Mouse DNA Research Articles

Farnesoid X receptor‐α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo

Hepatitis B virus (HBV) infection and bile acid (BA) metabolism are interdependent: infection modifies the expression of the BA nuclear receptor farnesoid X receptor (FXR)-α, and modulation of FXRα activity by ligands alters HBV replication. Mechanisms of HBV control by FXRα remain to be unveiled. FXRα silencing in HBV-infected HepaRG cells decreased the viral covalently closed circular (ccc)DNA pool size and transcriptional activity. Treatment with the FXRα agonist GW4064 inhibited FXRα proviral effect on cccDNA similarly for wild-type and hepatitis B viral X protein (HBx)-deficient virus, whereas agonist-induced inhibition of pregenomic and precore RNA transcription and viral DNA secretion was HBx dependent. These data indicated that FXRα acts as a proviral factor by 2 different mechanisms, which are abolished by FXRα stimulation. Finally, infection of C3H/HeN mice by a recombinant adeno-associated virus-2/8-HBV vector induced a sustained HBV replication in young mice in contrast with the transient decline in adult mice. Four-week GW4064 treatment of infected C3H/HeN mice decreased secretion of HBV DNA and HB surface antigen in adult mice only. These results suggest that the physiologic balance of FXRα expression and activation by bile acid is a key host metabolic pathway in the regulation of HBV infection and that FXRα can be envisioned as a target for HBV treatment.-Mouzannar, K., Fusil, F., Lacombe, B., Ollivier, A., Ménard, C., Lotteau, V., Cosset, F.-L., Ramière, C., André, P. Farnesoid X receptor α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.

Swi5–Sfr1 stimulates Rad51 recombinase filament assembly by modulating Rad51 dissociation

Eukaryotic Rad51 protein is essential for homologous-recombination repair of DNA double-strand breaks. Rad51 recombinases first assemble onto single-stranded DNA to form a nucleoprotein filament, required for function in homology pairing and strand exchange. This filament assembly is the first regulation step in homologous recombination. Rad51 nucleation is kinetically slow, and several accessory factors have been identified to regulate this step. Swi5-Sfr1 (S5S1) stimulates Rad51-mediated homologous recombination by stabilizing Rad51 nucleoprotein filaments, but the mechanism of stabilization is unclear. We used single-molecule tethered particle motion experiments to show that mouse S5S1 (mS5S1) efficiently stimulates mouse RAD51 (mRAD51) nucleus formation and inhibits mRAD51 dissociation from filaments. We also used single-molecule fluorescence resonance energy transfer experiments to show that mS5S1 promotes stable nucleus formation by specifically preventing mRAD51 dissociation. This leads to a reduction of nucleation size from three mRAD51 to two mRAD51 molecules in the presence of mS5S1. Compared with mRAD51, fission yeast Rad51 (SpRad51) exhibits fast nucleation but quickly dissociates from the filament. SpS5S1 specifically reduces SpRad51 disassembly to maintain a stable filament. These results clearly demonstrate the conserved function of S5S1 by primarily stabilizing Rad51 on DNA, allowing both the formation of the stable nucleus and the maintenance of filament length.

XenofilteR: computational deconvolution of mouse and human reads in tumor xenograft sequence data

BackgroundMouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analyses of their RNA and DNA profiles are challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin result in false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. However, currently available algorithms are limited and improvements in accuracy and ease of use are necessary.ResultsWe developed the R-package XenofilteR, which separates mouse from human sequence reads based on the edit-distance between a sequence read and reference genome. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human DNA sequence data. These analyses revealed that XenofilteR removes > 99.9% of sequence reads of mouse origin while retaining human sequences. This allowed for mutation analysis of xenograft samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic tumor mutations.ConclusionsXenofilteR accurately dissects RNA and DNA sequences from mouse and human origin, thereby outperforming currently available tools. XenofilteR is open source and available at https://github.com/PeeperLab/XenofilteR.

Prevention of Lipid Peroxidation-derived Cyclic DNA Adduct and Mutation in High-Fat Diet-induced Hepatocarcinogenesis by Theaphenon E.

