Quantum-Dot-Decorated Robust Transductable Bioluminescent Nanocapsules

Bioluminescence, due to its high sensitivity, has been exploited in various analytical and imaging applications. In this work, we report a highly stable, cell-transductable, and wavelength-tunable bioluminescence system achieved with an elegant and simple design. Using aqueous in situ polymerization on a bioluminescent enzyme anchored with polymerizable vinyl groups, we obtained nanosized core−shell nanocapsules with the enzyme as the core and a cross-linked thin polymer net as the shell. These nanocapsules possess greatly enhanced stability, retained bioactivity, and a readily engineered surface. In particular, by incorporating polymerizable amines in the polymerization, we endowed the nanocapsules with efficient cell-transduction and sufficient conjugation sites for follow-up modification. Following in situ polymerization, decorating the polymer shell with fluorescent quantum dots allowed us to access a continuous tunable wavelength, which extends the application of such bioluminescent nanocapsules, especially in deep tissue. In addition, the unique core−shell structure and adequate conjugation sites on surface enabled us to maximize the BRET efficiency by adjusting the QD/enzyme conjugation ratio

Identification and Functional Characterization of Glycosylation of Recombinant Human Platelet-Derived Growth Factor-BB in <i>Pichia pastoris</i>

<div><p>Yeast <i>Pichia pastoris</i> is a widely used system for heterologous protein expression. However, post-translational modifications, especially glycosylation, usually impede pharmaceutical application of recombinant proteins because of unexpected alterations in protein structure and function. The aim of this study was to identify glycosylation sites on recombinant human platelet-derived growth factor-BB (rhPDGF-BB) secreted by <i>P</i>. <i>pastoris</i>, and investigate possible effects of O-linked glycans on PDGF-BB functional activity. PDGF-BB secreted by <i>P</i>. <i>pastoris</i> is very heterogeneous and contains multiple isoforms. We demonstrated that PDGF-BB was O-glycosylated during the secretion process and detected putative O-glycosylation sites using glycosylation staining and immunoblotting. By site-directed mutagenesis and high-resolution LC/MS analysis, we, for the first time, identified two threonine residues at the C-terminus as the major O-glycosylation sites on rhPDGF-BB produced in <i>P</i>. <i>pastoris</i>. Although O-glycosylation resulted in heterogeneous protein expression, the removal of glycosylation sites did not affect rhPDGF-BB mitogenic activity. In addition, the unglycosylated PDGF-BB^ΔGly mutant exhibited the immunogenicity comparable to that of the wild-type form. Furthermore, antiserum against PDGF-BB^ΔGly also recognized glycosylated PDGF-BB, indicating that protein immunogenicity was unaltered by glycosylation. These findings elucidate the effect of glycosylation on PDGF-BB structure and biological activity, and can potentially contribute to the design and production of homogeneously expressed unglycosylated or human-type glycosylated PDGF-BB in <i>P</i>. <i>pastoris</i> for pharmaceutical applications.</p></div

Heterogeneous expression of glycosylated recombinant human PDGF-BB in <i>Pichia pastoris</i>.

<p>(A) Purified rhPDGF-BB proteins from six independent expression and purification experiments were analyzed by SDS-PAGE. Heterogeneous rhPDGF-BB bands could be observed under reducing conditions. (B) Purified rhPDGF-BB and rhIFN-ω produced by <i>P</i>. <i>pastoris</i> were treated with PNGase F to hydrolyze N-glycan residues. Two gels loaded with the same amount of each protein (5 μg) were simultaneously subjected to SDS-PAGE, and analyzed by Coomassie Blue staining (left panel) and glycosylation staining (right panel), respectively. There were no differences between rhPDGF-BB samples treated or not with PNGase F.</p

Identification of rhPDGF-BB isoforms.

<p>(A) Five isoforms detected by SDS-PAGE were transferred to a PVDF membrane for protein N-terminal sequencing. (B) Schematic representation of N-terminal sequencing results. Bands I, II, and III represent intact PDGF-BB, while bands IV and V are truncated isoforms generated by the cleavage at the Arg 27-Thr 28 site. (C) rhPDGF-BB (5 μg) was subjected to SDS-PAGE under reducing conditions and analyzed by western blotting, Coomassie Blue staining, and glycosylation staining, respectively. Two bands corresponding to isoforms III and IV stained by Coomassie Blue could be detected by antibody staining (marked with red lines). Glycosylation staining revealed another two bands corresponding to isoforms I and II (marked with red lines). (D) SDS-PAGE and subsequent glycosylation staining of higher rhPDGF-BB load (10 μg and 20 μg) revealed three bands; the third band with the lowest molecular weight was assumed to correspond to isoform IV.</p

2H7 recognizes the conserved domain of SasA and binds to <i>S</i>. <i>aureus</i>.

