High Nucleic Acid Research Articles

Layer-by-layer assembled antisense DNA microsponge particles for efficient delivery of cancer therapeutics.

Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.

Catchment-scale biogeography of riverine bacterioplankton.

Lotic ecosystems such as rivers and streams are unique in that they represent a continuum of both space and time during the transition from headwaters to the river mouth. As microbes have very different controls over their ecology, distribution and dispersion compared with macrobiota, we wished to explore biogeographical patterns within a river catchment and uncover the major drivers structuring bacterioplankton communities. Water samples collected across the River Thames Basin, UK, covering the transition from headwater tributaries to the lower reaches of the main river channel were characterised using 16S rRNA gene pyrosequencing. This approach revealed an ecological succession in the bacterial community composition along the river continuum, moving from a community dominated by Bacteroidetes in the headwaters to Actinobacteria-dominated downstream. Location of the sampling point in the river network (measured as the cumulative water channel distance upstream) was found to be the most predictive spatial feature; inferring that ecological processes pertaining to temporal community succession are of prime importance in driving the assemblages of riverine bacterioplankton communities. A decrease in bacterial activity rates and an increase in the abundance of low nucleic acid bacteria relative to high nucleic acid bacteria were found to correspond with these downstream changes in community structure, suggesting corresponding functional changes. Our findings show that bacterial communities across the Thames basin exhibit an ecological succession along the river continuum, and that this is primarily driven by water residence time rather than the physico-chemical status of the river.

Self-propelled continuous-flow PCR in capillary-driven microfluidic device: Microfluidic behavior and DNA amplification

Continuous-flow polymerase chain reaction (CF-PCR) in microfluidic devices has a great potential for on-site detection of various pathogens and food species because of their high speed deoxyribo nucleic acid (DNA) amplification. However, flow-controlled pumps such as syringe pumps are absolutely necessary, which cause complex and difficult operations. Here, we present a self-propelled CF-PCR (SP-CF-PCR) in a microfluidic device which requires no external pumps to control the flow. The PCR solution is simply dropped onto the inlet and is autonomously transported by capillary forces. One of the difficulties in addressing the capillary flow in PCR microfluidic device is that the temperature of the PCR solution is periodically switched. Differing from previous theoretical approaches which deal with single steady-state temperature zone, for the first time, the displacement of capillary flow during temperature switching was mathematically formulated and was simulated by using experimental values of viscosities and capillary pressures at each temperature as parameters for simulation. Basing on the excellent matching between simulated and experimental data of the capillary flow, we were able to reveal the optimized design of 150-μm-wide and 150-μm-deep microchannel that successfully transported over 1600mm for carrying out PCR within less than 14min solely by capillary forces. We also demonstrated the SP-CF-PCR for verifying our concept. The specific amplifications of 295bp of β-actin from human genome, 232bp of AH1pdm influenza virus and 95bp of 16S rDNA of Escherichia coli genomic DNA were successfully achieved, proving the application potentials of our device.

Application of Magnetic/Magnetic Fluorescent Microsphere Preparation in Genes Food Testing

Polymerase chain reaction (PCR) and fluorescence quantitative polymerase chain reaction (qRT - PCR) is a major means of detection of genetically modified food, whose key is to extract high quality nucleic acid in quick and high flux. The main nucleic acid extraction method is hexadecyl trimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) and kits. Because operation of CTAB and SDS method are cumbersome, require the use of organic solvent and centrifugal unceasingly, thus can not meet the requirement of the rapid development of high throughput; and because of the high price of kit method, its application in the daily inspection is also limited. At the same time, the development trend of genetically modified detection based on nucleic acid level is multiple tests; the key is the preparation of different kinds of fluorescent microspheres and specific probe of microspheres. To solve above problems, this paper put forward nucleic acid extraction method on the basis of the magnetic separation technology.

