The POR rs10954732 polymorphism decreases susceptibility to hepatocellular carcinoma and hepsin as a prognostic biomarker correlated with immune infiltration based on proteomics.

The effect of the cytochrome P450 oxidoreductase (POR) rs10954732 (G > A) polymorphism on hepatocellular carcinoma (HCC) susceptibility is unknown. Here we found that A allele carriers showed a 69% decrease in susceptibility to HCC with overall survival (OS) prolonged to 199%, accompanied by lower activity for cytochrome P450 2E1. A total of 222 differentially expressed proteins were mainly enriched in neutrophil and T cell activation and involved in the immune and inflammatory responses, constituting the altered immune tumor microenvironment related with A allele by proteomics analysis. Hepsin (HPN) showed significant down-regulation in HCC and up-regulation in A allele carriers. A lower HPN level was associated with increased susceptibility to HCC and a worse prognosis. Moreover, HPN is a potential independent prognostic biomarker for HCC and is strongly associated with clinicopathological features, tumor-infiltrating status of immune cells both in our discovery cohort and database surveys. Our findings provide a new potential mechanism by which HPN may play an important role in the susceptibility of rs10954732 A allele carriers to HCC and their prognosis through tumor immune infiltration, thus offering potential insights for future studies on tumor immunotherapy.

Fc-specific and covalent conjugation of a fluorescent protein to a native antibody through a photoconjugation strategy for fabrication of a novel photostable fluorescent antibody.

Fluorophore-antibody conjugates with high photobleaching resistance, high chemical stability, and Fc-specific attachment is a great advantage for immunofluorescence imaging. Here, an Fc-binding protein (Z-domain) carrying a photo-cross-linker (p-benzoylphenylalanine, Bpa) fused with enhanced green fluorescent protein (EGFP), namely photoactivatable ZBpa-EGFP recombinant, was directly generated using the aminoacyl-tRNA synthetase/suppressor tRNA technique without any further modification. By employing the photoactivatable ZBpa-EGFP, an optimal approach was successfully developed which enabled EGFP to site-selectively and covalently attach to native antibody (IgG) with approximately 90% conjugation efficiency. After characterizing the Fc-specific and covalent manner of the EGFP-photoconjugated antibody, its excellent photobleaching resistance for immunofluorescence imaging was demonstrated in a model study by monitoring the toll-like receptor 4 (TLR4) expression in HepG2 cells. The proposed approach here for the preparation of a novel fluorescent antibody is available and reliable, which would play an important role in fluorescence immunoassay, and is expected to be extended to the generation of other biomolecule-photoconjugated antibodies, such as other fluorescent proteins for multiplex immunofluorescence imaging or reporter enzymes for highly sensitive enzyme immunoassays.Graphical abstract.

Efficient copper(i)-catalyzed oxidative intermolecular 1,2-estersulfenylation of styrenes with peroxyesters and disulfides.

A simple and practical method for the synthesis of thio-substituted esters through copper(i)-catalyzed intermolecular 1,2-estersulfenylation of styrenes with peroxyesters and disulfides was developed. In this transformation, two new C-S bond and C-O bond were constructed simultaneously under a copper catalyst system, and the transformation exhibits a broad substrate scope and good functional group compatibility. In addition, this method can also be applied to arylthiols. It should be noted that peroxyesters not only acted as nucleophilic reagents but also as oxidants.

Role of Mitogen-Activated Protein Kinases in the Formation of Hypertrophic Scar with Model of Lipopolysaccharide Stimulated Skin Fibroblast Cells.

OBJECTIVE: Hypertrophic scar is common in burn patients, but treating result could not meet the expectation of the patients and doctors. We have found that certain concentration level of lipopolysaccharide (LPS) stimulated normal fibroblast cells have statistically similar with fibroblast cells from hypertrophic scar on the phenotype level, and with this work we are trying to figure out which Mitogen-Activated Protein Kinase (MAPK) is affected and how it is affected. METHODS: Experiments were conducted in May, 2017 at the first affiliated hospital of the Chinese PLA General Hospital, Beijing, China. We have cultured the cell line of human skin fibroblast cells and randomly divided cells into four groups: control group and three stimulation groups. We have rebuilt the LPS stimulated model of skin fibroblast cells in hypertrophic scar based on our previous work. Experimental groups were stimulated with 0.1ug/mL LPS concentration for 24 hours, 48 hours, and 72 hours, respectively. Then we performed western blot analysis of Erk, p-Erk, JNK, p-JNK, p38 and p-p38. We performed statistical analysis with SPSS 15.0. RESULTS: LPS can up regulate the MAPK/p38 pathway (p<0.05) and down regulate the MAPK/Erk and MAPK/JNK pathways (p<0.05). The changes of phosphorylated protein are time-related, with longer stimulation duration, significant difference is increased (p<0.05). CONCLUSION: MAPKs can play an important role in the formation of hypertrophic scar in the skin. Early intervention through the MAPKs could be a promising target in the prevention of the formation of hypertrophic scar.

