P16, have been shown to significantly boost HIV-1 infectivity by forming amyloid fibrils. Here, a new modified nanofibril peptide P16-D derived from P16 was demonstrated to have an enhanced ability to promote retroviral gene transfer. Moreover, the “networks” formed by P16-D nanofibrils could effectively capture and concentrate enveloped virus by low-speed centrifugation. In addition, the captured influenza virus H1N1 could elicit a stronger immune response in mice at a lower dose than that in the absence of the nanofibrils. The results implied a potential for P16-D to improve gene transfer rates and vaccine applications.

Humanization and directed evolution of the selenium-containing scFv phage abzyme

According to the binding site structure and the catalytic mechanism of the native glutathione peroxidase (GPX), three glutathione derivatives, GSH-S-DNP butyl ester (hapten Be), GSH-S-DNP hexyl ester (hapten He) and GSH-S-DNP hexamethylene ester (hapten Hme) were synthesized. By a four-round panning with a human synthetic scFv phage library against three haptens, the enrichment of the scFv phage particles with specific binding activity could be determined. Three phage particles were selected binding to each glutathione derivative, respectively. After a two-step chemical mutation to convert the serine residues of the scFv phage particles into selenocysteine residues, GPX activity could be observed and determined upto 3000 U μmol−1 in the selenium-containing scFv phage abzyme which was isolated by affinity capture against the hapten Be. Also the scFv phage abzymes elicited by different antigens displayed different catalytic activities. After a directed evolution by DNA shuffling to improve the affinity to the hapten Be, a secondary library with GPX activity was created in which the catalytic activity of the selenium-containing scFv phage abzyme could be increased 17%. This study might be helpful for new haptens or antigens design to optimize the abzymes with high binding activities and might also provide a novel scheme for GPX mimic candidates for drug development.

Effects of poly(I:C) and MF59 co-adjuvants on immunogenicity and efficacy of survivin polypeptide immunogen against melanoma

Malignant tumors pose a public health problem that jeopardizes human life and quality of living. At present, tumor vaccines in clinical research typically are aimed at stimulating the cellular immune response, while more effective vaccines should take into account the synergy between broad spectrum antibodies and high levels of cellular immunity. In this study, epitope peptides (68–81, 95–104, 80–88) of the tumor antigen survivin were chosen as immunogens and supplemented with poly(I:C) and/or MF59 adjuvant to evaluate the immune effects and anti‐melanoma activities. The results indicated that poly(I:C) and MF59 could assist the survivin epitope peptide immunogen to control the tumor size, quality, and volume in black melanoma mouse models. Analyses by antibody titering, antibody isotyping and ELISPOT suggested that the adjuvanted immunogen could induce humoral immunity in mice. Poly(I:C) and MF59 combined with survivin peptide 95–104 could effectively induce humoral immunity mediated by type 2 T helper (Th2) cells. This study provides a basis for candidate immunogen design based on survivin and provides support for tumor therapy that can induce a more balanced Th1/Th2 immune response.

Effects of insulin on transcriptional response and permeability in an in vitro model of human blood‐brain barrier

Alzheimers disease (AD) is the most prevalent form of dementia worldwide and is an emerging global epidemic. Active and passive immune therapies targeting beta amyloid (Aβ) have shown very limited evidence in human studies of clinical benefits from these approaches. Epidemiological studies have shown that subjects with type 2 diabetes (T2D) are at higher risk of developing AD. However, whether and how these two conditions are causally linked is unknown. With the purpose of confirming the relationship between T2D and AD, this study specifically focused on effects of insulin in an in vitro model of the human blood‐brain barrier (BBB) and on potential mechanisms of action in the treatment of AD. By using a series of assays to establish a BBB model, we demonstrated that insulin treatment alone could induce the increase of brain endothelial barrier properties. The transcriptional response of hCMEC/D3 cells to activation with different concentrations of insulin was determined by RT‐PCR, and expression levels of genes involved in the control of barrier permeability, including inter‐brain endothelial junctions, integrin‐focal adhesions complexes, and transporter system, were found to be altered by the treatment. Notably, the influence of insulin on expression of the ATP‐binding cassette (ABC) transporter which contributes to the clearance of Aβ was investigated. Insulin up‐regulated adherens junction and tight junction transmembrane proteins, as well as the ABC transporter. By treatment with insulin, the models have major advantages: it is fast, it has low cost, it is fit for considerable samples, and its conditions are under control.