Using coagulation to restrict microbial re-growth in tap water by phosphate limitation in water treatment

Extensive microbial re-growth in a drinking water distribution system can deteriorate water quality. The limiting factor for microbial re-growth in a tap water produced by a conventional drinking water treatment plant in China was identified by determining the microbial re-growth potential (MRP) by adding different nutrients to stimulate growth of a natural microbial consortium as inoculum and flow-cytometric enumeration. No obvious change of MRP was found in tap water after addition of carbon, whereas, a 1- to 2-fold increase of MRP was observed after addition of phosphate (P). This clearly demonstrated that microbial re-growth in this tap water was limited by P. Most of the re-grown microbial flora (>85%) consisted of high nucleic acid content cells.A subsequent investigation of the MRP in the actual water treatment plant demonstrated that coagulation was the crucial step for decreasing MRP and producing P-limited water. Therefore, a comparison concerning the control of MRP by three different coagulants was conducted. It showed that all the three coagulants efficiently reduced the MRP and shifted the limitation regime from C to P, but the required dose was different. The study shows that it is feasible to restrict microbial re-growth by P limitation using coagulation in water treatment.

T-cell receptor repertoires share a restricted set of public and abundant CDR3 sequences that are associated with self-related immunity.

The T-cell receptor (TCR) repertoire is formed by random recombinations of genomic precursor elements; the resulting combinatorial diversity renders unlikely extensive TCR sharing between individuals. Here, we studied CDR3β amino acid sequence sharing in a repertoire-wide manner, using high-throughput TCR-seq in 28 healthy mice. We uncovered hundreds of public sequences shared by most mice. Public CDR3 sequences, relative to private sequences, are two orders of magnitude more abundant on average, express restricted V/J segments, and feature high convergent nucleic acid recombination. Functionally, public sequences are enriched for MHC-diverse CDR3 sequences that were previously associated with autoimmune, allograft, and tumor-related reactions, but not with anti-pathogen-related reactions. Public CDR3 sequences are shared between mice of different MHC haplotypes, but are associated with different, MHC-dependent, V genes. Thus, despite their random generation process, TCR repertoires express a degree of uniformity in their post-genomic organization. These results, together with numerical simulations of TCR genomic rearrangements, suggest that biases and convergence in TCR recombination combine with ongoing selection to generate a restricted subset of self-associated, public CDR3 TCR sequences, and invite reexamination of the basic mechanisms of T-cell repertoire formation.

Purification and biocompatibility of fermented hyaluronic acid for its applications to biomaterials.

BackgroundHyaluronic acid (HA) is of importance due to its diverse applications in pharmaceuticals and medical devices such as dermal filler, adhesion barriers, carrier for cells and bioactive molecules as well as scaffold biomaterials for tissue engineering. Evaluations of purification and biocompatibility of HA are required for its applications to biomaterials.ResultsAfter synthesizing HA by fermentation of streptococcus zooepidemicus for 25 hr, extensively purification of the fermented broth was performed to remove impurities using a filtration process for insoluble components and cells, and diverse adsorbents for soluble impurities. Its in vitro biocompatibility has been evaluated by measurement of cell counting and assay of cell live and dead. 60% yield of white HA powder was obtained, having 15–17 dL/g intrinsic viscosity with a molecular weight of approximately 1,000 kDa. While low molecular weight impurities and insoluble impurities were successfully removed using a ultrafiltration membrane with 50 KDa molecular weight cut, endotoxins, high molecular weight proteins and nucleic acids were removed from the broth by employing adsorbents such as alumina and activated carbons. Alumina showed the best results for the removal of endotoxins, all of the activated carbons were very effective in the removal of high molecular weight proteins and nucleic acids. The purified HA solution showed excellent cell compatibility with no cell damages as observed by both measurement of cell proliferation and observation of cell viability.ConclusionsWe obtained high molecular weight HA with excellent biocompatibility as judged by both measurement of cell proliferation and viability, indicating high possibility of its applications to biomaterials.

Linking host prokaryotic physiology to viral lifestyle dynamics in a temperate freshwater lake (Lake Pavin, France).