Structure-Property Relationship Study of Donor and Acceptor 2,6-Disubstituted BODIPY Derivatives for High Performance Dye-Sensitized Solar Cells.

Seven donor and acceptor 2,6-disubstituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes have been synthesized and characterized. Including MPBTCA, which is a known compound, the seven BODIPY dyes have been characterized by varied physical methods, such as UV/Visible absorption spectroscopy, low energy photo-electron spectroscopy (AC-2), and HOMO-LUMO DFT/TDDFT calculation. All seven BODIPY dyes have absorption λmax around 535-545 nm, which is significantly longer than 499 nm of 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (PM 546). Having structural variation on donor group, acceptor group, donor π-spacer, acceptor π-spacer, and the substituent on boron, some BODIPY dyes exhibit small extinction coefficients or spectral integrals in solution (MPCtBTCA, MPBT-pyO, MPBTT-pyO, MTBTCA), broadening absorption spectral profile (MTBTCA), weak intramolecular charge transfer characteristics (MPBT-pyO, MPBTT-pyO, MTBTCA), too low LUMO energy level (PPBTCA), or insufficient dye-uptake by TiO2 FTO (MPBT-pyO, MPBTT-pyO, MTBTCA). Two of the seven BODIPY dyes, MPBTCA and MPBTTCA, do not show the adverse properties like other BODIPY dyes. With our improved TiO2 FTO (fluorine doped tin oxide) dyeing method, namely a solution dropping method, high performance dye-sensitized solar cells (DSCs) have been realized by MPBTCA and MPBTTCA photosensitizers. Power conversion efficiencies of 6.3 and 6.4 % have been achieved by MPBTCA and MPBTTCA DSCs, respectively. To the best of our knowledge, MPBTCA and MPBTTCA are the most efficient dyes for the donor and acceptor 2,6-disubstituted BODIPY DSCs so far.

A 4,5-quinolimide-based fluorescent sensor for the turn-on detection of Cd2+ with live-cell imaging.

A 4,5-quinolimide derivative, BNA, bearing the amide-DPA receptor, was synthesized as a turn-on fluorescent sensor for Cd2+. Under physiological conditions, BNA could distinguish Cd2+ from Zn2+, showing turn-on fluorescence behaviour and an increased fluorescence lifetime. BNA and Cd2+ formed a 1 : 1 stoichiometric complex, and the detection limit was measured to be as low as 11 nM. Furthermore, BNA was utilized for fluorescence imaging of Cd2+ in live cells. To the best of our knowledge, it is the first 4,5-quinolimide-based sensor for the detection of metal ions.

Curcumin induces apoptosis in p53-null Hep3B cells through a TAp73/DNp73-dependent pathway.

Curcumin has anticancer functions in various tumors. It has been shown to induce apoptosis through p53-dependent pathways. p73 gene is a member of the p53 family which encodes both a tumor suppressor (transactivation-competent p73 (TAp73)) and a putative oncogene (dominant-negative p73 (DNp73)); the former shares similarity with the tumor suppressor p53, and the latter behaves as dominant-negative proteins that interfere with the activity of TAp73. To understand the p73-dependent mechanisms that are engaged during curcumin-induced apoptosis, we established a p73 overexpression cell models using p53-deficient Hep3B cells (Hep3B(TAp73/DNp73)). Our results demonstrated that curcumin at concentrations of 40 and 80 µM induced DNA damage, increased TAp73/DNp73 ratio, and also led to apoptosis in the Hep3B(TAp73/DNp73) cells. The apoptotic cell death was concurrent with the loss of mitochondrial membrane potential; release of cytochrome c from mitochondria; and the cleavage of caspase 9, caspase 3, and poly(ADP-ribose) polymerase (PARP). These results demonstrated a p73-dependent mechanism for curcumin-induced apoptosis that involves the mitochondria-mediated pathway.