Obesity is associated with cancer risk and its link with liver cancer is particularly strong. Obesity causes non-alcoholic fatty liver disease (NAFLD) that could progress to hepatocellular carcinoma (HCC). Chronic inflammation likely plays a key role. We carried out a bioassay in the high-fat diet (HFD)-fed C57BL/6J mice to provide insight into the mechanisms of obesity-related HCC by studying γ-OHPdG, a mutagenic DNA adduct derived from lipid peroxidation. In an 80-week bioassay, mice received a low-fat diet (LFD), high-fat diet (HFD), and HFD with 2% Theaphenon E (TE) (HFD+TE). HFD mice developed a 42% incidence of HCC and LFD mice a 16%. Remarkably, TE, a standardized green tea extract formulation, completely blocked HCC in HFD mice with a 0% incidence. γ-OHPdG measured in the hepatic DNA of mice fed HFD and HFD+TE showed its levels increased during the early stages of NAFLD in HFD mice and the increases were significantly suppressed by TE, correlating with the tumor data. Whole-exome sequencing showed an increased mutation load in the liver tumors of HFD mice with G>A and G>T as the predominant mutations, consistent with the report that γ-OHPdG induces G>A and G>T. Furthermore, the mutation loads were significantly reduced in HFD+TE mice, particularly G>T, the most common mutation in human HCC. These results demonstrate in a relevant model of obesity-induced HCC that γ-OHPdG formation during fatty liver disease may be an initiating event for accumulated mutations that leads to HCC and this process can be effectively inhibited by TE. Cancer Prev Res; 11(10); 665-76. ©2018 AACR.

Detection of Mice DNA in Meatballs Using Real Time – PCR

Meat is one of perishable food and has the high of water content and protein content. Meatballs are processed food who are using meat as main ingredient and other ingredients, such tapioca flour, salt, pepper, and the other ingredients as food additives. Meatballs is the mostpopular because has tasty flavor, high protein content, especiallyessential amino acids.In Indonesia, until now there were still cases of counterfeiting of processed meat products. One of method is mixing the beef meat and other animalsmeat in the making process to reduce production costs, so that similar products can be obtained at a much lower cost. One common case is the mixing of beef meat with rat meat. This study aims to identify meatballs contaminated by rat meat. Meatball samples used do not have Halal certificate from LPPOM MUI. From the results of the study, obtained the green amplification curve with the highest peak showed positive control of rat DNA, followed by three other green curves from the sample that showed the occurrence of amplification, thus indicating samples positive containing rat DNA. In the positive sample, the rat DNA concentration in the sample is quite low which can be seen from the intensity of the peak produced on the amplification curve when compared with positive control. Keywords :DNA, Meat,Meatballs, Rat, RT – PCR

A simple but profound mutation in mouse DNA polymerase ε drives tumorigenesis.

Over 40 years ago, Loeb and colleagues proposed that errors in DNA replication produce a mutator phenotype that is involved in generating the multiple mutations required for tumor development. In this issue of the JCI, Li, Castrillon, and colleagues describe a mouse model containing a single base change in the gene encoding replicative DNA polymerase ε (POLE) that mimics the "ultramutator" phenotype recently reported in many human tumors. Their seminal accomplishment validates Loeb's hypothesis and the use of mutational signatures to understand the origins and potentially the treatment of human tumors, and it offers an exciting opportunity to further explore the mechanisms responsible for normal DNA replication fidelity and their perturbations.

Fetal and Placental DNA Stimulation of TLR9: A Mechanism Possibly Contributing to the Pro-inflammatory Events During Parturition.

While there is evidence for a relationship between cell-free fetal DNA (cffDNA) and parturition, questions remain regarding whether cffDNA could trigger a pro-inflammatory response on the pathway to parturition. We hypothesized that placental and/or fetal DNA stimulates toll-like receptor 9 (TLR9) leading to secretion of pro-inflammatory cytokines by macrophage cells. Four in vitro DNA stimulation studies were performed using RAW 264.7 mouse peritoneal macrophage cells incubated in media containing the following DNA particles: an oligodeoxynucleotide (ODN2395), intact genomic DNA (from mouse placentas, fetuses and adult liver), mouse DNA complexed with DOTAP (a cationic liposome forming compound), and telomere-depleted mouse DNA. Interleukin 6 (IL6) secretion was measured in the media by enzyme-linked immunosorbent assay; and the cell pellet was homogenized for protein content (picograms IL6/mg protein). Robust IL6 secretion was observed in response to ODN2395 (a CpG-rich TLR9 agonist), mouse DNA-DOTAP complexes, and telomere-depleted mouse DNA in concentrations of 5 to 15 μg/mL. In contrast, ODN A151 (containing telomere sequence motifs), intact genomic mouse DNA, and restriction enzyme-digested DNA had no effect on IL6 secretion. The IL6 response was significantly inhibited by chloroquine (10 μg/mL), thereby confirming the important role for TLR9 in the response by macrophage cells. DNA derived from mouse placentas and fetuses, and depleted of telomeric sequences, stimulates a robust pro-inflammatory response by macrophage cells, thereby supporting the hypothesis that cffDNA is able to stimulate an innate immune response that could trigger the onset of parturition. These findings are of clinical importance, as we search for effective treatment/prevention of preterm parturition.