In aquatic ecosystems, fluctuations in environmental conditions and prokaryotic host physiological states can strongly affect the dynamics of viral life strategies. The influence of prokaryote physiology and environmental factors on viral replication cycles (lytic and lysogeny) was investigated from April to September 2011 at three different strata (epi, meta, and hypolimnion) in the mixolimnion of deep volcanic temperate freshwater Lake Pavin (France). Overall, the euphotic region (epi and metalimnion) was more dynamic and showed significant variation in microbial standing stocks, prokaryotic physiological state, and viral life strategies compared to the aphotic hypolimnion which was stable within sampled months. The prokaryotic host physiology as inferred from the nucleic acid content of prokaryotic cells (high or low nucleic acid) was strongly regulated by the chlorophyll concentration. The predominance of the high nucleic acid (HNA) prokaryotes (cells) over low nucleic acid (LNA) prokaryotes (cells) in the spring (HNA/LNA = 1.2) and vice versa in the summer period (HNA/LNA = 0.4) suggest that the natural prokaryotic communities underwent major shifts in their physiological states during investigated time period. The increase in the percentage of inducible lysogenic prokaryotes in the summer period was associated with the switch in the dominance of LNA over HNA cells, which coincided with the periods of strong resource (nutrient) limitation. This supports the idea that lysogeny represents a maintenance strategy for viruses in unproductive or harsh nutrient/host conditions. A negative correlation of percentage of lysogenic prokaryotes with HNA cell abundance and chlorophyll suggest that lysogenic cycle is closely related to prokaryotic cells which are stressed or starved due to unavailability of resources for its growth and activity. Our results provide support to previous findings that changes in prokaryote physiology are critical for the promotion and establishment of lysogeny in aquatic ecosystems, which are prone to constant environmental fluctuations.

Peripheral and integral membrane binding of peptides characterized by time-dependent fluorescence shifts: focus on antimicrobial peptide LAH₄.

Positioning of peptides with respect to membranes is an important parameter for biological and biophysical studies using model systems. Our experiments using five different membrane peptides suggest that the time-dependent fluorescence shift (TDFS) of Laurdan can help when distinguishing between peripheral and integral membrane binding and can be a useful, novel tool for studying the impact of transmembrane peptides (TMP) on membrane organization under near-physiological conditions. This article focuses on LAH4, a model α-helical peptide with high antimicrobial and nucleic acid transfection efficiencies. The predominantly helical peptide has been shown to orient in supported model membranes parallel to the membrane surface at acidic and, in a transmembrane manner, at basic pH. Here we investigate its interaction with fully hydrated large unilamellar vesicles (LUVs) by TDFS and fluorescence correlation spectroscopy (FCS). TDFS shows that at acidic pH LAH4 does not influence the glycerol region while at basic pH it makes acyl groups at the glycerol level of the membrane less mobile. TDFS experiments with antimicrobial peptides alamethicin and magainin 2, which are known to assume transmembrane and peripheral orientations, respectively, prove that changes in acyl group mobility at the glycerol level correlate with the orientation of membrane-associated peptide molecules. Analogous experiments with the TMPs LW21 and LAT show similar effects on the mobility of those acyl groups as alamethicin and LAH4 at basic pH. FCS, on the same neutral lipid bilayer vesicles, shows that the peripheral binding mode of LAH4 is more efficient in bilayer permeation than the transmembrane mode. In both cases, the addition of LAH4 does not lead to vesicle disintegration. The influence of negatively charged lipids on the bilayer permeation is also addressed.

Molecular cloning, expression and polymorphism of goose LRP8 gene

1. The low-density lipoprotein receptor-related protein 8 (LRP8), a member of the low-density lipoprotein receptor (LDLR) gene family, participates in the supplying of lipid during follicular development. The objective of the study was to identify and characterise the LRP8 gene in goose.2. A 2867 bp fragment that covered the complete coding region (CDS) of goose (Anser cygnoides) LRP8 gene was cloned. It encoded a protein of 917 amino acid residues containing a 24-amino acid signal peptide and 5 functional domains. The goose LRP8 showed high nucleic acid and amino acid identities with those in other species.3. Similarly to duck LRP8 gene, two splice variants of LRP8, LRP8-1 (containing 8 ligand-binding repeats) and LRP8-2 (containing 7 ligand-binding repeats), were identified in goose.4. Semi-quantitative RT-PCR analysis indicates that the LRP8-1 transcript is expressed in heart, liver, spleen, lung, kidney, breast muscle, duodenum, hypothalamus, pituitary and ovary, negligible or absent in sebum and oviduct, and the LRP8-2 transcript is widely expressed in all examined tissues.5. A total of 7 SNPs were identified in the coding region of the goose LRP8 gene.