<p>ELISA was used to determine the concentration-dependent binding of 2H7 and an isotype control mAb to SasA or <i>S</i>. <i>aureus</i> USA300. (A to D) ELISA plates were coated with purified antigens (SRR1, NRR, NRR1, NRR2 and SRR1-NRR1) and blocked with 5% nonfat milk. (E) ELISA plate was coated with formalin-fixed <i>S</i>. <i>aureus</i> USA300. Rabbit serum (1%) was added to block the surface expressed protein A of <i>S</i>. <i>aureus</i>. Each point represents the antibody concentration and its absorbance at 450 nm. The data represent the means ± SEM (n = 3).</p

DataSheet_1_Single-dose of a replication-competent adenovirus-vectored vaccine provides sterilizing protection against Rift Valley fever virus challenge.docx

Rift Valley fever virus (RVFV) is one of the most important virulent pathogens causing severe disease in animals and humans. However, there is currently no approved vaccine to prevent RVFV infection in humans. The use of human adenovirus serotype 4 (Ad4) as a vector for an RVFV vaccine has not been reported. Here, we report the generation of a replication-competent recombinant Ad4 vector expressing codon-optimized forms of the RVFV glycoproteins Gn and Gc (named Ad4-GnGc). Intramuscular immunization with Ad4-GnGc elicited robust neutralizing antibodies against RVFV and cellular immune responses in mice. A single low-dose vaccination with Ad4-GnGc completely protected interferon-α/β receptor-deficient A129 mice from lethal RVFV infection. More importantly, Ad4-GnGc efficacy was not affected by pre-existing immunity to adenovirus serotype 5, which currently exists widely in populations. These results suggest that Ad4-GnGc is a promising vaccine candidate against RVFV.</p

2H7 promotes opsonophagocytic killing by mouse whole blood and human neutrophils.

(A) PBS-washed USA300 (3×104 CFU) was incubated with 200 μl heparinized whole blood collected from BALB/c mice (female, 6-week-old) in the presence of 10 μg/ml 2H7 or isotype control antibody. In addition, blood cells were first lysed in the presence of 0.5% saponin and a control experiment was conducted to examine if 2H7 mediate bacterial aggregations and result in a drop in apparent CFUs. (B) HL60 cells were cultured and differentiated to granulocytes. PBS-washed USA300 (1×104 CFU) was incubated with viable differentiated-HL60 cells (1×105), 4% Guinea pig serum (the cross-reactive antibodies were removed) and 10 μg/ml 2H7 or isotype control antibody. The samples were incubated at 37°C with shaking for 60 minutes. The CFUs of S. aureus were determined by plating on TSA. The relative killing was calculated as the percent difference in CFU between samples at 0 min and 60 min. The data represent the means ± SEM (n = 3). The statistical significance was measured using a two-tailed unpaired t test (**: p<0.01).</p

Treatment with 2H7 reduces the staphylococcal burden in kidneys.

BALB/c mice (female, 6-week-old, n = 9) were injected intraperitoneally with a single dose (15 mg/kg) of 2H7 or isotype control mAb 24 h prior to challenge. On day 2 following intraperitoneal challenge and day 4 following intravenous challenge, the infected mice were euthanized by CO2 inhalation. The staphylococcal loads in the both kidneys were measured by plating the homogenized renal tissue on TSA plates. (A) Mice were challenged intravenously with 1×107 CFUs of USA300. (B) Mice were challenged intraperitoneally with 1×109 CFUs of USA300. The data represent the log10 (CFU/g) means ± SEM. The statistical significance was analyzed with a two-tailed unpaired t test (*: p<0.05, **: p<0.01).</p

Monoclonal Antibody Targeting <i>Staphylococcus aureus</i> Surface Protein A (SasA) Protect Against <i>Staphylococcus aureus</i> Sepsis and Peritonitis in Mice

<div><p>Epidemic methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) imposes an increasing impact on public health. Due to multi-antibiotics resistance in MRSA strains, there is an urgent need to develop novel therapeutics such as effective monoclonal antibodies (mAbs) against MRSA infections. <i>Staphylococcus aureus</i> surface protein A (SasA), a large surface-located protein (~240 kDa), is one of MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) and a potential target for immunotherapeutic approaches against <i>S</i>. <i>aureus</i> infections. In the present study, we analyzed the sequence of SasA with bioinformatics tools and generated a protective monoclonal antibody (2H7) targeting the conserved domain of SasA. 2H7 was shown to recognize wild-type <i>S</i>. <i>aureus</i> and promote opsonophagocytic killing of <i>S</i>. <i>aureus</i>. In both sepsis and peritoneal infection models, prophylactic administration of 2H7 improved the survival of BALB/c mice challenged by <i>S</i>. <i>aureus</i> strain USA300 and ST239 (prevalent MRSA clones in North America and Asian countries, respectively) and enhanced bacterial clearance in kidneys. Additionally, 2H7 prophylaxis prevented the formation of intraperitoneal abscess in a murine model of peritoneal infection and therapeutic administration of 2H7 showed protective efficacy in a murine sepsis model. Our results presented here provide supporting evidences that an anti-SasA mAb might be a potential component in an antibody-based immunotherapeutic treatment of MRSA infections.</p></div

Identification of O-glycosylation sites in rhPDGF-BB using LC/MS.

<p>(A) Schematic representation of PDGF-B mutants. Monoisotopic mass of every mutant is shown. (B) Deconvoluted mass spectra of the wild-type PDGF and its mutants are presented with monoisotopic peaks; glycosylated isoforms are annotated.</p