Site-specific, covalent immobilization of BirA by microbial transglutaminase: A reusable biocatalyst for in vitro biotinylation.

A facile approach for the production of a reusable immobilized recombinant Escherichia coli biotin ligase (BirA) onto amine-modified magnetic microspheres (MMS) via covalent cross-linking catalyzed using microbial transglutaminase (MTG) was proposed in this study. The site-specifically immobilized BirA exhibited approximately 95% of enzymatic activity of the free BirA, and without a significant loss in intrinsic activity after 10 rounds of recycling (P > 0.05). In addition, the immobilized BirA can be easily recovered from the solution via a simple magnetic separation. Thus, the immobilized BirA may be of general use for in vitro biotinylation in an efficient and economical manner.

An efficient protocol to enhance the extracellular production of recombinant protein from Escherichia coli by the synergistic effects of sucrose, glycine, and Triton X-100.

Targeting recombinant proteins at highly extracellular production in the culture medium of Escherichia coli presents a significant advantage over cytoplasmic or periplasmic expression. In this work, a recombinant protein between ZZ protein and alkaline phosphatase (rZZ-AP) was constructed. Because rZZ-AP has the IgG-binding capacity and enzymatic activity, it can serve as an immunoreagent in immunoassays. However, only a very small portion of rZZ-AP is generally secreted into the aqueous medium under conventional cultivation procedure. Hence, we emphasized on the optimization of the culture procedures and attempted to dramatically enhance the yield of extracellular rZZ-AP from E. coli HB101 host cells by adding sucrose, glycine, and Triton X-100 in the culture medium. Results showed that the extracellular production of rZZ-AP in the culture medium containing 5% sucrose, 1% glycine, and 1% Triton X-100 was 18.6 mg/l, which was 18.6-fold higher than that without the three chemicals. And the ß-galactosidase activity test showed that the increased extracellular rZZ-AP was not due to cell lysis. Further analysis suggested a significant interaction effect among the three chemicals for the enhancement of extracellular production. Ultrastructural analysis indicated that the enhancement may be due to the influence of sucrose, glycine, and Triton X-100 on the periplasmic osmolality, permeability, or integrity of the cell wall, respectively. This proposed approach presents a simple strategy to enhance the extracellular secretion of recombinant proteins in the E. coli system at the process of cell cultivation.

[Effects of continuous sedation with propofol on peripheral blood mononuclear cell and intercellular adhesion molecule in beagles with combined burn-blast injuries].