Electromigration behavior of nucleic acids in capillary electrophoresis under pulsed-field conditions

We have presented a study focused on the migration pattern of double-stranded DNA (dsDNA) and RNA under pulsed field conditions. By calculating the dependence of nucleic acid mobility on its molecular size in a double logarithm plot, we found that (I) dsDNA molecules proceeded by a sigmoidal migration regime which was probably related to Ogston sieving, transition regime, and reptation model. Furthermore, the transition regime disappeared if DNA was resolved in a higher molecular mass HEC. (II) The migration pattern of RNA was relevant to the denaturant used for separation. When RNA was denatured by acetic acid, its mobility parabolically declined with its molecular size. The mobility was linearly decreased with the molecular size if urea was employed as denaturant. (III) RNA may migrate by Ogston, reptation without orientation mechanism when denatured by urea, whereas these two models were not suitable for RNA if denatured by acetic acid. Even though the electrophoretic conditions of PFCE were varied, the sigmoidal, linear, parabolic migration patterns could still be observed. (IV) Under certain modulation depth, the migration time (Tm) of acetic acid decreased with the increase of average separation voltage (Va), and when RNA denatured in 4.0M urea, Tm showed a linear correlation with Va. (V) The mobility of nucleic acids increased with the growth of artificial temperature in the capillary volume due to the decrease in the viscosity of the polymer. This is the first systematic and comparative research of high molecular mass nucleic acids in PFCE, which provides us deep insight into RNA and DNA migration behavior under pulsed electric field conditions.

Dynamics of picoplankton community from coastal waters to the open sea in the Central Adriatic

Flow cytometry was used to describe seasonal cycles of Prochlorococcus (Prochl), Synechococcus (Syn), picoeukaryotes and heterotrophic bacteria in the central Adriatic Sea along the trophic gradient from January to December 2010. All picoplankton parameters decreased from eutrophic to oligotrophic areas, while the biomass ratio of bacterial to autotrophic picoplankton showed an increase along the trophic gradient. Bacterial biomass ranged from 5.28 to 21.20 μg C l-1. Increased values were present during warmer seasons with the domination of low nucleic acid (LNA) group of bacteria. The high nucleic acid (HNA) bacterial group dominated during the winter and the spring. Bacterial production ranged from 0.09 -0.45 × 104 cells ml-1 h-1 .At coastal stations increased production was present during the winter and the spring and was more or less uniform at open sea stations. Biomasses of Syn and Prochl ranged from 0.16 to 11.47 µg C-1 l-1 and from 0.01 to 3.08 µg C l-1, respectively. They were elevated during the summer and the autumn at coastal stations and during the late winter at the open sea. Syn biomass always dominated over Prochl participating with 61.6-97.2% in biomass of cyanobacteria. Biomass of picoeukaryotes ranged from 1.21 to 21.85 µg C l-1 and was the highest during the winter. Their biomass notably prevailed in autotrophic picoplankton (APP) biomass over that of picocyanobacteria during the whole year. Autotrophic components (Prochl, Syn and picoeukaryotes) made greater contribution to the picoplankton biomass in mesotrophic and eutrophic areas, while heterotrophic bacteria became more important under oligotrophic conditions.

Optimization and Validation of a Real Time Reverse Transcriptase Polymerase Chain Reaction with RNA Internal Control to Detect Rubella RNA

BACKGROUND: According to a report from WHO, cases of rubella infection in Indonesia has increased up to 10-fold from 2007 to 2011. Despite no data of congenital rubella syndrome in the report, there are approximately 45,000 cases of babies born with heart failure and 0.1-0.3% live births with congenital deafness in Indonesia. Allegedly, rubella infection during pregnancy may play a role in this condition. This study aimed to optimize and validate a real-time reverse transcriptase polymerase chain reaction (RT-qPCR) method to detect rubella virus RNA as an aid for the diagnosis of congenital rubella infection.METHODS: Method optimization was conducted using nucleic acids extracted from Trimovax Merieux vaccine with the High Pure Viral Nucleic Acid Kit. One step RT-qPCR was performed with Quantifast Multiplex RTPCR+R Kit. Target synthetic DNA was designed and used to determine the sensitivity of the method. RNA internal control was synthesized to control the process of extraction and amplification.RESULTS: The analytical sensitivity of this method was as low as 5 copies target synthetic DNA/μl. The mean Coefficient of Variation (CV) % of the critical threshold (Ct) obtained were 2.71%, 1.20%, 1.62%, and 1.59% for within run, between run, between kit lots, and between operators, respectively. Recovery of the target synthetic DNA from amniotic fluid was 100.51% (by the log copies/μl) at the concentration of 1,000,000 copies/μl.CONCLUSION: RT-qPCR is successfully used for the detection of rubella virus RNA in vaccine and synthetic nucleic acid. With its high sensitivity, good precision and recovery, this method offers a means to improve the diagnosis of congenital rubella infection in developing countries like Indonesia.KEYWORDS: congenital rubella, RT-qPCR, prenatal diagnosis, amniotic fluid

