Single nucleotide polymorphisms in the D-loop region of mitochondrial DNA is associated with colorectal cancer outcome

<p>Single nucleotide polymorphisms (SNPs) in the displacement loop (D-Loop) of mitochondrial DNA (mtDNA) has been identified for their association with the risk and outcome in many cancers. We have identified risk associated D-loop SNPs for colorectal cancer previously, in the present study, we evaluate their prognostic value for postoperative survival of colorectal cancer (CRC). The minor haplotype of nucleotides 16290T and frequent haplotype of nucleotide 16298T in the hypervariable segment 1 (HV1) region of the D-loop were identified for their association with high survival rate of CRC. After adjusted with COX proportional hazard model, the nucleotide site of 16290 was identified as independent predictor for CRC (RR, 0.379; 95% CI, 0.171–0.839; <i>p</i> = 0.017). In conclusion, SNPs in the mtDNA D-Loop were found to be valuable markers for colorectal cancer outcome evaluation.</p

A schematic representation of the multiple mode of action of CpAMs on HBV capsid assembly and disassembly as well as their effects on cccDNA biosynthesis.

<p>The red arrowed lines indicate the normal processes in viral replication. The black arrowed lines indicate CpAMs-induced processes. See text for detailed explanation.</p

A schematic representation of the multiple mode of action of CpAMs on HBV capsid assembly and disassembly as well as their effects on cccDNA biosynthesis.

<p>The red arrowed lines indicate the normal processes in viral replication. The black arrowed lines indicate CpAMs-induced processes. See text for detailed explanation.</p

HBV capsid assembly modulator treatment confers HBV virion DNA sensitivity to DNase digestion.

<p><b>(A)</b> Each of the endogenous DNA polymerase reaction contains approximately 10⁸ HBV virions prepared from chronic HBV carriers and incubated in the presence or absence of dNTP and indicated concentrations of Bay 41–4109, ENAN-34017 or GLS4 at 37°C for 16 h. Viral DNA were extracted without or with prior DNase I digestion at 37°C for 30 min and detected by Southern blot hybridization with full-length riboprobe recognizing minus strand DNA. <b>(B)</b> Approximately 10⁸ HBV virions in each endogenous DNA polymerase reaction were incubated in the absence of dNTP (except for lanes 2, 3, 19 and 20) and absence or presence of the indicated concentrations of Bay 41–4109, ENAN-34017 or GLS4 at 37°C for 16 h. Viral DNA were extracted and analyzed as described above.</p

HBV capsid assembly modulators efficiently inhibit cccDNA formation during <i>de novo</i> HBV infection of C3A^hNTCP cells.

<p>C3A^hNTCP cells were infected with HBV at an MOI of 500 genome equivalents. The cells were mock-treated or treated with 100nM of MyrB, 1 μM of ETV, 2.5 μM of Bay 41–4109, 1 μM of GLS-4, 5 μM of ENAN-34017 or 1,000 IU/ml of IFN-α, starting from 24 h before infection until harvesting at 3 or 6 days post infection (dpi). HBV cccDNA <b>(A)</b>, pgRNA <b>(B)</b> and cytoplasmic core DNA <b>(C)</b> were quantified by real-time PCR assays. Differences in viral cccDNA, core DNA or pgRNA between mock-treated control and treated groups were statistically analyzed (t-test, * <i>p</i> <0.05, ** <i>p</i> <0.01, *** <i>p</i> <0.001). <b>(D)</b> Hybridization analyses of HBV replication intermediates in cells harvested at 6 dpi. <i>Upper pan</i>e<i>l</i>, Hirt DNA extracted from the cells harvested at 6 dpi were denatured at 88°C for 5 min to denature DP-rcDNA to single-stranded DNA and followed by restriction with EcoRI to convert cccDNA into unit-length double stranded linear DNA and detected by Southern blot hybridization (labeled as CCC/EcoRI). Unit-length HBV linear DNA served as a molecular weight marker. <i>Lower panel</i>, HBV RNAs, pre-genomic RNA (pgRNA), 2.4 and 2.1kb mRNA specifying envelope proteins were determined by Northern blot hybridization. 28S and 18S ribosomal RNA (rRNA) served as loading controls.</p

Quantitative analyses of the effects of CpAMs on cccDNA synthesis.

<p>HepAD38 cells were treated with 2 mM of PFA from day 2 to day 6 after removal of tet from medium. On day 6, PFA-containing medium were removed and the cells were mock-treated or treated with 1 μM of ETV, 1 μM of GLS4, 2.5 μM of Bay 41–4109 or 5 μM of ENAN-34017 and harvested at 0, 6, 12 and 24 h of treatment. (<b>A</b>) Hirt DNA were extracted and resolved in 1.5% agarose gel electrophoresis. HBV DNA were detected by Southern blot hybridization. (<b>B</b>) The amounts of cccDNA in each sample were quantified with Tyhoon FLA 7000 and plotted as arbitrary units. <b>(C)</b> Hirt DNA extracted from cells treated with the indicated compounds for 12 h after PFA removal were denatured at 88°C for 5 min and followed by EcoRI digestion. The DNA samples were then resolved in agarose gel and HBV DNA were detected by Southern blot hybridization (left panel). The amounts of cccDNA in each samples were quantified and normalized to the levels of mock-treated controls. The relative amounts of cccDNA were plotted (right panel). The relaxed circular (RC) DNA, double-stranded linear (DSL) DNA and cccDNA (CCC) species were indicated. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 (by t-test).</p

Differential effects of different chemotypes of CpAMs on integrity of mature double-stranded DNA-containing nucleocapsids.

