Mariko Esumi

2′,5′-Oligoadenylate synthetase-like gene highly induced by hepatitis C virus infection in human liver is inhibitory to viral replication in vitro

We found the 2,5-oligoadenylate synthetase-like (OASL) gene to be significantly elevated by high virus loads in human liver infected with hepatitis C virus (HCV). Here, we determined whether OASL inhibited HCV replication using an in vitro system. We constructed three expression vectors of OASL to produce isoform a (OASLa), isoform b (OASLb), and the C-terminal ubiquitin-like domain of isoform a (Ub). When Huh7 JFH-1 HCV replicon cells were separately transfected with these three vectors, colony formation of HCV-replicating cells was inhibited by 95%, 94%, and 65%, respectively. Both OASLa and OASLb were also inhibitory for cells as well as the virus because colony formation of OASL-producing cells was reduced to 41% and 8%, respectively. Stable Huh7 clones producing each of the three OASLs were established and assessed for their inhibition of HCV replication using luciferase reporter gene-containing JFH-1 replicon RNA. HCV replication was inhibited by 50-90% in several stable OASL clones. Association analysis in six Ub clones expressing different levels of Ub mRNA showed that the degree of inhibition of HCV replication was significantly associated with the amount of Ub present. In conclusion, OASL possesses two domains with HCV inhibitory activity. The N-terminal OAS-homology domain without OAS activity is inhibitory for cell growth as well as HCV replication, whereas C-terminal Ub is inhibitory only for HCV replication. Therefore, OASLa, a major isoform of this molecule induced in human liver, may mediate anti-HCV activity through two different domains.

Long-term oral administration of glucosamine or chondroitin sulfate reduces destruction of cartilage and up-regulation of MMP-3 mRNA in a model of spontaneous osteoarthritis in Hartley guinea pigs.

Histological and molecular changes were examined to investigate the effects of long‐term administration of glucosamine (GlcN) and chondroitin sulfate (CS) in a model of spontaneous osteoarthritis (OA) in Hartley guinea pigs. Three groups of female 3‐week‐old Hartley guinea pigs received GlcN, CS, and neither agent, respectively. Five animals in each group were sacrificed at 8, 12, and 18 months of age. At 8 months of age, Hartley guinea pigs had severe degeneration of knee joint cartilage, chondrocyte apoptosis, marked reduction of tissue total RNA, decreases of aggrecan and collagen type 2 mRNAs, and increases in MMP‐3 and MMP‐8 mRNAs. Long‐term administration of GlcN and CS reduced cartilage degeneration at 8 months of age. The marked loss of total RNA and the increase in MMP‐3 mRNA were also inhibited by GlcN and CS. Thus, long‐term oral administration of GlcN or CS inhibits OA progression, maintains total RNA and down‐regulates MMP‐3 mRNA in a spontaneous OA model in Hartley guinea pigs.

Identification and analysis of an early diagnostic marker for malignant melanoma: ZAR1 intra-genic differential methylation

BACKGROUNDnEpigenetic changes such as aberrant DNA methylation and histone modification have been shown to play an important role in the tumorigenesis of malignant melanoma.nnnOBJECTIVEnTo identify novel tumor-specific differentially methylated regions (DMRs) in human malignant melanoma.nnnMETHODSnThe aberrant methylation at 14 candidate human genomic regions identified through a mouse model study with quantitative DNA methylation analysis using the Sequenom MassARRAY system was performed.nnnRESULTSnThe CpG island Exon 1 region of the Zygote arrest 1 (ZAR1) gene, which is responsible for oocyte-to-embryo transition, showed frequent aberrant methylation of 28 out of 30 (93%) melanoma surgical specimens, 16 of 17 (94%) melanoma cell lines, 0% of 4 normal human epidermal melanocyte (NHEM) cell lines, 0% of 10 melanocytic nevi and 100% of 51 various cancer cell lines. According to the real-time RT-PCR, the ZAR1 gene was overexpressed in part of the hypermethylated cell lines, while its low expression with bivalent histone methylation status was seen in unmethylated cell lines.nnnCONCLUSIONnOur findings suggest that the ZAR1 intra-genic differentially methylated region would be a useful tumor marker for malignant melanoma and may be other type of cancers. The involvement of ZAR1 in the carcinogenesis of melanoma, still remains unclear, although we have examined tumorigenic capacities by exogenous full-length ZAR1 over-expression and siRNA knock-down experiments.

Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions.

The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for quantifying DNA available for PCR.

TMPRSS2 Is an Activating Protease for Respiratory Parainfluenza Viruses

ABSTRACT Here, we show that human parainfluenza viruses and Sendai virus (SeV), like other respiratory viruses, use TMPRSS2 for their activation. The membrane fusion proteins of respiratory viruses often possess serine and glutamine residues at the P2 and P3 positions, respectively, but these residues were not critical for cleavage by TMPRSS2. However, mutations of these residues affected SeV growth in specific epithelial cell lines, suggesting the importance of these residues for SeV replication in epithelia.

