Low DNA Concentrations Research Articles

CGAS is activated by DNA in a length-dependent manner.

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP-AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length-dependent manner. This is observed over a wide length-span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, in vitro studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length-dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.

Mechanical Contact Characteristics of PC3 Human Prostate Cancer Cells on Complex-Shaped Silicon Micropillars.

In this study we investigated the contact characteristics of human prostate cancer cells (PC3) on silicon micropillar arrays with complex shapes by using high-resolution confocal fluorescence microscopy techniques. These arrays consist of micropillars that are of various cross-sectional geometries which produce different deformation profiles in adherent cells. Fluorescence micrographs reveal that some DAPI (4′,6-diamidino-2-phenylindole)-stained nuclei from cells attached to the pillars develop nanometer scale slits and contain low concentrations of DNA. The lengths of these slits, and their frequency of occurrence, were characterized for various cross-sectional geometries. These DNA-depleted features are only observed in locations below the pillar’s top surfaces. Results produced in this study indicate that surface topography can induce unique nanometer scale features in the PC3 cell.

Abstract 5401: Assessment of whole genome amplification for whole exome sequencing in detecting genetic mutation

Abstract Whole exome sequencing (WES) has been widely used for studying genetic mutations in DNA coding regions to elucidate cancer etiology and for identifying biomarkers to optimize chemotherapy. However, these studies can be limited when only small amount of DNA is available. Whole genome amplification (WGA) technology can be combined with WES to make these studies technically possible. Here, we evaluated WGA using a phi 29 polymerase prior to library preparation for WES in samples with various DNA concentrations. WES was performed by targeted exon amplification followed by massively parallel sequencing. We compared the base calls of single nucleotide variants for individual same samples with or without WGA prior to library construction to determine the concordance rate of variant calls. We also assessed genetic variant call rate in the same samples with or without WGA. The number of variants obtained ranged from 159,851,999 to 390,784 in the non-amplified samples and from 353,215 to 384,118 in the same amplified samples. The average concordance rate of identical variants in the samples with or without WGA was 96.5%. Our results indicate that WGA in prior to WES can increase detection efficiency of single nucleotide variants in samples with relatively small amount of DNA and/or low DNA concentration. Citation Format: Crystal Xue, Laura Gardner, Guanglong Jiang, Fei Shen, Bryan Schneider. Assessment of whole genome amplification for whole exome sequencing in detecting genetic mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5401. doi:10.1158/1538-7445.AM2017-5401

Abstract 4376: Next-generation sequencing of gastric cancer samples from Rwanda: a feasibility study

Abstract Background: Gastric cancer is associated with high morbidity and mortality around the world. In developing countries, patients often present with advanced disease creating a challenge for effective treatment. Next generation sequencing is identifying mutations in more than 50% of gastric cancers in developed countries. Chronic infections and other geographic differences may lead to variations in somatic mutations that could lead to different approaches to treatment. In this pilot study we sought to determine if an overseas collaboration between the University of Rwanda College of Medicine and Health Sciences and the Geisel School of Medicine at Dartmouth was feasible and effective at determining molecular profiles of gastric cancer. Methods: Patients referred for endoscopy at University Teaching Hospital of Kigali, Rwanda were consented to participate prior to the procedure. Biopsy tissues were processed for routine pathologic assessment and those confirmed as gastric adenocarcinoma were included. Formalin-fixed paraffin embedded tissues, H&E stained slides and tissue blocks were transported from Rwanda to New Hampshire, USA by visiting professors participating in the Human Resources for Health Program between May 2015 and July 2016. DNA and RNA from 39 samples were extracted using at least 7 FFPE unstained slides according to AllPrep RNA/DNA FFPE Kit Protocol. Library preparation was performed using 5 ng gDNA using the Pillar NGS SLIMampTM Lung and Colon Hot Spot Panel. Library quanitification was performed using Qubit, and samples with at least 4 nM were normalized, pooled and sequenced on the v3 cartridge on the Illumina's MiSeq system. For data analysis, FASTq files were uploaded to Pillar Biosciences, where sequence alignment, annotation, and variant classification were performed. Results: Eighteen samples had insufficient tissue for nucleic acid extraction. Of the 39 samples processed for sequencing, 17 had low library quantification due to low DNA concentration or poor quality. Mutations were detected in 9/22 samples (41%). One sample contained 3 mutations while the majority of mutated cases had 1-2 mutations identified. A total of 12 mutations were identified: TP53 (4), SMAD4 (2), ERBB4 (2) (S341L and D228N), PTEN (2)(K267RfsTer9 and C136Y), FBXW7 (1) and KRAS (1). Conclusions: Overseas collaboration for next generation sequencing of gastric cancer samples is feasible. Quality control is a common challenge and may improve with larger sized biopsies or tissue blocks. Frequency of mutations is lower than expected. ERBB4 mutations identified are not known to be pathogenic. The PTEN mutations observed are considered pathogenic and this may be a feasible target for therapy. A fusion panel and amplification profiling may be more effective in identifying potential targets for therapeutic trials. Limitations: Small sample size. Panel limited to mutations. Citation Format: Mary D. Chamberlin, Francine B. De Abreu, Torrey L. Gallagher. Next-generation sequencing of gastric cancer samples from Rwanda: a feasibility study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4376. doi:10.1158/1538-7445.AM2017-4376