OBJECTIVE: To observe the effects of continuous sedation with propofol on peripheral blood mononuclear cell (PBMC) and intercellular adhesion molecule 1 (ICAM-1) in beagles with combined burn-blast injuries. METHODS: A total of 32 male beagles were randomly divided into 4 groups of normal control (NC), combined injury control (CC), propofol 1 (P1) and propofol 2 (P2) (n = 8 each). Except for NC group, the other 3 groups were subject to severe combined burn-blast injury. And sodium lactate Ringer's solution was infused after trauma according to the Parkland formula, including NC group. At the same time, P1 and P2 groups received continuous intravenous infusions of 2 mg×kg(-1)×h(-1), 5 mg×kg(-1)×h(-1) doses of propofol respectively for 72 hours. The serum concentrations of ICAM-1 and lymphocyte function associated antigen-1 (LFA-1) were measured by enzyme-linked immunosorbent assay (ELISA) at 6, 24, 48, 72 h post-injury. Flow cytometry was used to detect the major histocompatibility complex (MHC) antigen expression on CD14(+) monocytes, CD4(+)/CD8(+) T lymphocyte rate and PBMC apoptosis rate. RESULTS: The level of ICAM-1 in CC group ((10.5 ± 1.1), (10.8 ± 1.3), (12.3 ± 1.4) ng/ml) was significantly higher than that in NC group ((7.4 ± 1.4), (7.4 ± 1.1), (7.4 ± 1.6) ng/ml) at 12, 24, 48 h post-injury (all P < 0.05). The level of ICAM-1 in P1 group was significantly lower than that in CC group ((10.7 ± 1.3) vs (12.3 ± 1.4) ng/ml) while the level of ICAM-1 in P2 group was significantly lower than that in P1 group at 72 h post-injury ((8.8 ± 1.4) vs (10.7 ± 1.3) ng/ml) (both P < 0.05). The level of LFA-1 in CC group ((7.3 ± 1.3), (8.4 ± 1.3), (9.6 ± 1.7) ng/ml) was significantly higher than that in NC group ((5.1 ± 1.2), (5.4 ± 1.3), (5.8 ± 1.2) ng/ml) at 24, 48, 72 h post-injury (all P < 0.05). MHC antigen expression on the CD14(+) monocytes of P2 group was obviously higher than that of CC and P1 groups ((46 ± 13)% vs (26 ± 15)% and (32 ± 12)%, both P < 0.05). The CD4/CD8 rate in P1 and P2 was significantly higher than that in CC group (1.71 ± 0.26, 1.82 ± 0.31 and 1.81 ± 0.24, 1.96 ± 0.24 vs 1.41 ± 0.34, 1.34 ± 0.26) at 48, 72 h post-injury (all P < 0.05). At 72 h post injury, the PBMC apoptosis rate in CC and P1 group was obviously higher than that of the NC group ((2.57 ± 0.21)% and (1.64 ± 0.10)% vs (0.81 ± 0.11)%) (both P < 0.01); the apoptosis rate in P2 group was significantly lower than that in P1 group ((1.09 ± 0.15)% vs (1.64 ± 0.10)%) (P < 0.01). CONCLUSION: Propofol may improve the immune function after combined burn-blast injuries through suppressing an excessive release of ICAM-1 and PBMC apoptosis in a concentration-dependent manner.

Fabrication of cysteine-modified antibodies with Fc-specific conjugation for covalent and oriented immobilization of native antibodies.

Covalent and oriented immobilization of antibodies (Abs) can substantially improve the sensitivity and stability of solid-phase immunoassays. By modifying the natural Abs with functional groups that provide unique handles for further conjugation, Abs could be immobilized onto the solid matrices with uniform orientation. Herein, an effective approach for Fc-specific modification of Abs was developed for the oriented and covalent immobilization of Abs. Twelve photoreactive Z-domain variants, incorporated with a photoactivable probe (p-benzoyl-L-phenylalanine, Bpa) at different positions and carrying a C-terminal Cys-tag (i.e. ZBpa-Cys variants), were individually constructed and produced in Escherichia coli and tested for photo-cross-linking to various IgGs. The different ZBpa-Cys variants demonstrated large differences in photo-conjugation efficiency for the tested IgGs. The conjugation efficiencies of 17thZBpa-Cys ranged from 90 % to nearly 100 % for rabbit IgG and mouse IgG2a, IgG2b and IgG3. Other variants, including 5thZBpa-Cys, 18thZBpa-Cys, 32thZBpa-Cys, and 35thZBpa-Cys, also displayed conjugation efficiencies of 61 %-83 % for mouse IgG1, IgG2a and IgG3. Subsequently, the photo-modified Abs, namely IgG-Cys conjugates, were covalently immobilized onto a maleimide group-functionalized solid-phase carrier on the basis of the reaction of sulfhydryl and maleimide. Thus, a generic platform for the controlled and oriented immobilization of Abs was developed, and the efficacy and potential of the proposed approach for sensitive immunoassays was demonstrated by detecting human α-fetoprotein.

Adenosine diphosphate ribosylation factor 6 inhibition protects burn sepsis induced lung injury through preserving vascular integrity and suppressing ASC inflammasome transmission.

