Sadayori Hoshina

A novel magnetic crystal-lipid nanostructure for magnetically guided in vivo gene delivery.

Cancer gene therapy requires a safe and effective gene delivery system. Polymer- and lipid-coated magnetic nanocrystals have been used to deliver silencing RNA, but synthesizing these magnetic vectors is difficult. Here, we show that a new nanoparticle formulation can be magnetically guided to deliver and silence genes in cells and tumours in mice. This formulation, termed LipoMag, consists of an oleic acid-coated magnetic nanocrystal core and a cationic lipid shell. When compared with the commercially available PolyMag formulation, LipoMag displayed more efficient gene silencing in 9 of 13 cell lines, and better anti-tumour effects when systemically administered to mice bearing gastric tumours. By delivering an optimized sequence of a silencing RNA that targets the epidermal growth factor receptor of tumour vessels, the intended therapeutic benefit was achieved with no evident adverse immune reaction or untoward side effects.

Bovine lactoferrin potently inhibits liver mitochondrial 8-OHdG levels and retrieves hepatic OGG1 activities in Long-Evans Cinnamon rats.

BACKGROUND/AIMSnTo assess the effect of lactoferrin on oxidative liver damage and its mechanism, we used Long-Evans Cinnamon (LEC) rats that spontaneously develop fulminant-like hepatitis and lethal hepatic failure.nnnMETHODSnFour-week-old female LEC rats were divided into the untreated and treated groups. The latter was fed bovine lactoferrin at 2% mixed with conventional diet.nnnRESULTSnThe cumulative survival rates were 75.0% vs. 100% at 14 weeks, 37.5% vs. 91.7% at 15 weeks, and 12.5% vs. 91.7% at 16 weeks, respectively, for untreated and treated rats (P=0.0008). The 8-OHdG levels in liver mitochondrial DNA and malondialdehyde in plasma and liver tissues were significantly lower in treated than untreated rats (P<0.001, =0.017 and 0.034, respectively). Mitochondrial DNA mutations were more common in untreated rats. OGG1 mRNA and protein expression levels were significantly lower in untreated than treated rats (P=0.003 and 0.007, respectively). Hypermethylation of the second CpG island located upstream of OGG1 gene was observed in untreated rats.nnnCONCLUSIONSnOur findings indicated that lactoferrin inhibits oxidative liver damage in LEC rats. Lactoferrin could be potentially useful for the treatment of oxidative stress-induced liver diseases.

Application of chimeric RNA–DNA oligonucleotides to the detection of pathogenic microorganisms using surface plasmon resonance

Abstract Chimeric oligonucleotides consisting of 21 bases of ribonucleic acid (RNA) with six bases of deoxyribonucleic acid (DNA) at the 3′-hydroxyl terminus (chimeric RNA–DNA primer) and the recombinant thermostable DNA polymerase derived from Thermus thermophilus (rTth DNA polymerase) were utilized to efficiently amplify DNA fragments using the conventional polymerase chain reaction (PCR). The reaction required the use of both DNA polymerase and reverse transcriptase during each thermal cycle to form a double-stranded DNA in which one terminus was an RNAxa0:xa0DNA hybrid. Due to the ability of rTth DNA polymerase to function as both the DNA polymerase and reverse transcriptase, a chimeric RNA–DNA primer was shown to serve as a primer in the conventional PCR procedure. We further demonstrate the advantages of using these PCR products to identify microorganisms using surface plasmon resonance (SPR) technology. This detection system was able to distinguish Shiga toxin-producing Escherichia coli O157xa0:xa0H7 strains from other bacteria such as Salmonella typhimurium and S. enteritidis.

Genetic identification of Staphylococcus aureus by polymerase chain reaction using single-base-pair mismatch in 16S ribosomal RNA gene

Staphylococcus aureus is the most predominant and important pathogen in clinical microbiology. A DNA amplification assay using the polymerase chain reaction (PCR) was designed to identify S. aureus through a single‐base‐pair mismatch in the sequences of staphylococcal 16S ribosomal RNA (16S rRNA) genes. It was able to detect and identify S. aureus without requiring additional analytical techniques. Twenty‐eight staphylococcal and non‐staphylococcal strains were tested to verify the specificity of the assay, and only S. aureus strains gave a positive reaction. It may be possible to provide immediate and exact information for the identification of S. aureus.