Rapid and annealing-free self-assembly of DNA building blocks for 3D hydrogel chaperoned by cationic comb-type copolymers

The construction and self-assembly of DNA building blocks are the foundation of bottom-up development of three-dimensional DNA nanostructures or hydrogels. However, most self-assembly from DNA components is impeded by the mishybridized intermediates or the thermodynamic instability. To enable rapid production of complicated DNA objects with high yields no need for annealing process, herein different DNA building blocks (Y-shaped, L- and L′-shaped units) were assembled in presence of a cationic comb-type copolymer, poly (L-lysine)-graft-dextran (PLL-g-Dex), under physiological conditions. The results demonstrated that PLL-g-Dex not only significantly promoted the self-assembly of DNA blocks with high efficiency, but also stabilized the assembled multi-level structures especially for promoting the complicated 3D DNA hydrogel formation. This study develops a novel strategy for rapid and high-yield production of DNA hydrogel even derived from instable building blocks at relatively low DNA concentrations, which would endow DNA nanotechnology for more practical applications.

Low concentration DNA extraction and recovery using a silica solid phase.

DNA extraction from clinical samples is commonly achieved with a silica solid phase extraction column in the presence of a chaotrope. Versions of these protocols have been adapted for point of care (POC) diagnostic devices in miniaturized platforms, but commercial kits require a high amount of input DNA. Thus, when the input clinical sample contains less than 1 μg of total DNA, the target-specific DNA recovery from most of these protocols is low without supplementing the sample with exogenous carrier DNA. In fact, many clinical samples used in the development of POC diagnostics often exhibit target DNA concentrations as low as 3 ng/mL. With the broader goal of improving the yield and efficiency of nucleic acid-based POC devices for dilute samples, we investigated both DNA adsorption and recovery from silica particles by using 1 pg– 1 μg of DNA with a set of adsorption and elution buffers ranging in pH and chaotropic presence. In terms of adsorption, we found that low pH and the presence of chaotropic guanidinium thiocyanate (GuSCN) enhanced DNA-silica adsorption. When eluting with a standard low-salt, high-pH buffer, > 70% of DNA was unrecoverable, except when DNA was initially adsorbed with 5 M GuSCN at pH 5.2. Unrecovered DNA was either not initially adsorbed or irreversibly bound on the silica surface. Recovery was improved when eluting with 95°C formamide and 1 M NaOH, which suggested that DNA-silica-chaotrope interactions are dominated by hydrophobic interactions and hydrogen bonding. While heated formamide and NaOH are non-ideal elution buffers for practical POC devices, the salient results are important for engineering a set of optimized reagents that could maximize nucleic acid recovery from a microfluidic DNA-silica-chaotrope system.