BACKGROUND: Severe burns are devastating injuries with significant immune dysfunction and result in substantial mortality and morbidity due to sepsis induced organ failure. Acute lung injury is the most common type of organ injury in sepsis, however, the mechanisms of which are poorly understood and effective therapeutic measures are limited. This study is aimed to investigate the effect of a small Guanosine triphosphatase (GTPase), Adenosine diphosphate ribosylation factor 6 (ARF6), on burn sepsis induced lung injury, and discuss the possible mechanisms. METHODS: Burn sepsis was established in male C57BL/6 mice. Mice were anesthetised by intramuscular injection of ketamine and xylazine hydrochloride, then 30% TBSA full thickness burn followed by sub-eschar injection of lipopolysaccharide. Animals were treated with intraperitoneal injection of a small molecule inhibitor of ARF6: NAV-2729, or vehicle, right after the burn and sepsis stimuli were inflicted. Lung tissues were harvested for histopathological observation and the acute lung injury scores were calculated. Organ permeability, Vascular Endothelial Cadherin (VE-cadherin) expression, inflammatory cytokine levels and myeloperoxidase activity in lung tissues were detected. Rat pulmonary microvascular endothelial cells (PMVECs) were stimulated by burn sepsis serum with or without 10 µM NAV-2729. The ARF6 activation, VE-cadherin expression, inflammasome activity, adapter protein apoptosis speck-like protein containing a caspase recruiting domain (ASC) specks and cytokines secretion were determined. Student's t test was used for comparison between two groups. Multiple comparisons among groups were performed by using analysis of variance, with Tukey's test for the post hoc test. RESULTS: NAV-2729 treatment attenuated burn sepsis induced lung injury and promoted survival of burn septic mice by preserving VE-cadherin expression in endothelial cell adherent junction and limited vascular hyperpermeability in lung tissues. Moreover, inflammatory cytokine expression and inflammatory injury in lung tissues were alleviated. Mechanistically, NAV-2729 enhanced vascular integrity by inhibiting ARF6 activation and restoring VE-cadherin expression in PMVECs. In addition, NAV-2729 inhibited ARF6-dependent phagocytosis of ASC specks, thus preventing inflammation propagation mediated by cell-to-cell transmission of ASC specks. CONCLUSIONS: ARF6 inhibition preserved vascular integrity by restoring expression of VE-cadherin and suppressed the spread of inflammation by affecting phagocytosis of ASC specks, thus protected against sepsis induced lung injury and improve survival of burn septic animals. The findings of this study implied potential therapeutics by which ARF6 inhibition can protect lung function from septic induced lung injury and improve outcomes in burn sepsis.

Effect of lipopolysaccharide on the biological characteristics of human skin fibroblasts and hypertrophic scar tissue formation.

Burn injury-mediated destruction of the skin barrier normally induces microbial invasion, in turn leading to the development of systemic infection and occasional septic shock by the release of endotoxins. The objective of this work was to study the influence of lipopolysaccharide (LPS) on the biological characteristics of normal skin fibroblasts and to elucidate the influence of LPS in the initial stage of skin wound healing. Twenty patients with hypertrophic scar in proliferative stage were selected randomly and primary cultures were established from fibroblasts derived from their hypertrophic scar tissue and normal skin. Normal skin fibroblasts of passage 3 were stimulated with different concentrations of LPS. LPS stimulated the proliferation and collagen synthesis of fibroblasts within a certain extent of concentrations (0.005-0.5 µg/mL) (P < 0.05), whereas at a concentration of 1 µg/mL inhibited the proliferation and collagen synthesis of fibroblasts (P < 0.05). Collagen synthesis by normal skin fibroblasts after LPS stimulation mimicked those derived from hypertrophic scar tissue. LPS of 0.1 µg/mL had significant effect on normal skin fibroblasts-continuous passage of these fibroblasts resulted in ultrastructural pattern similar to fibroblasts derived from hypertrophic scar tissue, and the findings was substantiated by hematoxylin and eosin staining and immunohistochemistry detection of proliferation cell nuclear antigen, type I procollagen and α-smooth muscle actin. Our results suggest that LPS might convert normal skin fibroblasts to hypertrophic scar tissue fibroblasts and participate in the formation of hypertrophic scar; hence, appropriate concentration of LPS may have no effect or be beneficial to skin wound healing, whereas excessive concentration of LPS may delay the time of wound healing.

Immobilization of unraveled immunoglobulin G using well-oriented ZZ-His protein on functionalized microtiter plate for sensitive immunoassay.