<p>HBV capsids prepared from HepAD38 cells were mock treated <b>(A)</b> or incubated with 5 μM ENAN-34017 <b>(B)</b>, 3 μM Bay 41–4109 <b>(C)</b> or 3 μM GLS4 <b>(D)</b> in a reaction containing 150 mM NaCl, 50 mM Tris-HCl, pH8.0, 10 mM MgCl₂, 1 mM DTT and 0.1% NP-40 for at 37°C for 16 h. The reactions were then fractioned by sucrose gradient centrifugation. HBV capsids in each of viral DNA-containing fractions, as detected by a qPCR assay, were determined by a 1.5% native agarose gel electrophoresis-based particle gel assay. HBV DNA in the fractions were detected by Southern blot hybridization with a riboprobe that specifically hybridizes to negative strand of HBV DNA. The relaxed circular (RC) DNA, double-stranded linear (DSL) DNA and full-length single stranded (SS) DNA species were indicated. Sucrose concentration (w/v %) of each of the fractions is provided. The mature forms of HBV DNA species (RC and DSL) are highlighted in red-dash lined box.</p

Design, Synthesis, and Biological Evaluation of <i>N</i>-Alkylated Deoxynojirimycin (DNJ) Derivatives for the Treatment of Dengue Virus Infection

We recently described the discovery of oxygenated <i>N</i>-alkyl deoxynojirimycin (DNJ) derivative <b>7</b> (CM-10–18) with antiviral activity against dengue virus (DENV) infection both in vitro and in vivo. This imino sugar was promising but had an EC₅₀ against DENV in BHK cells of 6.5 μM, which limited its use in in vivo. Compound <b>7</b> presented structural opportunities for activity relationship analysis, which we exploited and report here. These structure–activity relationship studies led to analogues <b>2h</b>, <b>2l</b>, <b>3j</b>, <b>3l</b>, <b>3v</b>, and <b>4b</b>–<b>4c</b> with nanomolar antiviral activity (EC₅₀ = 0.3–0.5 μM) against DENV infection, while maintaining low cytotoxicity (CC₅₀ > 500 μM, SI > 1000). In male Sprague–Dawley rats, compound <b>3l</b> was well tolerated at a dose up to 200 mg/kg and displayed desirable PK profiles, with significantly improved bioavailability (<i>F</i> = 92 ± 4%)

Design, Synthesis, and Biological Evaluation of <i>N</i>-Alkylated Deoxynojirimycin (DNJ) Derivatives for the Treatment of Dengue Virus Infection

We recently described the discovery of oxygenated <i>N</i>-alkyl deoxynojirimycin (DNJ) derivative <b>7</b> (CM-10–18) with antiviral activity against dengue virus (DENV) infection both in vitro and in vivo. This imino sugar was promising but had an EC₅₀ against DENV in BHK cells of 6.5 μM, which limited its use in in vivo. Compound <b>7</b> presented structural opportunities for activity relationship analysis, which we exploited and report here. These structure–activity relationship studies led to analogues <b>2h</b>, <b>2l</b>, <b>3j</b>, <b>3l</b>, <b>3v</b>, and <b>4b</b>–<b>4c</b> with nanomolar antiviral activity (EC₅₀ = 0.3–0.5 μM) against DENV infection, while maintaining low cytotoxicity (CC₅₀ > 500 μM, SI > 1000). In male Sprague–Dawley rats, compound <b>3l</b> was well tolerated at a dose up to 200 mg/kg and displayed desirable PK profiles, with significantly improved bioavailability (<i>F</i> = 92 ± 4%)

Inhibition of cccDNA formation by HBV capsid assembly modulators is time- and concentration-dependent.

<p><b>(A)</b> C3A^hNTCP cells were infected with HBV at an MOI of 500 genome equivalents. The cells were mock-treated or treated with 100 nM of MyrB, 1 μM of ETV, 2.5 μM of Bay 41–4109, 1 μM of GLS-4, 5 μM of ENAN-34017 or 1,000 IU/ml of IFN-α., starting from 24 h before infection, at infection or 24 h post infection until harvesting at 3 days post infection. HBV cccDNA was quantified by a real-time PCR assay. <b>(B)</b> C3A^hNTCP cells were infected with HBV at an MOI of 500 genome equivalents. The cells were mock-treated or treated with the indicated concentrations of Bay 41–4109, GLS4 or ENAN-34017, starting from 24 h before infection until harvesting at 3 days post infection. HBV cccDNA were quantified by a real-time PCR assay. Differences in viral cccDNA between mock-treated control and treated group cultures under each treatment schedule were statistically analyzed (t-test, * <i>p</i> <0.05, ** <i>p</i> <0.01, *** <i>p</i> <0.001).</p