Frequent occurrence of CYP2D6*10 duplication allele in a Japanese population

Allele-specific long-polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (RFLP) and haplotype analysis using XbaI and EcoRI were used to determine whether gene duplication of CYP2D6*10 exists in a Japanese population of 162 healthy subjects. Based on the results of PCR and haplotype analysis, the frequencies of CYP2D6*1X2, CYP2D6*2X2 and CYP2D6*10X2 in the Japanese population were estimated to be 0.3, 0.3 and 0.6%, respectively. The results suggest that duplicated alleles of CYP2D6*10 exist in the Japanese population and that it may be one of the factors affecting the capacity of Japanese to metabolize various CYP2D6 substrate drugs.     

Near-infrared theranostic photoimmunotherapy (PIT): repeated exposure of light enhances the effect of immunoconjugate

Armed antibody-based targeted molecular therapies offer the possibility of effective tumor control with a minimum of side effects. Photoimmunotherapy (PIT) employs a monoclonal antibody-phototoxic phthalocyanine dye, IR700 conjugate, that is activated by focal near-infrared (NIR) light irradiation after antibody binding to the targeted tumor cell surface leading to rapid necrotic cell death. Therapy by single NIR light irradiation was effective without significant side effects; however, recurrences were seen in most treated mice probably because of inhomogeneous distribution of panitumumab-IR700 immunoconjugate in the tumor, leading to ineffective PIT. We describe here an optimized regimen of effective PIT method for the same HER1-overexpressing tumor model (A431) with fractionated administration of panitumumab-IR700 conjugate followed by systematic repeated NIR light irradiation to the tumor based on timing of antibody redistribution into the remnant tumor under the guidance of IR700 fluorescence signal. Eighty percent of the A431 tumors were eradicated with repeated PIT without apparent side effects and survived tumor-free for more than 120 days even after stopping therapy at day 30. Therapeutic effects were monitored using IR700 fluorescent signal. PIT is a promising highly selective and clinically feasible theranostic method for treatment of mAb-binding tumors with minimal off-target effects.     

Time allocation of Orius sauteri in attacking Thrips palmi on an eggplant leaf

Orius sauteri (Poppius) (Heteroptera: Anthocoridae) is a polyphagous predator used as a biological control agent of palm thrips, Thrips palmi (Karny) (Thysanoptera: Thripidae). We studied O. sauteri's searching efficiency, time allocation on a leaf, leaving tendency, and attacking of prey. Approximately 78% of the encountered prey was eaten. Searching for prey was concentrated for 86% of the time on the lower leaf side, where palm thrips are usually found. Patch residence times on empty leaves were different from those on leaves with T. palmi larvae. Walking activity was not affected by the thrips density, and walking took place during 64% of the total search time. The leaving tendency of O. sauteri was affected by the time from patch entry and the presence or absence of palm thrips, but not by the thrips density. If prey were present, the leaving tendency decreased (multiplication factor 0.327), resulting in longer giving‐up times than when no prey was present. The fact that the leaving tendency increases when patch exploitation lasts longer suggests that not much time is wasted on patches where encounters with prey are scarce.     

A Bead-based Normalization for Uniform Sequencing depth (BeNUS) protocol for multi-samples sequencing exemplified by HLA-B

Background

Human leukocyte antigen (HLA) is a group of genes that are extremely polymorphic among individuals and populations and have been associated with more than 100 different diseases and adverse drug effects. HLA typing is accordingly an important tool in clinical application, medical research, and population genetics. We have previously developed a phase-defined HLA gene sequencing method using MiSeq sequencing.

Results

Here we report a simple, high-throughput, and cost-effective sequencing method that includes normalized library preparation and adjustment of DNA molar concentration. We applied long-range PCR to amplify HLA-B for 96 samples followed by transposase-based library construction and multiplex sequencing with the MiSeq sequencer. After sequencing, we observed low variation in read percentages (0.2% to 1.55%) among the 96 demultiplexed samples. On this basis, all the samples were amenable to haplotype phasing using our phase-defined sequencing method. In our study, a sequencing depth of 800x was necessary and sufficient to achieve full phasing of HLA-B alleles with reliable assignment of the allelic sequence to the 8 digit level.

Conclusions

Our HLA sequencing method optimized for 96 multiplexing samples is highly time effective and cost effective and is especially suitable for automated multi-sample library preparation and sequencing.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-645) contains supplementary material, which is available to authorized users.     

Possible Association between Dysfunction of Vitamin D Binding Protein (GC Globulin) and Migraine Attacks

To identify the genetic causality of migraine and acute, severe melalgia, we performed a linkage analysis and exome sequencing in a family with four affected individuals. We identified a variant (R21L) in exon 2 of the GC globulin gene, which is involved in the transportation of vitamin D metabolites and acts as a chemotaxic factor; this variant was co-segregated within the family. To investigate the relationship between GC globulin and melalgia, we investigated the cytokine levels in serum samples from the patients and control subjects using a cytokine antibody array. GC globulin can bind to both MCP-1 and RANTES in human serum but has a higher affinity to MCP-1. In cell culture systems, MCP-1 was able to bind to overexpressed wild-type GC globulin but not to the GC globulin variant, and the GC globulin binding affinity to MCP-1 was significantly lower in sera from the patients than in sera from control subjects. A higher concentration of MCP-1 was also observed in sera from the patients. Thus, the dysfunctional GC globulin affected cytokine release, especially the release of MCP-1, and MCP-1 might play important roles in melalgia and migraine.     