Highly efficient protein immobilization is extremely crucial for solid-phase immunoassays. We present a strategy for oriented immobilization of functionally intact immunoglobulin G (IgG) on a polystyrene microtiter plate via iminodiacetic acid (IDA)-Ni(2+) and ZZ-His protein interaction. We immobilized a ZZ-EAP (Escherichia coli alkaline phosphatase)-His fusion protein, which exhibits Fc binding, His tag, and intrinsic AP activities, and analyzed it against the interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP to investigate the specificity and efficacy of this method. We compared the IDA-Ni(2+)-(ZZ-His) method with ZZ-EAP random immobilization using sandwich enzyme-linked immunosorbent assay, and the results showed that the former method had an enhanced signal, 10-fold higher sensitivity, and a wider linear range. Thus, the proposed method allows a broad range of oriented immobilized functionally intact IgG antibodies on polystyrene plates using only one type of IDA-Ni(2+) chelate surface because the ZZ protein can bind to the Fc region of various IgGs.

Comparative characterization of recombinant ZZ protein-alkaline phosphatase and its application in enzyme immunoassays.

A functional fusion protein, which consists of an antibody and an enzyme that can be used in enzyme immunoassays, has been constructed. However, a quantitative comparison of the characteristics of fusion proteins and chemical conjugates of the parents, which are functionally produced in a uniform microbial system, has not been adequately achieved. In this study, a fusion protein between the ZZ protein and Escherichia coli alkaline phosphatase (AP) and the parental ZZ protein and AP for chemical conjugate was functionally produced in the same bacterial system. A detailed examination of the ZZ-AP fusion protein and the effect of the ZZ-AP chemical conjugate on IgG affinity and enzymatic activity were performed. Compared with the parents, the equilibrium dissociation constant of ZZ-AP conjugate decreased by 32 % and catalytic activity decreased by 24 %, whereas the ZZ-AP fusion retained full parental activities and exhibited an approximately tenfold higher sensitivity than that of ZZ-AP conjugate in enzyme-linked immunosorbent assay. Thus, ZZ-AP fusion is a promising immunoreagent for IgG detection and a potential biolinker between antibodies and reporter enzymes (i.e., IgG-ZZ-AP fusion complex) in immunoassays.

Hydroxyapatite/Poly (Butylene Succinate)/Metoprolol Tartrate Composites with Controllable Drug Release and a Porous Structure for Bone Scaffold Application.

Nowadays, it is a challenge for a bone scaffold to achieve controllable drug release and a porous structure at the same time. Herein, we fabricated hydroxyapatite/poly (butylene succinate)/metoprolol tartrate (HA/PBS/MPT) composites via melt blending, aiming to provide the option of an in situ pore-forming strategy. The introduction of HA not only significantly improved the hydrophilicity of the PBS matrix by reducing the hydrophilic contact angle by approximately 36% at a 10% content, but also damaged the integrity of the PBS crystal. Both were beneficial for the penetration of phosphate-buffered saline solution into matrix and the acceleration of MPT release. Accompanied with MPT release, porous structures were formed in situ, and the HA inside the matrix was exposed. With the increase in HA content, the MPT release rate accelerated and the pore size became larger. The in vitro cytocompatibility evaluation indicated that HA/PBS/MPT composites were conductive to the adhesion, growth, and proliferation of MC3T3-E1 cells due to the HA being exposed around the pores. Thus, the MPT release rate, pore size, and cell induction ability of the HA/PBS/MPT composites were flexibly and effectively adjusted by the composition at the same time. By introducing HA, we innovatively achieved the construction of porous structures during the drug release process, without the addition of pore-forming agents. This approach allows the drug delivery system to combine controllable drug release and biocompatibility effectively, offering a novel method for bone repair material preparation. This work might provide a convenient and robust strategy for the fabrication of bone scaffolds with controllable drug release and porous structures.

Preparation of a bio-immunoreagent between ZZ affibody and enhanced green fluorescent protein for immunofluorescence applications.

In the present study, we constructed plasmid pUC-ZZ-EGFP to express Pro-ZZ-EGFP using ZZ peptide (a synthetic artificial IgG-Fc-fragment-binding protein derived from the B domain of staphylococcal protein A) and enhanced green fluorescent protein (EGFP). Without induction with isopropyl-ß-D: -thiogalactopyranoside, the chimeric protein was effectively expressed in Escherichia coli HB101. Its affinity constant binding IgG was 2.6 × 10(8) M(-1) obtained by competitive enzyme-linked immunosorbent assay, indicating that the ZZ peptide retains the native structure in Pro-ZZ-EGFP. The application of immunofluorescence assay for detecting the Mycoplasma pneumoniae IgG antibody, Pro-ZZ-EGFP, exhibited a good signal comparable in brightness and fluorescence pattern with the signal generated using the fluorescein isothiocyanate-labeled anti-human IgG. The result indicates that Pro-ZZ-EGFP possesses great potential for clinical immunofluorescence IgG test as an alternative versatile fluorescent antibody.