Passive cigarette smoking changes the circadian rhythm of clock genes in rat intervertebral discs

We aimed to elucidate the molecular changes in intervertebral discs (IVDs) caused by passive smoking. Rats were subjected to 8 weeks of passive smoking; thereafter, their lumbar vertebrae were harvested. The annulus fibrosus and cartilage endplate (AF/CEP) were harvested together, and the nucleus pulposus (NP) was isolated separately. The expression of 27,342 rat genes was analyzed. In 3 “nonsmoking” rats, 96 of 112 genes whose expression varied ≥10‐fold between the AF/CEP and NP were more highly expressed in the AF/CEP. With these differentially expressed genes, we uncovered novel AF/CEP and NP marker genes and indicated their possible novel functions. Although passive smoking induced less marked alteration in the gene expression profiles of both the AF/CEP and NP, multiple clock‐related genes showed altered expression. These genes were expressed with a circadian rhythm in IVD cells, and most genes showed a phase shift of −6 to −9u2009h induced by passive smoking. Some clock‐related genes showed abolished oscillation in the NP. Passive smoking also changed the expression levels of proteases and protease inhibitors and reduced the expression of NP marker genes. Thus, passive smoking induces changes in the circadian rhythm of a peripheral clock (IVD clock) that might be involved in molecular events related to IVD degeneration.

Protein from the fraction remaining after RNA extraction is useful for proteomics but care must be exercised in its application

Simultaneous isolation of mRNA and proteins from a single small biopsy specimen can be useful for integrated omics studies. Here, we have improved the method for extracting protein from the fraction remaining after RNA isolation by TRIzol reagent, for application in protein and proteome analyses. Protein yield was reduced by half, but the patterns developed on 2D gels were equivalent to conventional urea extractions. Thus, although quantitative profiles of individual proteins were different from conventionally-isolated samples, overall profiles were similar. Therefore, this particular protein source is useful for proteomics but care must be exercised in its application.

Transmembrane serine protease TMPRSS2 activates hepatitis C virus infection

The human liver reacts to hepatitis C virus (HCV) with a balanced response consisting of host anti‐ and proviral activities. To explore these subtle host responses, we used oligonucleotide microarrays to investigate the differential gene expression between two groups of liver samples with high and low HCV loads (>100‐fold difference). We identified and validated 26 genes that were up‐regulated in livers with high HCV loads, including transmembrane protease serine 2 (TMPRSS2). Trypsin inhibitors inhibited the infection of Huh7‐25‐CD81 cells with cell‐culture–derived HCV (HCVcc) of Japanese fulminant hepatitis 1 isolate at the postbinding and entry step, and trypsin enhanced HCVcc infection at an early stage of infection. Several major transmembrane serine proteases, in particular, furin and hepsin, were detected in Huh7‐25‐CD81 cells, but TMPRSS2 was not. Huh7‐25‐CD81 cell clones stably expressing TMPRSS2‐ WT (wild type) and inactive TMPRSS2‐mutant genes showed positive and negative enhancement of their susceptibility to HCVcc infection, respectively. The enhanced susceptibility of TMPRSS2‐WT Huh7‐25‐CD81 cells was confirmed by knockdown of TMPRSS2 using small interfering RNA. The cell‐surface protease activity of TMPRSS2‐WT cells was markedly active in the cleavage of QAR and QGR, corresponding to amino acid residues at P3 to P1. Conclusion: The cell‐surface activity of a trypsin‐like serine protease, such as TMPRSS2, activates HCV infection at the postbinding and entry stage. Host transmembrane serine proteases may be involved in the sensitivity, persistence, and pathogenesis of HCV infection and be possible targets for antiviral therapy. (Hepatology 2015;61:438‐447)

Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation

Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: ‘microgenomics’. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.

β-Glucuronidase is a suitable internal control gene for mRNA quantitation in pathophysiological and non-pathological livers

The level of expression of housekeeping genes is in general considered stable, and a representative gene such as glyceraldehyde-3-phosphate dehydrogenase is commonly used as an internal control for quantitating mRNA. However, expression of housekeeping genes is not always constant under pathological conditions. To determine which genes would be most suitable as internal controls for quantitative gene expression studies in human liver diseases, we quantified 12 representative housekeeping genes in 27 non-cancerous liver tissues (normal, chronic hepatitis C with and without liver cirrhosis). We identified β-glucuronidase as the most suitable gene for studies on liver by rigorous statistical analysis of inter- and intra-group comparisons. We conclude that it is important to determine the most appropriate control gene for the particular condition to be analyzed.