Y.P. Hou

Rational design of Polymeric Hybrid Micelles to Overcome Lymphatic and Intracellular Delivery Barriers in Cancer Immunotherapy

Poor distribution of antigen/adjuvant to target sites and inadequate induction of T cell responses remain major challenges in cancer immunotherapy because of the lack of appropriate delivery systems. Nanocarrier-based antigen delivery systems have emerged as an innovative strategy to improve vaccine efficacy. Here we present polymeric hybrid micelles (PHMs) as a simple and potent antigen/adjuvant co-delivery system with highly tunable properties. PHMs consist of two amphiphilic diblock copolymers, polycaprolactone-polyethylenimine (PCL-PEI) and polycaprolactone-polyethyleneglycol (PCL-PEG). PHMs with different proportions of cationic PCL-PEI were prepared and loaded with tyrosinase-related protein 2 (Trp2) peptide and adjuvant CpG oligodeoxynucleotide to generate the Trp2/PHM/CpG co-delivery system. Lymphatic and intracellular antigen delivery as a function of PCL-PEI proportion was evaluated in vitro and in vivo. PHMs containing 10% (w/w) PCL-PEI (Trp2/PHM10/CpG) showed the optimal balance of good distribution to lymph nodes, strong immunization effect after subcutaneous administration, and low toxicity to dendritic cells. In a mouse model of B16F10 melanoma, Trp2/PHM10/CpG showed significantly higher antigen-specific cytotoxic T lymphocyte activity and greater anticancer efficacy than Trp2/PHM0/CpG without PCL-PEI or a mixture of free Trp2 and CpG. These results provide new insights into how cationic segments affect the efficiency of antigen delivery by cationic nanocarriers. They also suggest that PHMs can serve as a structurally simple and highly tunable platform for co-delivery of antigen and adjuvant in cancer immunotherapy.

Typing of pentanucleotide STR polymorphisms

Pentanucleotide tandem repeat markers are of interest for forensic science because they may present less stutter in electrophoretic patterns. We focused on the analysis of the DNA sequence for each allele at two pentanucleotide STR loci, D6S957 and D10S2325, in order to understand their structures in the human genome and to construct allelic ladders, necessary for forensic DNA typing. In order to evaluate the forensic applicability of both pentanucleotide tandem repeat loci and to construct a preliminary database, the genotype distributions and allele frequencies in different ethnic groups were investigated. The population samples included Caucasians (Germans) and Asians (Chinese). The Amp-FLP technique was employed for DNA typing. An example of each allele and new alleles were sequenced. Allele determination for each pentanucleotide STR locus was carried out by comparison with a sequenced allelic ladder made in-house. Both pentanucleotide STR markers provided easily interpretable results. No evidence of deviation from Hardy–Weinberg equilibrium was observed. In 64 confirmed father/mother/child trios, no mutation event was observed. Using a maximum likelihood method, the mutation rate at both STR loci was indirectly estimated as 2.510 5 , suggesting both pentanucleotide STR markers would be useful for

Development a hyaluronic acid ion-pairing liposomal nanoparticle for enhancing anti-glioma efficacy by modulating glioma microenvironment

Abstract Glioma, one of the most common brain tumors, remains a challenge worldwide. Due to the specific biological barriers such as blood–brain barrier (BBB), cancer stem cells (CSCs), tumor associated macrophages (TAMs), and vasculogenic mimicry channels (VMs), a novel versatile targeting delivery for anti-glioma is in urgent need. Here, we designed a hyaluronic acid (HA) ion-pairing nanoparticle. Then, these nanoparticles were encapsulated in liposomes, termed as DOX-HA-LPs, which showed near-spherical morphology with an average size of 155.8 nm and uniform distribution (PDI = 0.155). HA was proven to specifically bind to CD44 receptor, which is over-expressed on the surface of tumor cells, other associated cells (such as CSCs and TAMs) and VMs. We systematically investigated anti-glioma efficacy and mechanisms in vivo and in vitro. The strong anti-glioma efficacy could attribute to the accumulation in glioma site and the regulation of tumor microenvironment with depletion of TAMs, inhibition of VMs, and elimination of CSCs.