IQGAP1 and vimentin are key regulator genes in naturally occurring hepatotumorigenesis induced by oxidative stress

To identify key genes involved in the complex multistep process of hepatotumorigenesis, we reduced multivariate clinicopathological variables by using the Long-Evans Cinnamon rat, a model with naturally occurring and oxidative stress-induced hepatotumorigenesis. Gene expression patterns were analyzed serially by profiling liver tissues from rats of a naive status (4 weeks old), through to those with chronic hepatitis (26 and 39 weeks old) to tumor development (67 weeks old). Of 31 099 probe sets used for microarray analysis, 87 were identified as being upregulated in a stepwise manner during disease progression and tumor development. Quantitative real-time reverse transcription-polymerase chain reaction and statistical analyses verified that IQGAP1 and vimentin mRNA expression levels increased significantly throughout hepatotumorigenesis. A hierarchical clustering algorithm showed both genes clustered together and in the same cluster group. Immunohistochemical and western blot analyses showed similar increases in protein levels of IAGAP1 and vimentin. Finally, pathway analyses using text-mining technology with more comprehensive and recent gene-gene interaction data identified IQGAP1 and vimentin as important nodes in underlying gene regulatory networks. These findings enhance our understanding of the multistep hepatotumorigenesis and identification of target molecules for novel treatments.

Clinical course and predictive factors of virological response in long-term lamivudine plus adefovir dipivoxil combination therapy for lamivudine-resistant chronic hepatitis B patients†

The aims of this study were to assess the long‐term efficacy of lamivudine (LAM) plus adefovir dipivoxil (ADV) combination therapy in patients with chronic hepatitis B resistant to LAM, to identify predictive factors of complete viral response (HBV‐DNA <2.6u2009logu2009copies/ml at 12 months of combination therapy), and to analyze amino acid substitutions associated with treatment resistance in the hepatitis B virus (HBV) genome. Seventy‐two patients who received ADV in addition to LAM for breakthrough hepatitis were enrolled. Undetectable HBV‐DNA was observed in 61%, 74%, 81%, 84%, and 85% at 12, 24, 36, 48, and 60 months of combination therapy, respectively. On multivariate analysis, undetectable HBV‐DNA during the preceding LAM monotherapy (Pu2009<u20090.0001), alanine aminotransferase valueu2009≥u2009the upper limit of normalu2009×u20096 (Pu2009=u20090.006) and HBV‐DNA levelu2009<u20096.0u2009logu2009copies/ml at the initiation of combination therapy (Pu2009=u20090.007) were independent significant predictors of complete viral response. The cumulative rate of undetectable HBV‐DNA was significantly higher in patients with response to the preceding LAM monotherapy than in those with poor response to it. Breakthrough hepatitis occurred in three patients without complete viral response and with poor response to the preceding LAM monotherapy, and rtA181A/V substitution was detected in one of the three patients. In conclusion, undetectable HBV‐DNA during the LAM monotherapy was the strongest independent predictor of complete viral response to the following combination therapy. The efficacy of LAM plus ADV combination therapy may be determined by viral response to the preceding LAM monotherapy. J. Med. Virol. 83:953–961, 2011.

Malakoplakia of the Kidney

b alakoplakia is a rare inflammatory disease associated with Escherichia coli nfection. Despite some characteristic histoogical features, including the presence of large istiocytes with clusters of Michaelis-Gutann (MG) bodies, malakoplakia of the kidey is difficult to diagnose because of its rarity nd varying histological picture. We report the ase of a 57-year-old man who had granulomaous interstitial nephritis caused by kidney arenchymal malakoplakia. Of note, using olymerase chain reaction (PCR), we were ble to site-specifically amplify E coli DNA rom his kidney biopsy specimens. Malakoplaia of the kidney should be considered in the ifferential diagnosis of granulomatous intertitial nephritis, and application of molecular iological approaches to the analysis of kidney issues may be useful in the diagnosis of this isease.

A macrolide antibiotic, roxithromycin, inhibits the growth of human myeloid leukemia HL60 cells by producing multinucleate cells

The antiproliferative effect of roxithromycin (RXM) was studied using human myeloid leukemia HL60 cells. RXM inhibited the growth of HL60 cells in a concentration-dependent manner, and significantly inhibited growth at concentrations above 75 μM. This growth inhibition was not associated with specific cell cycle arrest and DNA synthesis was not impaired. In addition, the number of viable cells remained almost unchanged in the presence of 100 νM RXM. RXM induced growth inhibition at least partly by the formation of multinucleate cells. Both flowcytometric and morphological examination revealed that more than 40% of the RXM-treated cells were binucleate. These findings demonstrate that RXM is a potent new modulator of cell cycle progression in HL60 cells and suggest that the inhibition of cytokinesis by this drug may provide a new model for studying mitosis.

Hepatitis B virus variants in carriers with hematologic malignancies in whom fulminant hepatic failure develops after chemotherapy

Abstract Three patients who were carriers of hepatitis B virus (HBV) and had hematologic malignancies, experienced fulminant hepatic failure after withdrawal of chemotherapy. Before treatment, each patient had inactive HBV infection with undetectable viral DNA polymerase; two patients were negative for hepatitis B e antigen. After chemotherapy, DNA polymerase increased and HBV variants with mutations in the precore region prevailed in their sera. These results implicate precore mutants thriving after chemotherapy and restored immune responses of patients in the pathogenesis of severe hepatic failure.