Elastic turbulence in entangled semi-dilute DNA solutions measured with optical coherence tomography velocimetry

The flow instabilities of solutions of high molecular weight DNA in the entangled semi-dilute concentration regime were investigated using optical coherence tomography velocimetry, a technique that provides high spatial (probe volumes of 3.4 pL) and temporal resolution (sub μs) information on the flow behaviour of complex fluids in a rheometer. The velocity profiles of the opaque DNA solutions (high and low salt) were measured as a function of the distance across the gap of a parallel plate rheometer, and their evolution over time was measured. At lower DNA concentrations and low shear rates, the velocity fluctuations were well described by Gaussian functions and the velocity gradient was uniform across the rheometer gap, which is expected for Newtonian flows. As the DNA concentration and shear rate were increased there was a stable wall slip regime followed by an evolving wall slip regime, which is finally followed by the onset of elastic turbulence. Strain localization (shear banding) is observed on the boundaries of the flows at intermediate shear rates, but decreases in the high shear elastic turbulence regime, where bulk strain localization occurs. A dynamic phase diagram for non-linear flow was created to describe the different behaviours.

Detection of fetal epigenetic biomarkers through genome-wide DNA methylation study for non-invasive prenatal diagnosis.

The discovery of cell-free DNA fetal (cff DNA) in maternal plasma during pregnancy provides a novel perspective for the development of non-invasive prenatal diagnosis (NIPD). Against the background of maternal DNA, the use of the relatively low concentration of cff DNA is limited in NIPD. Therefore, in order to overcome the complication of the background of maternal DNA and expand the scope of cff DNA application in clinical practice, it is necessary to identify novel universal fetal-specific DNA markers. The GeneChip Human Promoter 1.0R Array set was used in the present study to analyze the methylation status of 12 placental tissue and maternal peripheral blood whole-genome DNA samples. In total, 5 fetus differential hypermethylation regions and 6 fetus differential hypomethylation regions were identified. In order to verify the 11 selected methylation regions and detect the differential CpG sites in these regions, a bisulfate direct sequencing strategy was used. In total, 87 fetal differential methylation CpG sites were identified from 123 CpG sites. The detection of fetal differential methylation DNA regions and CpG sites may be instrumental in the development of efficient NIPD and in the expansion of its application in other disorders.

The complete mitogenome of Orcula dolium (Draparnaud, 1801); ultra-deep sequencing from a single long-range PCR using the Ion-Torrent PGM

BackgroundWith the increasing capacity of present-day next-generation sequencers the field of mitogenomics is rapidly changing. Enrichment of the mitochondrial fraction, is no longer necessary for obtaining mitogenomic data. Despite the benefits, shotgun sequencing approaches also have disadvantages. They do not guarantee obtaining the complete mitogenome, generally require larger amounts of input DNA and coverage is low compared to sequencing with enrichment strategies. If the mitogenome could be amplified in a single amplification, additional time and costs for sample preparation might outweigh these disadvantages.ResultsA sequence of the complete mitochondrial genome of the pupilloid landsnail Orcula dolium is presented. The mitogenome was amplified in a single long-range (LR) PCR and sequenced on an Ion Torrent PGM (Life Technologies). The length is 14,063 nt and the average depth of coverage is 1112 X. This is the first published mitogenome for a member of the family Orculidae. It has the typical metazoan makeup of 13 protein coding genes (PCGs), 2 ribosomal RNAs (12S and 16S) and 22 transfer RNAs (tRNAs). Orcula is positioned between Pupilla and the Vertiginidae as the sister-group of Gastrocopta and Vertigo, together. An ancestral gene order reconstruction shows that Orthurethra in contrast to other Stylommatophora, have tRNA-H before tRNA-G and that the gene order in the ‘non-achatinoid’ clade is identical to that of closely related non-stylommatophoran taxa.ConclusionsWe show it is feasible to ultra-deep sequence a mitogenome from a single LR-PCR. This approach is particularly relevant to studies that have low concentrations of input DNA. It results in a more efficient use of NGS capacity (only the targeted fraction is sequenced) and is an effective selection against nuclear mitochondrial inserts (NUMTS). In contrast to previous studies based in particular on 28S, our results indicate that phylogeny reconstructions based on complete mitogenomes might be more suitable to resolve deep relationships within Stylommatophora. Ancestral gene order reconstructions reveal rearrangements that characterize systematic groups.

Evaluation of assays for quantification of DNA in canine plasma as an indirect marker of NETosis.