Polymorphisms of six STR loci on chromosome 22 in a Chinese population

Abstract To develop more STR markers for forensic DNA typing in Chinese populations, six STR loci on chromosome 22 were investigated. An Amp-FLP technique was employed for genotyping individuals from a Han population sample from North and South China. Hardy–Weinberg equilibrium was checked using the chi-square test and no significant deviation was found at any of the six loci. The results showed that the differences of genotype distributions at two loci were significant between two populations, but the distributions of genotypes at the other four loci were similar in both populations. Five of the six loci showed good levels of polymorphism in both populations, with heterozygosities of more than 0.59. The discrimination power and the exclusion probability were more than 0.82 and 0.40, respectively, making them suitable candidate markers for forensic applications. At another locus, only two alleles were found. The heterozygosity of this locus was less than 0.36 and the discrimination power and exclusion probability were less than 0.62 and 0.12, respectively. A two-step mutation was observed at one of six loci.

Single nucleotide polymorphisms detected by temperature-modulated high-performance liquid chromatography

Abstract Single nucleotide polymorphisms (SNPs) are new generation markers. An open question in research on SNPs is how to find novel SNPs in a nature population. To this end, we developed an approach of high-throughput genotyping by applying the temperature-modulated heteroduplex analysis (TMHA) based upon ion-pair reversed-phase high-performance liquid chromatography. This approach allowed a very efficient resolution of identically sized PCR products with a single nucleotide change. This approach was also known as denaturing high-performance liquid chromatography (DHPLC). However, the terms of both TMHA and DHPLC should be considered carefully. The term of DHPLC may lead to confusion in annealing and denaturing. Since TMHA can be carried out using gel electrophoresis and HPLC, there is a need to distinguish HPLC from gel electrophoresis. Therefore, we suggest a new term for this technology, which is temperature-modulated high-performance liquid chromatography (TmHPLC). This term not only provides a clear definition for this new method, but also includes both TMHA and HPLC. More importantly, TmHPLC implies that the temperature in HPLC is modulated according to Tm of the DNA fragments. Our results show that TmHPLC is a sensitive, accurate and cost effective approach to screening sequence variation in human genome.

A new useful STR locus for forensic analysis

Abstract In order to get more available STR loci for forensic DNA analysis in Chinese populations, we investigated 10 tetranucleotide STR loci on chromosome 9 selected from GDB (D9S917, D9S254, D9S1121, D9S1118, D9S301, D9S1122, D9S922, D9S252, D9S2026, D9S934). Some of these were then chosen for further evaluation. 1039 EDTA-blood samples of unrelated individuals and some biological materials obtained from crime scene were used in this study. Amp-FLP techniques were employed for DNA typing. All alleles were cloned in plasmids and sequenced with an ABI 310. For each locus, the cloned alleles were amplified and the PCR products were mixed to make an allelic ladder for DNA typing. The software POPGENE was used to analyze the allele frequencies and heterozygosities of 10 STR loci in Chinese Han population. The results showed that compared with the other nine loci, allele frequencies of D9S1118 had a preferable distribution in Chinese Han population. The exclusion probability was 0.6405, while the discrimination power was 0.9424. Analyzing biological materials from crime scenes showed that typing D9S1118 had a higher success rate. All of these results suggest that D9S1118 as a marker could be used for forensic DNA analysis in Chinese populations.

Polymorphism of two new Y-STR loci in a Chinese population

Abstract Human chromosome Y-specific short tandem repeat (Y-specific STR) markers have useful properties for forensic applications. However, there is a need to develop more Y-specific STR markers because the discriminating power of each STR locus is limited. In the present study, we describe our results on two new Y-specific STR markers, which were initially reported by White et al. [Genomics 57 (1999) 433] as Y-GATA-C4 and Y-GATA-A10. The distributions of alleles and haplotypes for both Y-specific STR loci were studied in a Chinese Han population sample. Allele determination was carried out by comparison with sequenced allelic ladders which were made in-house. Following the recommendations of the International Society of Forensic Genetics, the allele classification for both Y-specific STR loci was based on the number of repeat motifs. The results show that the haplotype diversity, the power of discrimination and the exclusion probability in the Chinese population for both Y-specific STR loci were 0.9090. The implication from this study is that Y-GATA-C4 and Y-GATA-A10 are useful Y-specific STR markers for forensic DNA typing in Chinese populations.