Short-term variability in bacterial abundance, cell properties, and incorporation of leucine and thymidine in subarctic sea ice

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 71:57-73 (2013) - DOI: https://doi.org/10.3354/ame01667 Short-term variability in bacterial abundance, cell properties, and incorporation of leucine and thymidine in subarctic sea ice H. Kaartokallio1,*, D. H. Søgaard2,3, L. Norman4, S. Rysgaard2,5,6, J.-L. Tison7, B. Delille8, D. N. Thomas1,4,5 1Marine Research Center, Finnish Environment Institute, Erik Palmenin aukio 1 00560, Helsinki, Finland 2Greenland Climate Research Centre, c/o Greenland Institute of Natural Resources, Kivioq 2, Nuuk 3900, Greenland 3University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark 4School of Ocean Sciences, Bangor University, Menai Bridge, Anglesey LL59 5AB, UK 5Arctic Research Centre, Aarhus University, C.F. Møllers Allé 8, Bldg. 1110, 8000 Aarhus C, Denmark 6Centre for Earth Observation Science, 535 Wallace Building, University of Manitoba, Winnipeg R3T 2N2, Canada 7Glaciology Unit, Department of Earth and Environmental Science, Université Libre de Bruxelles, ULB CP 160/03 50 Avenue F. D. Roosevelt, 1050 Bruxelles, Belgium 8Unité d’Océanographie Chimique, MARE, Université de Liège, Allé du 6 Août, 17–4000 Liège, Belgium *Email: hermanni.kaartokallio@ymparisto.fi ABSTRACT: Sea ice is a biome of immense size and provides a range of habitats for diverse microbial communities, many of which are adapted to living at low temperatures and high salinities in brines. We measured simultaneous incorporation of thymidine (TdR) and leucine (Leu), bacterial cell abundance and cell population properties (by flow cytometry) in subarctic sea ice in SW Greenland. Short-term temporal variability was moderate, and steep environmental gradients, typical for sea ice, were the main drivers of the variability in bacterial cell properties and activity. Low nucleic acid (LNA) bacteria, previously linked to oligotrophic ecotypes in marine habitats, were more abundant in the upper ice layers, whereas high nucleic acid (HNA) bacteria dominated in lower ice, where organic carbon was in high concentrations. Leu incorporation was saturated at micromolar concentrations, as known from freshwater and marine biofilm systems. Leu:TdR ratios were high (up to >300) in lowermost ice layers, and when compared to published respiration measurements, these results suggest non-specific Leu incorporation. There was evidence of polyhydroxyalkanoate (PHA)-containing bacteria in the sea ice, shown by brightly fluorescing intracellular inclusions after Nile Blue A staining. High Leu saturating concentrations coupled with the occurrence of PHA-producing organisms further highlight the similarity of sea ice internal habitats to biofilm-like systems rather than to open-water systems. KEY WORDS: Sea ice · Bacteria · Temporal variability · Leucine · Thymidine · Flow cytometry · Polyhydroxyalkanoates Full text in pdf format PreviousNextCite this article as: Kaartokallio H, Søgaard DH, Norman L, Rysgaard S, Tison JL, Delille B, Thomas DN (2013) Short-term variability in bacterial abundance, cell properties, and incorporation of leucine and thymidine in subarctic sea ice. Aquat Microb Ecol 71:57-73. https://doi.org/10.3354/ame01667 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 71, No. 1. Online publication date: November 27, 2013 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2013 Inter-Research.