Neutrophil extracellular traps (NET), consisting of a filamentous DNA/chromatin-histone scaffold originating from neutrophils are part of the innate immune response, may be released under a variety of inflammatory conditions and are associated with an increased risk for thrombosis. The purpose of this study was to evaluate a SYTOX green fluorescence assay and a histone-DNA complex (hisDNA) ELISA for quantification of NET-related DNA in canine plasma. The influence of variations in blood sample handling on assay results was tested. Accuracy of the SYTOX green fluorescence and the hisDNA ELISA was evaluated with dilutional linearity using serial dilutions. Interference was assessed by addition of purified bilirubin or hemoglobin. Precision was determined by calculating the intra- and inter-assay CV. Preanalytic sample handling did not influence DNA measurements by either assay. Citrate and EDTA plasma samples were equivalent. For the DNA fluorescence assay, dilutional linearity was poor due to autofluorescence, which was corrected by addition of canine plasma to the diluent. The presence of bilirubin and hemoglobin also increased autofluorescence, and resulted in falsely low concentrations of DNA. On the hisDNA ELISA, pigmentemia had no effect. Both assays as modified in this study are suitable for measuring DNA in canine EDTA or citrate plasma. However, performance of the fluorescence assay was impacted by pigmentemia, and it was less sensitive than the ELISA in detecting the presence of nucleosome material in the plasma.

A fast and efficient purification platform for cell-based influenza viruses by flow-through chromatography

Since newly emerging influenza viruses with pandemic potentials occurred in recent years, the demand for producing pandemic influenza vaccines for human use is high. For the development of a quick and efficient vaccine production, we proposed an efficient purification platform from the harvest to the purified bulk for the cell-based influenza vaccine production. This platform based on flow-through chromatography and filtration steps and the process only involves a few purification steps, including depth filtration, inactivation by formaldehyde, microfiltration, ultrafiltration, anion-exchange and ligand-core chromatography and sterile filtration. In addition, in the proposed chromatography steps, no virus capture steps were employed, and the purification results were not affected by the virus strain variation, host cells and culturing systems. The results from different virus strains which produced by Vero or MDCK cells in different culturing systems also obtained 33–46% HA recovery yields by this platform. The overall removal rates of the protein and DNA concentration in the purified bulk were over 96.1% and 99.7%, respectively. The low residual cellular DNA concentrations were obtained ranged from 30 to 130pg per human dose (15µg/dose). All influenza H5N1 purified bulks met the regulatory requirements for human vaccine use.

A Small Regulatory RNA Contributes to the Preferential Colonization of Escherichia coli O157:H7 in the Large Intestine in Response to a Low DNA Concentration.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 (O157) is one of the most notorious human pathogens, causing severe disease in humans worldwide. O157 specifically colonizes the large intestine of mammals after passing through the small intestine, and this process is influenced by differential signals between the two regions. Small regulatory RNAs (sRNAs) are able to sense and respond to environmental changes and regulate diverse physiological processes in pathogenic bacteria. Although some sRNAs of O157 have been extensively investigated, whether these molecules can sense differences between the small and large intestine and influence the preferential colonization in the large intestine by O157 remains unknown. In this study, we identified a new sRNA, Esr055, in O157 which senses the low DNA concentration in the large intestine and contributes to the preferential colonization of the bacteria in this region. The number of O157 wild-type that adhered to the colon is 30.18 times higher than the number that adhered to the ileum of mice, while the number of the ΔEsr055 mutant that adhered to the colon decreased to 13.27 times higher than the number adhered to the ileum. Furthermore, we found that the expression of Esr055 is directly activated by the regulator, DeoR, and its expression is positively affected by DNA, which is significantly more abundant in the ileum than in the colon of mice. Additionally, combining the results of informatics predictions and transcriptomic analysis, we found that several virulence genes are up-regulated in the ΔEsr055 mutant and five candidate genes (z0568, z0974, z1356, z1926, and z5187) may be its direct targets.