Development and parasitization of an aphid’s secondary parasitoid,Dendrocerus laticeps (Hymenoptera: Megaspilidae), on Aphidius colemani (Hymenoptera: Braconidae)

Banker-plant systems using Aphidius species have been employed to control pest aphids in greenhouses growing eggplant and sweet pepper in Japan. However, a strong negative correlation between the occurrence of secondary parasitoids and aphid-control success by this system was reported by Nagasaka et al. (2010). To control secondary parasitoid populations, detailed knowledge of their ecological characteristics is needed. In this paper, the development and parasitization ability of Dendrocerus laticeps (Hedicke) (Hymenoptera: Megaspilidae), a major secondary ectoparasitoid of Aphidius species in Japan, were examined under 25 °C laboratory conditions. The maximum female longevity was about 11 days when honeydew was available. Parasitization occurred only on mummified aphids, and the age of mummified aphids did not influence the ecological value of offspring. The maximum parasitization ability was 18.9 hosts/day estimated by Holling’s disc equation. Possible lifetime fecundity was 100.35. The intrinsic rate of natural increase estimated by the bootstrap method ranged from 0.211 to 0.321, depending on the assumptions made about larval mortality. The countermeasures for avoiding the negative impacts of D. laticeps in a banker-plant system include the development of a refuge for mummified aphids and increasing the temperature around the banker plants.     

Improved loop-mediated isothermal amplification for HLA-DRB1 genotyping using RecA and a restriction enzyme for enhanced amplification specificity

Our aim was to test and develop the use of loop-mediated isothermal amplification (LAMP) for HLA-DRB1 genotyping. Initially, we found that the conventional LAMP protocols produced non-specific and variable amplification results depending on the sample DNA conditions. Experiments with different concentrations of DNase in the reaction mixture with and without T4 DNA ligase-treated samples suggested that the strand displacement activity of DNA polymerase in LAMP, at least in part, started from randomly existing nicks because T4 DNA ligase treatment of sample DNA resulted in no amplification. Such non-specific amplification due to the randomly existing nicks was improved specifically by the addition of RecA of Escherichia coli and a restriction enzyme, for example, PvuII, to the reaction mixture. We applied the modified LAMP (mLAMP) (1) to detect specific HLA-DRB1 alleles by using only specific primers for amplification or (2) for genotyping in multiple samples with a multi-probe typing system. In the latter case, HLA-DRB1 genotyping was developed by combining the mLAMP with amplicon capture using polymorphic region-specific probes fixed onto the bottom of the wells of a 96-well plate and the captured amplicons visualized as a black spot at the bottom of the well. The multi-probe human leukocyte antigen (HLA) typing method and the specific HLA allele detection method could be applied for point-of-care testing due to no requirement for specific and expensive instruments.     

Change in the activity of Cl-,HCO3(-)-ATPase in microsome fraction during early development of the sea urchin, Hemicentrotus pulcherrimus

In sea urchin embryos, primary mesenchyme cells, descendants from micromeres produced at the 16-cell stage, form spicules or CaCO3 deposits in their skeletal vacuoles, at the post-gastrula stage. Micromeres isolated at the 16-cell stage also differentiate into spicule-forming cells during their culture at the same time schedule as in the embryos. The present study was planned to observe change in the activity of Cl-,HCO3(-)-ATPase, which was expected to contribute to the carbonate supply for CaCO3 deposition, during development. ATP-hydrolysis in the microsome fraction, obtained from embryos of the sea urchin, Hemicentrotus pulcherrimus, and from micromere-derived cells in culture was stimulated by Cl- and HCO3- in the presence of ouabain and EGTA. The ATP-hydrolysis was inhibited by ethacrynic acid, an inhibitor of Cl-,HCO3(-)-ATPase. The activity of Cl-,HCO3(-)-ATPase in embryos and in micromere-derived cells increased during development, keeping pace with the rate of calcium deposition in spicules. Formation of calcified spicules in the cultured micromere-derived cells was inhibited by ethacrynic acid. These results indicate that Cl-,HCO3(-)-ATPase plays an important role in the mechanism of CaCO3 deposition in the primary mesenchyme cells.     

Female mating strategy in an enclosed group of Japanese macaques

Female Japanese macaques (Macaca fuscata) are noted for mating with multiple males and for their ability to exert mate choice. In a captive group of Japanese macaques housed at the Primate Research Institute of Kyoto University, Japan, behavioral and endocrine data were combined to examine female mating strategies. During one breeding season, daily behavioral observations were conducted on females who exhibited copulatory behavior. Blood was collected from females twice weekly and their ovulatory periods estimated by analyzing hormone profiles. Females began mating shortly before ovulation, peaked at ovulation, and continued receiving ejaculations for up to ten weeks after conception. Females were more responsible than males for inbreeding avoidance with matrilineal kin. Males sometimes approached females from their own matriline, but females avoided such males and expressed mate choice behavior preferentially toward non-matrilineal males. Over the entire mating season, females did not choose non-matrilineal males on the basis of displays, dominance rank, age, weight, or weight change during the mating season. When females were likely to conceive, however, they expressed mate choice behavior toward males who displayed most frequently. Female mating strategy may include both mate choice at ovulation and other, non-procreative functions.