Regulation of bacterial metabolic activity by dissolved organic carbon and viruses

AbstractThe regulation of bacterial metabolic activity by viruses and dissolved organic carbon (DOC) was examined using natural microbial communities in three treatments (active viruses, inactive viruses, and virus free) at two contrasting coastal sites (pristine vs. eutrophic) with substantial differences in environmental conditions during the wet and dry seasons. Our results showed that net growth rates and production of bacterioplankton were reduced primarily by viruses via repressing metabolically active bacteria with high nucleic acid (HNA) content which had a high capacity for incorporating carbon, while bacterial respiration was primarily regulated by DOC lability. The quality of organic matter played a more important role in regulating bacterial growth efficiency (BGE) than the supply of organic matter in eutrophic coastal waters. The lack of HMW‐DOC and high carbon demand in the virus‐free treatment resulted in a significant increase in cell‐specific bacterial respiration, which was responsible for the lowest bacterial growth efficiency among the three treatments. The presence of viruses did not necessarily lower bacterial growth efficiency since virus‐induced mortality alleviated bacterial carbon demand and enhanced carbon cycling. Virus‐induced mortality was greater in relatively pristine waters than eutrophic waters, likely since the high supply of substrates alleviated the pressure of viral infection, through extracellular proteases produced by bacteria, which might result in the hydrolytic destruction or modification of viral capsids. An important implication of our results was that the input of riverine DOC and nutrients improved bacterial metabolic activity by alleviating virus‐induced mortality of bacteria in estuarine and coastal waters.

Impact of water mass mixing on the biogeochemistry and microbiology of the Northeast Atlantic Deep Water

The extent to which water mass mixing contributes to the biological activity of the dark ocean is essentially unknown. Using a multiparameter water mass analysis, we examined the impact of water mass mixing on the nutrient distribution and microbial activity of the Northeast Atlantic Deep Water (NEADW) along an 8000 km long transect extending from 62°N to 5°S. Mixing of four water types (WT) and basin scale mineralization from the site where the WT where defined to the study area explained up to 95% of the variability in the distribution of inorganic nutrients and apparent oxygen utilization. Mixing-corrected average O2:N:P mineralization ratios of 127(±11):13.0(±0.7):1 in the core of the NEADW suggested preferential utilization of phosphorus compounds while dissolved organic carbon mineralization contributed a maximum of 20% to the oxygen demand of the NEADW. In conjunction with the calculated average mineralization ratios, our results indicate a major contribution of particulate organic matter to the biological activity in the NEADW. The variability in prokaryotic abundance, high nucleic acid containing cells, and prokaryotic heterotrophic production in the NEADW was explained by large scale (64-79%) and local mineralization processes (21-36%), consistent with the idea that deep-water prokaryotic communities are controlled by substrate supply. Overall, our results suggest a major impact of mixing on the distribution of inorganic nutrients and a weaker influence on the dissolved organic matter pool supporting prokaryotic activity in the NEADW.

Binding Of Immune Serine Proteases To Nucleic Acids Enhances Their Nuclear Localization and Promotes Their Cleavage Of Nucleic Acid-Binding Protein Substrates