Using cytochrome b to identify nests and museum specimens of cryptic songbirds

Understanding demography of vertebrate populations requires quantifying reproductive success. For taxa that are difficult to distinguish, estimates of reproductive success can be biased if species are misidentified or if breeding concludes before identification is confirmed. We surveyed desert grasslands where three species of cryptic sparrows breed: Peucaea botterii, P. cassinii, and Aimophila ruficeps (Emberizidae). Nests, eggs, and nestlings of these species are similar, making it difficult to differentiate nests without observing adults, which can be challenging. We collected seven types of material from nests for DNA analysis: maternal cells from exterior surfaces of unhatched eggs, epithelial cells from the oropharyngeal cavity of nestlings, eggshells, feathers, feather sheaths, feces, and fecal sacs. From these materials, we amplified and sequenced a fragment of the diagnostic locus, mitochondrial cytochrome b (cyt b), and analyzed the data in a phylogenetic framework to classify nests to species. We validated our classification by sequencing the same locus from feathers of museum specimens. Overall, 72% of samples from nests yielded high-quality sequences. We identified to species 44 of 51 nests and identified museum specimens with archival ages of up to 47 years. Our study extends previous research by demonstrating the efficacy of standard kits, inexpensive reagents, low DNA concentrations, and diverse materials in classifying nests of grassland sparrows. Compared to more invasive methods of acquiring DNA, the approaches we describe are less likely to affect nesting behavior and bias estimates of nesting success of birds, issues especially important for species of conservation concern.

Optimization of DNA extraction for advancing coral microbiota investigations

BackgroundDNA-based sequencing approaches are commonly used to identify microorganisms and their genes and document trends in microbial community diversity in environmental samples. However, extraction of microbial DNA from complex environmental samples like corals can be technically challenging, and extraction methods may impart biases on microbial community structure. MethodsWe designed a two-phase study in order to propose a comprehensive and efficient method for DNA extraction from microbial cells present in corals and investigate if extraction method influences microbial community composition. During phase I, total DNA was extracted from seven coral species in a replicated experimental design using four different MO BIO Laboratories, Inc., DNA Isolation kits: PowerSoil®, PowerPlant® Pro, PowerBiofilm®, and UltraClean® Tissue & Cells (with three homogenization permutations). Technical performance of the treatments was evaluated using DNA yield and amplification efficiency of small subunit ribosomal RNA (SSU ribosomal RNA (rRNA)) genes. During phase II, potential extraction biases were examined via microbial community analysis of SSU rRNA gene sequences amplified from the most successful DNA extraction treatments.ResultsIn phase I of the study, the PowerSoil® and PowerPlant® Pro extracts contained low DNA concentrations, amplified poorly, and were not investigated further. Extracts from PowerBiofilm® and UltraClean® Tissue and Cells permutations were further investigated in phase II, and analysis of sequences demonstrated that overall microbial community composition was dictated by coral species and not extraction treatment. Finer pairwise comparisons of sequences obtained from Orbicella faveolata, Orbicella annularis, and Acropora humilis corals revealed subtle differences in community composition between the treatments; PowerBiofilm®-associated sequences generally had higher microbial richness and the highest coverage of dominant microbial groups in comparison to the UltraClean® Tissue and Cells treatments, a result likely arising from using a combination of different beads during homogenization.ConclusionsBoth the PowerBiofilm® and UltraClean® Tissue and Cells treatments are appropriate for large-scale analyses of coral microbiota. However, studies interested in detecting cryptic microbial members may benefit from using the PowerBiofilm® DNA treatment because of the likely enhanced lysis efficiency of microbial cells attributed to using a variety of beads during homogenization. Consideration of the methodology involved with microbial DNA extraction is particularly important for studies investigating complex host-associated microbiota.

Protective effects of leucine on redox status and mitochondrial-related gene abundance in the jejunum of intrauterine growth-retarded piglets during early weaning period

ABSTRACTThe aim of this study was to investigate the effects of dietary supplementation with 0.35% l-leucine on redox status and gene abundance relating to mitochondrial biogenesis and function in the jejunum of intrauterine growth-retarded (IUGR) piglets during early weaning period. According to a 2 × 2 factorial arrangement, 16 IUGR and 16 normal body weight (NBW) piglets were fed a basal diet without l-leucine supplementation or a basal diet plus 0.35% l-leucine supplementation from the age of 14 to 35 d. The results showed that compared with NBW piglets, IUGR piglets had a lower (p < 0.05) jejunal DNA concentration, a reduced (p < 0.05) manganese superoxide dismutase (MnSOD) and total antioxidant capability (T-AOC) activities and mitochondrial DNA content in the jejunum. Leucine supplementation increased (p < 0.05) MnSOD and T-AOC activities and decreased (p < 0.05) the malondialdehyde content in the jejunum of IUGR piglets. The mRNA gene abundance of nuclear respiratory factor-1 (NRF1), mitochondrial transcription factor A (TFAM), ATP synthase (ATPs), cytochrome c oxidase V (CcOX V), cytochrome c and glucokinase in the jejunum of IUGR piglets was reduced (p < 0.05) compared with NBW piglets. However, NRF1, peroxisome proliferation-activated receptor gamma coactivator-1 alpha, TFAM, ATPs and CcOX I mRNA gene abundance in the jejunum of IUGR piglets were increased (p < 0.05) by diets supplemented with leucine. These data indicate that leucine supplementation has therapeutic potential for attenuating intestinal oxidative stress and mitochondrial dysfunction in IUGR piglets during the early period of life via increasing enzyme activities and up-regulating mRNA gene abundance.