Convolutional Prototype Network for Open Set Recognition.

Despite the success of convolutional neural network (CNN) in conventional closed-set recognition (CSR), it still lacks robustness for dealing with unknowns (those out of known classes) in open environment. To improve the robustness of CNN in open-set recognition (OSR) and meanwhile maintain its high accuracy in CSR, we propose an alternative deep framework called convolutional prototype network (CPN), which keeps CNN for representation learning but replaces the closed-world assumed softmax with an open-world oriented and human-like prototype model. To equip CPN with discriminative ability for classifying known samples, we design several discriminative losses for training. Moreover, to increase the robustness of CPN for unknowns, we interpret CPN from the perspective of generative model and further propose a generative loss, which is essentially maximizing the log-likelihood of known samples and serves as a latent regularization for discriminative learning. The combination of discriminative and generative losses makes CPN a hybrid model with advantages for both CSR and OSR. Under the designed losses, the CPN is trained end-to-end for learning the convolutional network and prototypes jointly. For application of CPN in OSR, we propose two rejection rules for detecting different types of unknowns. Experiments on several datasets demonstrate the efficiency and effectiveness of CPN for both CSR and OSR tasks.

Directional immobilization of antibody onto magnetic nanoparticles by Fc-binding protein-assisted photo-conjugation for high sensitivity detection of antigen.

Immobilized antibodies with site-specific, oriented, and covalent pattern are of great significance to improve the sensitivity of solid-phase immunoassay. Here, we developed a novel antibody conjugation strategy that can immobilize antibodies in a directional and covalent manner. In this study, an IgG-Fc binding protein (Z domain) carrying a site-specific photo-crosslinker, p-benzoyl-L-phenylalanine, and a single C-terminal cysteine (Cys) handle was genetically engineered. Upon UV irradiation, the chimeric protein enables the Cys handle to couple with the native antibody in Fc-specific and covalent conjugation pattern, resulting in a novel thiolated antibody. Thus, an approach for the covalent, directional immobilization of antibodies to maleimide-modified magnetic nanoparticles (MNPs) was developed on the basis of the crosslinking between sulfhydryl and maleimide groups. The antibody-conjugated MNPs were applied in MNP-based enzyme-linked immunosorbent assay (ELISA) for the detection of carcinoembryonic antigen. The MNP-based ELISA presented a quantification linear range of 0.1-100 ng mL-1 and detection limit of 0.02 ng mL-1, which was approximately 100 times more sensitive than the traditional microplate ELISA (2.0 ng mL-1). Thus, the proposed antibody immobilization approach can be used in surface functionalization for the sensitive detection of various biomarkers.

Development of site-specific antibody-conjugated immunoliposomes for sensitive detection of disease biomarkers.

Liposome-based immunoassay (LIA) is an attractive protocol for amplifying the detection signals because of the excellent ability of liposomes to encapsulate signal marker compounds. The antigen-binding activity of the conjugated antibodies on the liposomal surface is crucial for the specificity and sensitivity of LIA. We present here a general platform to ensure that antibodies can conjugate onto the surface of liposomes in a site-specific and oriented manner. A His-handle-modified antibody with Fc region-specific and covalent conjugation was first fabricated using a photoactivatable ZBpa-His tag that was engineered using the aminoacyl-tRNA synthetase/suppressor tRNA technique. Based on the high affinity between the His tag and divalent metal ions, the novel His-modified antibody was oriented onto the surface of nickel ion-modified liposomes encapsulating horseradish peroxidase. With the prostate-specific antigen as a model, the detection efficiency of the new immunoliposomes was evaluated by chemiluminescence immunoassay. The immunoliposomes exhibited a limit of detection of 0.2 pg mL-1, which was a six time improvement compared with that of the chemical-coupled antibody-liposome conjugates. Thus, the proposed immunoliposomes are expected to hold potential applications for the sensitive detection of various biomarkers in complicated serum samples.