DNA-release by Streptococcus pneumoniae autolysin LytA induced Krueppel-like factor 4 expression in macrophages

The recruitment of myeloid cells to the lung is of utmost importance for the elimination of invading pathogens. We investigated the Streptococcus pneumoniae-dependent induction mechanism of KLF4 in macrophages as a potential regulator of the macrophage immune response. We demonstrated that only viable pneumococci, which have direct contact to the host cells and release LytA-dependent DNA, induced KLF4. Exogenous supplementation of pneumococcal, other bacterial, eukaryotic foreign (human) or self (mouse) DNA to autolysis-deficient pneumococci restored (at least in part) pneumococci-related KLF4 induction. Experiments using TLR9, TRIF and MyD88 knockout macrophages revealed that TLR9, TRIF and MyD88 were partly involved in the S. pneumoniae-induced KLF4 expression. BMMs missing important DNA receptor related molecules (ASC−/−, STING−/−) showed no differences in pneumococci-related KLF4 expression. Similar results were observed with IFNAR−/− BMMs and Type I IFN stimulated cells. LyzMcre mediated knockdown of KLF4 in BMMs resulted in a decreased secretion of proinflammatory cytokines and enhanced IL-10 release. In summary, we showed that pneumococci-related KLF4 induction in macrophages is mediated via a PAMP-DAMP induction mechanism involving a hitherto unknown host cell DNA sensor leading to a more proinflammatory macrophage phenotype.

Effects of Erythropoietin on Acute and Chronic Phases of Experimental Chagas Disease on Mice

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects about seven million people worldwide, mostly in Latin America. However, this disease has become cosmopolitan due to migration of people infected by the protozoan. The most important clinical manifestation of the disease is cardiomyopathy, which develops in approximately 30% of individuals with chronic infection. Despite reducing parasite load, trypanocidal drugs have no efficacy on the progression of lesions, doing little to decrease morbidity. Erythropoietin (Epo), a key‐regulator of erythropoiesis, also has a cardioprotective effect by reducing the processes of apoptosis, inflammation and myocardial ischemia through the formation of new blood vessels. However, it is unknown whether this protein can be effectively used in treatment of Chagas cardiomyopathy. Thereby, this study aims to investigate the possible cardioprotective effect of Epo on experimental Chagas disease. C57BL/6 mice were divided into five groups (n = 8 in each): not infected and not treated (NINT); infected with T. cruzi, not treated (INT); treated with Epo before infection (EpoI); infected and treated with Epo in the acute phase of the disease (IEpoA) and infected and treated with Epo in the chronic phase (IEpoC). Mice were infected by intraperitoneal route (i.p.) with 105 trypomastigotes of the Colombiana strain of T. cruzi. Mice received 2000 U/kg of Epo i.p. on alternate days as follow: during 30 days before the infection for the group EpoI (protective effect of Epo); from 1st to 30th days postinfection (dpi) for the group IEpoA and from 120th to 150th dpi for the group IEpoC (therapeutic effect of Epo). The animals were euthanized at 180 dpi. Histopathological analysis of heart, spleen and large intestine were performed. The parasite load in blood, heart, spleen and large intestine was determined by qPCR. Histopathological analysis demonstrated necrosis of cardiomyocytes (NC), multifocal inflammatory infiltrates (MII) in the perivascular and interstitial regions in heart of mice and damage in spleen tissue of INT (p<0.006). Administration of Epo reduced both NC and MII in the perivascular region in heart with less effect for the IEpoA (p<0.006). No cardiac fibrosis was observed in any group, neither lesions in large intestine. Similar profiles of blood parasitaemia were observed in groups who received Epo, with a higher number of parasites in EpoI and IEpoC groups. The latter presented parasitic load significantly higher than INT. In cardiac tissue, qPCR detected T. cruzi DNA in all infected mice without significant difference between groups. In spleen tissue the parasite load was extremely low with no significant difference between groups. For the large intestine, the number of parasite was similar for all infected group in spite of treatment with Epo. Our results show that Epo has no trypanocidal effect but may have allowed the presence of a greater number of parasites in the blood. However, histopathological analysis from heart suggests a cardioprotective effect of Epo on Chagas disease.Support or Funding InformationThis work was funded by FAPDFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vikings in the South: Voyages to Iberia and the Mediterranean, by Ann Christys

