Prunus mume (mei), which was domesticated in China more than 3,000 years ago as ornamental plant and fruit, is one of the first genomes among Prunus subfamilies of Rosaceae been sequenced. Here, we assemble a 280M genome by combining 101-fold next-generation sequencing and optical mapping data. We further anchor 83.9% of scaffolds to eight chromosomes with genetic map constructed by restriction-site-associated DNA sequencing. Combining P. mume genome with available data, we succeed in reconstructing nine ancestral chromosomes of Rosaceae family, as well as depicting chromosome fusion, fission and duplication history in three major subfamilies. We sequence the transcriptome of various tissues and perform genome-wide analysis to reveal the characteristics of P. mume, including its regulation of early blooming in endodormancy, immune response against bacterial infection and biosynthesis of flower scent. The P. mume genome sequence adds to our understanding of Rosaceae evolution and provides important data for improvement of fruit trees.

Phase transition behavior of unimolecular dendritic three-layer nanostructures with dual thermoresponsive coronas is studied. Successive reversible addition-fragmentation transfer (RAFT) polymerizations of N-isopropylacrylamide (NIPAM) and 2-(dimethylamino)ethyl methacrylate (DMA) were conducted using fractionated fourth-generation hyperbranched polyester (Bolton H40) based macroRAFT agent. At lower temperatures (<20 degrees C), dendritic macromolecules H40-poly(N-isopropylacrylamide)-poly(2-(dimethylamino)ethyl methacrylate) (H40-PNIPAM-PDMA) exist as unimolcular core-shell-corona nanostructures with hydrophobic H40 as the core, swollen PNIPAM as the inner shell, and swollen PDMA as the corona. PNIPAM and PDMA homopolymers undergo phase transitions at their lower critical solution temperatures (LCST), which are found to be 32 degrees C for PNIPAM and 40-50 degrees C for PDMA, respectively. Upon continuously heating through the LCSTs of PNIPAM and PDMA, such dendritic unimolecular micelles exhibit two-stage thermally induced collapse. This process is reversible with a two-stage reswelling upon cooling. Laser light scattering, micro-differential scanning calorimetry, and excimer fluorescence measurements are used to investigate the double phase transitions.

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Recombinant adeno-associated virus (AAV) has become an attractive vector system for a number of gene therapy paradigms. However, the utility of AAV vectors is often limited by the absence of heparan sulfate proteoglycan (HSPG), the virus's primary attachment receptor, on the desired target cell population. In order to achieve HSPG-independent gene delivery, several groups have shown that the endogenous tropism of AAV can be expand by genetically altering the viral capsid. However, the parameters of this developing technology have yet to be defined and it has not yet been determined if these modified vectors actually infect cells via these engineered interactions. Previously we constructed a series of insertion mutants spanning the AAV capsid protein gene and identified specific sites that can tolerate the insertion of small exogenous peptides. Here we describe a number of sites within the AAV capsid gene that can be used for the insertion of integrin-targeting peptide epitopes. Incorporation of an Arg-Gly-Asp (RGD)-containing peptide at these sites enables AAV to infect integrin-expressing cells independent of HSPG. Mutant AAV vectors displaying these peptide ligands can be produced to wild-type titer and have been shown to specifically interact with the targeted integrin receptors and mediate infection via this interaction. We report significant increases in gene transfer to Raji, K562, and SKOV-3 cell lines that express integrin, but little HSPG, suggesting that rAAV vectors displaying RGD peptides may be of great utility for treatment of neoplasms characterized by the deficiency of HSPG expression. We have also demonstrated that due to their expanded tropism, these novel vectors are capable of efficient transduction of AAV2-resistant tumors in vivo suggesting that they may offer significant therapeutic advantages.