Rescue PCR: Reagent-rich PCR recipe improves amplification of degraded DNA extracts

Minimizing the inadvertent co-extraction of polymerase chain reaction (PCR) inhibitors and/or subduing their influence are two of the most pervasive challenges in the study of ancient DNA (aDNA). Some commonly employed methods to circumvent inhibition include dilution of DNA extracts and/or removal of inhibitors via silica-based treatments. While these methods have been shown to be effective, they may not be useful for all aDNA extracts. Samples with very low copy number, for instance, may not benefit from such methods, as dilutions lower DNA concentration in tandem with the inhibitors, and some DNA loss is expected to follow silica-based treatments. Therefore, the development of additional options to overcome PCR inhibition is at a premium. In this study, we present evidence that a reagent-rich PCR protocol, where all reagents are increased in equal relative proportion can increase amplification success when DNA concentration is reduced relative to inhibitors. The reagent-rich PCR recipe, termed rescue PCR, increased amplification success by 51% for the 112 extracts used in the study. Rescue PCR represents a simple and robust addition to the suite of options currently available to work with DNA in the presence of inhibition, especially ancient, degraded, and low copy number DNA extracts.

Direct Detection of Low Concentration DNA in High Ionic Strength Solution with AlGaN/GaN High Electron Mobility Transistors

In this research, the direct detection of DNA sequences in physiological salt concentration by FET based biosensor has been investigated. FET having high transconductance gain, can significantly improve the S/N ratio and sensitivity. As the complementary DNA concentration, ranging from 1fM to 100fM, is introduced, the current gain of the device changes accordingly. The sensor can efficiently detect target DNA down to a low concentration of 1 fM.

Genetic aspects of HPV infection detection in tumor and adjacent tissues of patients with laryngeal squamous cell carcinoma.

The article describes methods for the human papillomavirus (HPV) detection in tumor and adjacent (morphologically intact) tissues of patients with laryngeal squamous cell carcinoma (LSSC) in terms of viral pathogenesis. Comparative evaluation of the principles and techniques for HPV detection was performed. Advantages and disadvantages of the HPV detection methods are described. Approaches for DNA and HPV oncoproteins E6-E7 identification are substantiated. The results of our research into the qualitative and quantitative detection of HPV in the tumor and adjacent tissues of patients with Lssc are described. The research was conducted using commercial test systems Amplisens HPV HR screen-titre-FL and Amplisens HPV HR genotype-FL. Based on these results we developed the algorithm of HPV detection in samples of tumor tissue of patients with Lssc. The need for typing HPV-positive tissue samples with low concentration of HPV DNA was discussed.

Whole Metagenome Analysis of Extreme Environment Samples by Quasi-Linear Whole Genome Amplification