Ann Christys has provided a tremendous service to students of the Viking Age with this short book, which aims to integrate viking activity in the South with the much better known theatres of war and peaceful exchange in western Europe, Byzantium, and the East. Christys’s knowledge of early medieval Iberia and its Arabic writers, Latin charters and Romance chronicles is not shared by most students of the viking world, and her study is a very welcome addition to the field. While vikings made themselves ‘part of the history of Iberia’ (p. 95), details of what they did, and where, have been elusive. Christys lays out what is known: that Northmen raided the northern and western coasts of the Iberian peninsula, attacked Lisbon and Seville, and may have threatened the Umayyad capital at Cordoba; studies of mouse DNA (sic) suggest that their ships travelled as far as Madeira (p. 7). The most significant episodes seem to have taken place in 844–5, 859–62 and the 960s. Motives are uncertain: in contrast to other regions, there is no archaeology of trade (in the form of camps or trading sites, for example) and no physical or onomastic evidence of more than ephemeral settlement. Christys wonders whether slaves were in fact the greatest draw (pp. 11–12). Her verdict is that viking activity was ‘probably small-scale, although perhaps more frequent than our sources admit’ (p. 95).

Graphdiyne Nanoparticles with High Free Radical Scavenging Activity for Radiation Protection.

Numerous carbon networks materials comprised of benzene moieties, such as graphene and fullerene, have held great fascination for radioprotection because of their acknowledged good biocompatibility and strong free radical scavenging activity derived from their delocalized π-conjugated structure. Recently, graphdiyne, a new emerging carbon network material consisting of a unique chemical structure of benzene and acetylenic moieties, has gradually attracted attention in many research fields. Encouraged by its unique structure with strong conjugated π-system and highly reactive diacetylenic linkages, graphdiyne might have free radical activity and can thus be used as a radioprotector, which has not been investigated so far. Herein, for the first time, we synthesized bovine serum albumin (BSA)-modified graphdiyne nanoparticles (graphdiyne-BSA NPs) to evaluate their free radical scavenging ability and investigate their application for radioprotection both in cell and animal models. In vitro studies indicated that the graphdiyne-BSA NPs could effectively eliminate the free-radicals, decrease radiation-induced DNA damage in cells, and improve the viability of cells under ionizing radiation. In vivo experiments showed that the graphdiyne-BSA NPs could protect the bone marrow DNA of mice from radiation-induced damage and make the superoxide dismutase (SOD) and malondialdehyde (MDA) (two kinds of vital indicators of radiation-induced injury) recover back to normal levels. Furthermore, the good biocompatibility and negligible systemically toxicity responses of the graphdiyne-BSA NPs to mice were verified. All these results manifest the good biosafety and radioprotection activity of graphdiyne-BSA NPs to normal tissues. Therefore, our studies not only provide a new radiation protection platform based on graphdiyne for protecting normal tissues from radiation-caused injury but also provide a promising direction for the application of graphdiyne in the biomedicine field.

Endosomal Toll-Like Receptors (TLRs) mediate enhancement of IL-17A production triggered by Epstein-Barr virus (EBV) DNA in mice.

EBV has long-been associated with autoimmune disorders. We have previously demonstrated that EBV DNA increases the production of IL-17A in mice. This property may play a role in the association of EBV with autoimmune diseases. The objective of this study was to elucidate mechanisms through which EBV DNA modulates IL-17A levels in mice. To study the potential role of endosomal receptors in detecting EBV DNA, chloroquine, an endosomal maturation inhibitor, was used to treat mouse peripheral blood mononuclear cells (PBMCs) in the presence or absence of EBV DNA. IL-17A levels were then assessed by ELISA. Subsequently, to determine whether TLR3, 7 or 9 played a role in this pathway, specific inhibitors were used for these TLRs both in mouse PBMCs and in vivo in BALB/c mice treated with the viral DNA; IL-17A levels were then similarly assessed. IL-17A production was enhanced from mouse PBMCs cultured with EBV DNA; pre-incubation of PBMCs with chloroquine significantly reduced its production. When cells were cultured with EBV DNA and a TLR3, 7 or 9 inhibitor, a significant decrease in IL-17A levels was detected. A similar decrease in the EBV DNA-triggered IL-17A production in mice was observed when animals were treated with the TLR inhibitors. Endosomal TLRs appear to be involved in recognizing EBV DNA and subsequently triggering IL-17A production in mice. Targeting these receptors in EBV positive subjects with autoimmunity may be useful pending investigations assessing whether they play a similar role in humans.

