Silencing of p53 and CDKN1A establishes sustainable immortalized megakaryocyte progenitor cells from human iPSCs.

Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.

Genetic susceptibility to postherniotomy pain. The influence of polymorphisms in the Mu opioid receptor, TNF-α, GRIK3, GCH1, BDNF and CACNA2D2 genes.

BACKGROUND AND AIMS: Despite improvements in surgical technique, 5%-8% of patients undergoing herniorrhaphy still suffer from clinically relevant persistent postherniotomy pain. This is a problem at both individual and society levels. The aim of this study was to determine whether or not a single nucleotide polymorphism in a specific gene contributes to the development of persistent pain after surgery. METHODS: One hundred individuals with persistent postherniotomy pain, along with 100 without pain matched for age, gender and type of surgery were identified in a previous cohort study on patients operated for groin hernia. All patients underwent a thorough sensory examination and blood samples were collected. DNA was extracted and analysed for single nucleotide polymorphism in the Mu opioid receptor, TNF-α, GRIK3, GCH1, BDNF and CACNA2D2 genes. RESULTS: Patients with neuropathic pain were found to have a homozygous single nucleotide polymorph in the TNF-α gene significantly more often than pain-free patients (P=0.036, one-tailed test). CONCLUSIONS: SNP in the TNF-α gene has a significant impact on the risk for developing PPSP. IMPLICATIONS: The result suggests the involvement of genetic variance in the development of pain and this requires further investigation.

Combined analysis of circulating ß-endorphin with gene polymorphisms in OPRM1, CACNAD2 and ABCB1 reveals correlation with pain, opioid sensitivity and opioid-related side effects.

BACKGROUND: Opioids are associated with wide inter-individual variability in the analgesic response and a narrow therapeutic index. This may be partly explained by the presence of single nucleotide polymorphisms (SNPs) in genes encoding molecular entities involved in opioid metabolism and receptor activation. This paper describes the investigation of SNPs in three genes that have a functional impact on the opioid response: OPRM1, which codes for the µ-opioid receptor; ABCB1 for the ATP-binding cassette B1 transporter enzyme; and the calcium channel complex subunit CACNA2D2. The genotyping was combined with an analysis of plasma levels of the opioid peptide ß-endorphin in 80 well-defined patients with chronic low back pain scheduled for spinal fusion surgery, and with differential sensitivity to the opioid analgesic remifentanil. This patient group was compared with 56 healthy controls. RESULTS: The plasma ß-endorphin levels were significantly higher in controls than in pain patients.A higher incidence of opioid-related side effects and sex differences was found in patients with the minor allele of the ABCB1 gene. Further, a correlation between increased opioid sensitivity and the major CACNA2D2 allele was confirmed. A tendency of a relationship between opioid sensitivity and the minor allele of OPRM1 was also found. CONCLUSIONS: Although the sample cohort in this study was limited to 80 patients it appears that it was possible to observe significant correlations between polymorphism in relevant genes and various items related to pain sensitivity and opioid response. Of particular interest is the new finding of a correlation between increased opioid sensitivity and the major CACNA2D2 allele. These observations may open for improved strategies in the clinical treatment of chronic pain with opioids.

Semi-quantitative discrimination of HBV mutants using allele-specific oligonucleotide hybridization with Handy Bio-Strand.

The analysis of hepatitis B virus (HBV) mutations is important for understanding HBV progression and for deciding on appropriate clinical treatments. However, it is difficult to determine the quantitative abundance of various mutants in heterogeneous mixtures by conventional methods such as direct sequencing or the TaqMan assay. In this study, we investigated the possibility of using both allele-specific oligonucleotide hybridization (ASOH) and allele-specific oligonucleotide competitive hybridization (ASOCH) with the Handy Bio-Strand system for the quantitative identification of three well-defined HBV variants: the basal core promoter (BCP) mutations (nt1762 and nt1764), the pre-core (PC) mutation (nt1896), and variance at nt1858. Using standardized mixtures of wild-type and mutant DNA, optimal hybridization conditions for ASOH and ASOCH were determined. Next, the performance of these methods was evaluated using actual serum DNAs from HBV patients. Excellent reproducibility was obtained both in the analysis of internal positive controls and in the semi-quantitative categorization of heterogeneous viral mixtures into five abundance groups (0%, 25%, 50%, 75%, and 100% mutant virus). Combined with real-time PCR to determine the HBV viral load, this hybridization method offers a new tool with applications both in HBV clinical research and treatment.