Microbiomes are gaining a lot of importance owing to its impact in the human health and other life processes. The significance of microbes in the deep sea is often undermined but it holds the key for the environmental changes that affects our planet. Recently scientists are investigating the diversity of microbial life in the sea to learn its role in the ocean processes, its interactions with other marine life, and its importance to humans. Emergence of cutting edge genomics and molecular biology tools has propelled the field of metagenomics to study the microbial populations without the need to culture them. In spite of these growth studies of extreme environmental samples with very low nucleic acid concentration remains still a major challenge. A whole genome amplification method with less bias is required to get over this problem. Commercially available single cell whole genome amplification kits don’t perform well with the low concentration of samples. A minimum of 100ng of DNA is the input requirement for these kits and some of the extreme environment samples can only yield 1-10ng of DNA. Our aim was to devise an efficient and cost effective workflow that could work with very low concentration of metagenomic DNA. We have adopted a quasi-linear whole genome amplification methodology, Multiple Annealing and Looping Based Amplification Cycles (MALBAC) that was earlier used in the single cell genomic studies.MALBAC methodology was carried out using 4ng of the deep sea sediment DNA and it was compared against the NEB Next Ultra DNA Library prep kit using 125ng of deep sea sediment DNA. The data generated were analysed and compared for the microbial diversity and gene function annotations. The results obtained through MALBAC methodology were comparable to that of the commercial kit based procedure utilizing nearly 125ng of DNA.The MALBAC based whole metagenome analysis of deep sea sample from Arabian Sea revealed the immense microbial diversity of unexplored extreme environment. The study also identified the functional genes coding for valuable enzymes and proteins that could be used in the biotech industry. This study has showed that MALBAC is a cost effective procedure for extreme environment samples containing very low concentration of DNA.

A Novel Function of a CXC-Type Chemokine CXCL14 As a Specific Carrier of CpG DNA into Dendritic Cells for Activating Toll-like Receptor 9-Mediated Adaptive Immunity

CXCL14 is a primordial CXC-type chemokine that induces migration of immature dendritic cells (DCs), tissue resident macrophages, and natural killer cells. It has been reported that CXCL14 plays multiple roles in tumor suppression, exacerbation of autoimmune arthritis, and induction of obesity-associated insulin resistance. However, underlying molecular mechanisms of these phenomena remain to be elucidated. Although CXCL14 binds to the CXCL12 receptor CXCR4 with high affinity, chemoattractive activity of CXCL14 is much weaker than CXCL12.In this study, we newly discovered that CXCL14 specifically binds to CpG DNA and activates Toll-like receptor 9 (TLR9), thereby inducing inflammatory cytokines such as IL-6, IL-12 p40, and TNFa in mouse bone marrow-derived dendritic cells (BMDCs). Cell surface expression levels of MHC class-II and CD86 in BMDCs were also enhanced by the combination of CpG DNA and CXCL14. In this experimental setting, CXCL14 interacted with certain classes of CpG DNA, but not with RNA ligands for TLR3 and TLR8. In addition, this CpG DNA-cooperative activity was not present in CXCL8 and CXCL12, excluding a nonspecific interaction between CpG DNA and an alkaline chemokine.In BMDCs and inguinal lymph node DCs, intracellular transport of a low concentration of CpG DNA was greatly enhanced by the addition of CXCL14. Confocal microscopical analyses revealed that CpG DNA and CXCL14 mainly co-localized in EEA1+ endosome and LAMP1+ lysosomal compartments where TLR9 is present. Furthermore, we demonstrated that CXCL14 binds to CpG DNA in vitro in the neutral pH condition with high affinity (Kd=9.8 nM). This interaction was completely dissociated in pH 6.0, implying that CpG DNA can be released and passed to TLR9 in the endosome and lysosome. Consistent with our hypothesis, induction of IL-12 p40, MHC class-II, and CD86 by the combination of CpG DNA and CXCL14 was not observed in BMDCs derived from TLR9 knockout (KO) mice. Moreover, after systemic administration of CpG DNA, plasma concentration of IL-12 p40 and frequency of MHC-class II+CD11c+CD8+DCs in spleen were significantly decreased in CXCL14-deficient mice when compared to littermate control mice.Taken together, these results demonstrated that CXCL14 serves as a specific carrier for CpG DNA into conventional DCs for activating TLR9-mediated adaptive immune system. This is also the first demonstration of a DNA sensing function of chemokine. The combination of CXCL14 and CpG DNA would be a promising vaccine adjuvant for enhancing immnunosurveillance against pathogens and malignant cancers. DisclosuresTanegashima:Tokyo Metropolitan Institute of Medical Science: Patents & Royalties. Takahashi:Tokyo Metropolitan Institute of Medical Science: Patents & Royalties. Tsuji:Tokushima University: Patents & Royalties. Shigenaga:Tokushima University: Patents & Royalties. Otaka:Tokushima University: Patents & Royalties.