Computational approach to discriminate human and mouse sequences in patient-derived tumour xenografts

BackgroundPatient-Derived Tumour Xenografts (PDTXs) have emerged as the pre-clinical models that best represent clinical tumour diversity and intra-tumour heterogeneity. The molecular characterization of PDTXs using High-Throughput Sequencing (HTS) is essential; however, the presence of mouse stroma is challenging for HTS data analysis. Indeed, the high homology between the two genomes results in a proportion of mouse reads being mapped as human.ResultsIn this study we generated Whole Exome Sequencing (WES), Reduced Representation Bisulfite Sequencing (RRBS) and RNA sequencing (RNA-seq) data from samples with known mixtures of mouse and human DNA or RNA and from a cohort of human breast cancers and their derived PDTXs. We show that using an In silico Combined human-mouse Reference Genome (ICRG) for alignment discriminates between human and mouse reads with up to 99.9% accuracy and decreases the number of false positive somatic mutations caused by misalignment by >99.9%. We also derived a model to estimate the human DNA content in independent PDTX samples. For RNA-seq and RRBS data analysis, the use of the ICRG allows dissecting computationally the transcriptome and methylome of human tumour cells and mouse stroma. In a direct comparison with previously reported approaches, our method showed similar or higher accuracy while requiring significantly less computing time.ConclusionsThe computational pipeline we describe here is a valuable tool for the molecular analysis of PDTXs as well as any other mixture of DNA or RNA species.

AGK2, A SIRT2 Inhibitor, Inhibits Hepatitis B Virus Replication In Vitro And In Vivo.

Sirtuin 2 (SIRT2) is a nicotinamide adenine dinucleotide (NAD +)-dependent class III histone deacetylase. We have reported that HBx (hepatitis B virus X protein)-elevated SIRT2 promotes HBV replication and hepatocarcinogenesis. However, the potential anti-HBV effect of AGK2, a selective inhibitor of SIRT2, has not been reported. Here, the role of AGK2 on HBV replication was examined in the HepAD38 and HepG2-NTCP cell lines. The HBV genome was stably integrated in HepAD38 cell line which expresses HBV under the control of tetracycline. The HepG2-NTCP cells expressing the sodium taurocholate cotransporting polypeptide (NTCP) receptor are susceptible to HBV infection. We found that AGK2 exhibited a robust anti-HBV activity with minimal hepatotoxicity. AGK2 inhibited the expression of HBV total and 3.5kb RNAs, DNA replicative intermediates and HBV core protein (HBc). Moreover, AGK2 treatment suppressed the secretion of the hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg). Importantly, AGK2 treatment inhibited serum HBV DNA, HBeAg and HBsAg levels as well as hepatic HBV DNA, RNA and HBc in the HBV transgenic mice. The results indicated that AGK2, as a SIRT2 inhibitor, might be a new therapeutic option for controlling HBV infection.

DDM1 and Lsh remodelers allow methylation of DNA wrapped in nucleosomes.

Cytosine methylation regulates essential genome functions across eukaryotes, but the fundamental question of whether nucleosomal or naked DNA is the preferred substrate of plant and animal methyltransferases remains unresolved. Here, we show that genetic inactivation of a single DDM1/Lsh family nucleosome remodeler biases methylation toward inter-nucleosomal linker DNA in Arabidopsis thaliana and mouse. We find that DDM1 enables methylation of DNA bound to the nucleosome, suggesting that nucleosome-free DNA is the preferred substrate of eukaryotic methyltransferases in vivo. Furthermore, we show that simultaneous mutation of DDM1 and linker histone H1 in Arabidopsis reproduces the strong linker-specific methylation patterns of species that diverged from flowering plants and animals over a billion years ago. Our results indicate that in the absence of remodeling, nucleosomes are strong barriers to DNA methyltransferases. Linker-specific methylation can evolve simply by breaking the connection between nucleosome remodeling and DNA methylation.