Quantification and improvement of error rate during ligase detection reaction.

We estimated the actual error rate during ligase detection reaction (LDR), and confirmed that DNA sequences around 3' ends are greatly influenced on the specificity of LDR. Its specificity is increased about 1000 times by introducing a mismatch base near the 3' ends.

Noncovalent immobilization of streptavidin on in vitro- and in vivo-biotinylated bacterial magnetic particles.

Biotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) or biotin carboxyl carrier protein (BCCP) on the surface of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. BAP-displaying BacMPs (BAP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. Then the in vitro biotinylation of BAP-BacMPs was confirmed using alkaline phosphatase-labeled antibiotin antibody. In contrast, BacMPs displaying the intact 149 residues of AMB-1 BCCP (BCCP-BacMPs) and displaying the COOH-terminal 78 residues of BCCP (BCCP78-BacMPs) were biotinylated in AMB-1 cells. The in vivo biotinylation of BCCP-BacMPs and BCCP78-BacMPs was thought to be performed by endogenous AMB-1 biotin ligase. Streptavidin was introduced onto biotinylated BacMPs by simple mixing. In an analysis using tetramethyl rhodamine isocyanate-labeled streptavidin, approximately 15 streptavidin molecules were shown to be immobilized on a single BCCP-BacMP. Furthermore, gold nanoparticle-BacMP composites were constructed via the biotin-streptavidin interaction. The conjugation system developed in this work provides a simple, low-cost method for producing biotin- or streptavidin-labeled magnetic nanoparticles. Various functional materials can be site selectively immobilized on these specially designed BacMPs. By combining the site-selective biotinylation technology and the protein display technology, more innovative and attractive magnetic nanomaterials can be constructed.

Fully automated immunoassay for detection of prostate-specific antigen using nano-magnetic beads and micro-polystyrene bead composites, 'Beads on Beads'.

Magnetic beads have served as a conventional bioassay platform in biotechnology. In this study, a fully automated immunoassay was performed using novel nano- and microbead-composites constructed by assembling nano-magnetic beads onto polystyrene microbeads, designated 'Beads on Beads'. Nano-sized bacterial magnetic particles (BacMPs) displaying the immunoglobulin G (IgG)-binding domain of protein A (ZZ domain) were used for the construction of 'Beads on Beads' via the interaction of biotin-streptavidin. The efficient assembly of 'Beads on Beads' was performed by gradual addition of biotin-labeled BacMPs onto streptavidin-coated polystyrene microbeads. Approximately 2000 BacMPs were uniformly assembled on a single microbead without aggregation. The constructed 'Beads on Beads' were magnetized and separated from the suspension by using an automated magnetic separation system with a higher efficiency than BacMPs alone. Furthermore, fully automated detection of prostate-specific antigens was performed with the detection limit of 1.48 ng mL(-1). From this preliminary assay, it can be seen that 'Beads on Beads' could be a powerful tool in the development of high-throughput, fully automated multiplexed bioassays.

Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G).

We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.

Pretreatment of polyamide monofilament with hydrochloric acid improves sensitivity of three-dimensional microarray, Bio-Strand.

A single-nucleotide-polymorphism-typing method using a novel three-dimensional DNA microarray, Bio-Strand, is promising because it is rapid, inexpensive and easily automated. It has been developed with the intent to overcome the drawbacks of conventional DNA microarrays, which use flat surfaces and impermeable materials such as glass slides; Bio-Strand as a novel DNA microarray, with its permeability, has a significantly improved stability compared with conventional DNA microarrays that use impermeable materials. In this study, we have developed a simple method of pretreating a polyamide monofilament to increase its surface area and to make it permeable, which makes Bio-Strand more sensitive and stable, allowing it to be adapted for clinical diagnostic applications. The fluorescence signal obtained with a nylon 6 monofilament pretreated under optimal conditions (hydrolysis by 5 M HCl/ethanol followed by washing with 50% ethanol and 100% ethanol) was significantly stronger than that obtained with an untreated monofilament.