Recombinant adeno-associated virus (AAV) vectors are of interest in the context of gene therapy because of their ability to mediate efficient transfer and stable expression of therapeutic genes in a wide variety of tissues. However, AAV-mediated gene delivery to specific cell populations is often precluded by the widespread distribution of heparan sulfate proteoglycan (HSPG), the primary cellular receptor for the virus. Conversely, an increasing number of cell types are being identified that do not express HSPG and are therefore poor targets for AAV-mediated gene transfer. To address these issues, we have developed strategies to physically modify AAV vectors and allow efficient, HSPG-independent, receptor-targeted infection. We began by generating a series of 38 virus capsid mutants containing peptide insertions at 25 unique sites within the AAV capsid protein. The mutant viruses were characterized on the basis of their phenotypes and grouped into three classes: class I mutants (4 of 38) did not assemble particles; class II mutants (14 of 38) assembled noninfectious particles; and class III mutants (20 of 38) assembled fully infectious particles. We examined the HSPG-binding characteristics of the class II mutants and showed that a defect in receptor binding was a common reason for their lack of infectivity. The display of foreign peptide epitopes on the surface of the mutant AAV particles was found to be highly dependent on the inclusion of appropriate scaffolding sequences. Optimal scaffolding sequences and five preferred sites for the insertion of targeting peptide epitopes were identified. These sites are located within each of the three AAV capsid proteins, and thus display inserted epitopes 3, 6, or 60 times per vector particle. Modified AAV vectors displaying a 15-amino acid peptide, which binds to the human luteinizing hormone receptor (LH-R), were generated and assessed for their ability to target gene delivery to receptor-bearing cell lines. Titers of these mutant vectors were essentially the same as wild-type vector. The LH-R-targeted vector was able to transduce ovarian cancer cells (OVCAR-3) in an HSPG-independent manner. Furthermore, transduction was shown to proceed via the LH-R and therefore treatment of OVCAR-3 cells with progesterone, to increase LH-R expression, accordingly increased LH mutant-mediated gene transfer. This technology may have a significant impact on the use of AAV vectors for human gene therapy.

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A triblock copolymer, poly(ethylene glycol)-b-poly(glycerol monomethacrylate)-b-poly(2-(diethylamino)ethyl methacrylate) (PEG−PGMA−PDEA), was synthesized via atom transfer radical polymerization (ATRP) by successive polymerization of glycerol monomethacrylate (GMA) and 2-(diethylamino)ethyl methacrylate (DEA) using a PEG-based ATRP macroinitiator. Reacting the obtained triblock copolymer with varying amounts of cinnamoyl chloride in anhydrous pyridine yielded PEG−(PCGMA-co-PGMA)−PDEA triblock copolymer with photo-cross-linkable moieties, where PCGMA is poly(3-cinnamoyl glycerol monomethacrylate) and the mean degree of cinnamoylation ranges from 5 to 50 mol % relative to the PGMA block. All PEG−(PCGMA-co-PGMA)−PDEA triblock copolymers molecularly dissolve in aqueous media at acidic pH; upon addition of NaOH, micellization occurred above pH 7−8 to form three-layer “onionlike” micelles comprising PDEA cores, PCGMA-co-PGMA inner shells, and PEG outer coronas. The pH-induced micellization kinetics of PEG113−(CGMA0.5-co-GMA0.5)50−DEA65 triblock copolymers was investigated by stopped-flow light scattering upon a pH jump from 3 to 10, and compared to that of PEG113−PGMA50−PDEA65. Facile cross-linking of the PCGMA-co-PGMA inner shell was then conducted via UV irradiation. The PDEA cores of the resulting shell cross-linked (SCL) micelles exhibited reversible pH-responsive behavior. The extent of pH-induced swelling/shrinking and the colloidal stability of SCL micelles were mainly determined by the extent of cross-linking. The dissociation kinetics of the triblock copolymer micelles before and after shell cross-linking was also investigated employing the stopped-flow technique. It was found that SCL micelles prepared at higher degrees (>20 mol %) of cross-linking exhibited excellent colloidal stability to external pH changes.

An amphiphilic, hyperbranched polymer suitable for use in controlled drug delivery is reported. This polymer was obtained by modification of the hyperbranched aliphatic polyester Boltorn H20 (H20) with succinic anhydride and then glycidyl methacrylate, and formed nanoparticles in aqueous solution. The critical association concentration was 7.4 x 10(-3) g . L(-1), as determined by fluorescence spectroscopy using pyrene as a molecular probe. A static/dynamic laser light scattering (LLS) study revealed that the average particle size was 39.4 nm with a low particle size distribution (PDI=0.04), and that each particle was composed of about 350 amphiphilic molecules. Daidzein, a hydrophobic traditional Chinese medicine, was encapsulated during particle formation and the release properties were determined. The optimal feeding concentration of daidzein to hyperbranched polyester was 4.9 x 10(-5) g . mL(-1) to 5.0 x 10(-3) g . mL(-1) with a loading efficiency of 76.1%. In the presence of the enzyme Lipase PS, the drug loaded nanoparticles degraded in a random one-by-one manner and released the drug over a few days. This system is therefore a novel controlled drug release system based on nanoparticles formed of hyperbranched polyester. Encapsulation of daidzein by hyperbranched polyester particles.