Usability of human Infinium MethylationEPIC BeadChip for mouse DNA methylation studies

BackgroundThe advent of array-based genome-wide DNA methylation methods has enabled quantitative measurement of single CpG methylation status at relatively low cost and sample input. Whereas the use of Infinium Human Methylation BeadChips has shown great utility in clinical studies, no equivalent tool is available for rodent animal samples. We examined the feasibility of using the new Infinium MethylationEPIC BeadChip for studying DNA methylation in mouse.ResultsIn silico, we identified 19,420 EPIC probes (referred as mEPIC probes), which align with a unique best alignment score to the bisulfite converted reference mouse genome mm10. Further annotation revealed that 85% of mEPIC probes overlapped with mm10.refSeq genes at different genomic features including promoters (TSS1500 and TSS200), 1st exons, 5′UTRs, 3′UTRs, CpG islands, shores, shelves, open seas and FANTOM5 enhancers. Hybridization of mouse samples to Infinium Human MethylationEPIC BeadChips showed successful measurement of mEPIC probes and reproducibility between inter-array biological replicates. Finally, we demonstrated the utility of mEPIC probes for data exploration such as hierarchical clustering.ConclusionsGiven the absence of cost and labor convenient genome-wide technologies in the murine system, our findings show that the Infinium MethylationEPIC BeadChip platform is suitable for investigation of the mouse methylome. Furthermore, we provide the “mEPICmanifest” with genomic features, available to users of Infinium Human MethylationEPIC arrays for mouse samples.

A virus-like particle of the hepatitis B virus preS antigen elicits robust neutralizing antibodies and T cell responses in mice

The preS antigen of hepatitis B virus (HBV) corresponds to the N-terminal polypeptide in the large (L) antigen in addition to the small (S) antigen. The virus-like particle (VLP) of the S antigen is widely used as a vaccine to protect the population from HBV infection. The presence of the S antigen and its antibodies in patient blood has been used as markers to monitor hepatitis B. However, there is very limited knowledge about the preS antigen. We generated a preS VLP that is formed by a chimeric protein between preS and hemagglutinin (HA), and the matrix protein M1 of influenza virus. The HBV preS antigen is displayed on the surface of preS VLP. Asn112 and Ser98 of preS in VLP were found to be glycosylated and O-glycosylation of Ser98 has not been reported previously. The preS VLP shows a significantly higher immunogenicity than recombinant preS, eliciting robust anti-preS neutralizing antibodies. In addition, preS VLP is also capable of stimulating preS-specific CD8+ and CD4+ T cell responses in Balb/c mice and HBV transgenic mice. Furthermore, preS VLP immunization provided protection against hydrodynamic transfection of HBV DNA in mice. The data clearly suggest that this novel preS VLP could elicit robust immune responses to the HBV antigen, and can be potentially developed into prophylactic and therapeutic vaccines.

A simple and sensitive two-tube multiplex PCR assay for simultaneous detection of ten meat species

A sensitive and reliable technique for meat species identification is required to prevent food adulteration, particularly in meat production. This work developed an optimized multiplex PCR assay for simultaneous identification of five commonly consumed and five commonly banned meat species in meat products. We designed primers that specifically amplified mitochondrial ATPase subunit 8 gene regions of different lengths of bovine, ovine, swine, chicken, turkey, cat, dog, mouse and human DNAs. The developed multiplex PCR assay proved to be specific, sensitive down to 30pg DNA per reaction, reproducible and economical. It could be used with a variety of raw meats and processed food samples and is easily applicable in a routine laboratory analysis without specific sophisticated equipment.

A novel orally available small molecule that inhibits hepatitis B virus expression

The hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834). RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir. Unlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09 log10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice. We have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues. We discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses.

Diet affects epigenetics of aging

Explorers may have stopped seeking the mythical fountain of youth, but biologists have found one practice that can increase longevity across multiple species: calorie restriction. Eating a nutritionally complete, reduced-calorie diet is linked to increased life span in mice and monkeys. However, the mechanism by which calorie restriction promotes longevity has eluded scientists. New research suggests that the mechanism may involve changing patterns of DNA methylation. Over time, our cells add and remove methyl groups to and from cytosine nucleotide bases, an epigenetic process called methylation drift. The link between age-related methylation drift and age-related diseases, such as cancer and diabetes, prompted a team of geneticists to investigate the role methylation plays in aging and calorie restriction across species. Led by Jean-Pierre Issa at Temple University, the team used powerful deep-sequencing analyses to measure methylation drift in the DNA of mice, rhesus macaques, and humans. They also assessed the degree