When released from cytotoxic T lymphocytes and natural killer cells, Granzyme (Gzm) serine proteases induce programmed cell death of pathogen-infected cells and tumor cells. The Gzms rapidly accumulate in the target cell nucleus by an unknown mechanism. Many of the known substrates of GzmA and GzmB, the most abundant killer cell proteases, bind to DNA or RNA. Gzm substrates predicted by unbiased proteomics studies are also highly enriched for nucleic acid binding proteins. Here we show by fluorescence polarization assays that Gzms bind DNA and RNA with nanomolar affinity. We hypothesized that Gzm binding to nucleic acids enhances nuclear accumulation in target cells and facilitates their cleavage of nucleic acid-binding substrates. In fact, RNase treatment of cell lysates reduced cleavage of RNA binding protein (RBP) targets by GzmA and GzmB. Moreover, adding RNA to recombinant RBP substrates greatly enhanced in vitro cleavage by GzmB, but adding RNA to non-nucleic acid binding proteins did not. For example, exogenous RNA enhanced GzmB cleavage of recombinant hnRNP C1 (an RBP) but not LMNB1 (a non-RBP). In addition, GzmB cleaved the RNA-binding HuR protein efficiently only when it was bound to an HuR-binding RNA oligonucleotide, but not in the presence of an equal amount of non-binding RNA. Thus, nucleic acids facilitate Gzm cleavage of nucleic acid binding substrates.To evaluate whether nucleic acid binding influences Gzm trafficking in target cells, we incubated fixed target cells with RNase and then added Gzms. RNA degradation in target cells reduced Gzm cytosolic localization and increased nuclear accumulation. Similarly, pre-incubating Gzms with exogenous competitor DNA reduced Gzm nuclear localization. The Gzms form a monophyletic clade with other immune serine proteases including neutrophil elastase (NE) and cathepsin G (CATG). Upon neutrophil activation, NE translocates to the nucleus to drive the formation of neutrophil extracellular traps (NETs). NE and CATG, but not non-immune serine proteases such as trypsin and pancreatic elastase, also bind DNA with high affinity and localize to the nucleus of permeabilized cells. Consistent with this finding, competitor DNA also blocks the nuclear localization of NE. Moreover NE and CATG localization to NETs depends on DNA binding. Thus the antimicrobial activity of NETs may depend in part upon the affinity of these proteases for DNA. Our findings indicate that high affinity nucleic acid binding is a conserved and functionally important property of serine proteases involved in cell-mediated immunity. Disclosures:Lieberman:Alnylam Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Sieving polymer synthesis by reversible addition fragmentation chain transfer polymerization

Replaceable sieving polymers are the fundamental component for high resolution nucleic acids separation in CE. The choice of polymer and its physical properties play significant roles in influencing separation performance. Recently, reversible addition fragmentation chain transfer (RAFT) polymerization has been shown to be a versatile polymerization technique capable of yielding well defined polymers previously unattainable by conventional free radical polymerization. In this study, a high molecular weight PDMA at 765 000 gmol-1 with a PDI of 1.55 was successfully synthesized with the use of chain transfer agent - 2-propionic acidyl butyl trithiocarbonate (PABTC) in a multi-step sequential RAFT polymerization approach. This study represents the first demonstration of RAFT polymerization for synthesizing polymers with the molecular weight range suitable for high resolution DNA separation in sieving electrophoresis. Adjustment of pH in the reaction was found to be crucial for the successful RAFT polymerization of high molecular weight polymer as the buffered condition minimizes the effect of hydrolysis and aminolysis commonly associated with trithiocarbonate chain transfer agents. The separation efficiency of PABTC-PDMA was found to have marginally superior separation performance compared to a commercial PDMA formulation, POP™-CAP, of similar molecular weight range.

A cross-sectional study of genetic diversity of the envelope glycoprotein of HIV-1 in Baja California, Mexico

BackgroundMexico had 4598 reported cases of AIDS as of December, 2012, and 4% of the patients infected with HIV-1 in Mexico live in Baja California. The aim of this study was to analyse the predominant HIV-1 virus and characterise the extent of viral genetic diversity in populations from Ensenada and Tijuana cities in Baja California, Mexico. MethodsWe collected clinical samples from February, 2012, to April, 2013, in three different public hospitals (one in Ensenada and two in Tijuana). Only patients with HIV-1 infections confirmed by Western blot who were naive to antiretroviral treatment (ART) were included in the study. All patients answered a questionnaire addressing sociodemographic and behavioural issues, gave signed informed consent, and allowed the extraction of 5 mL of whole blood. Findings22 patients infected with HIV-1 were included in the study (mean age 37 years [range 18–50]) and 82% of the patients were men. 54% represented men who self-reported homosexual or bisexual behaviour. The mean plasma viral load was 239 946 HIV-1 RNA copies per mL (range 1950–1 862 087). The predominant route of transmission was sexual in 72% of the patients. Plasma RNA was extracted from whole blood with the High Pure Viral Nucleic Acid kit. The HIV-1 V3–V5 region of the gp120 env gene (HXB2 position 7065–7634) was amplified by nested polymerase chain reaction (PCR). Only five (23%) samples were successfully amplified and sequenced. We obtained a 579 bp sequence showing 116 variable sites. Reference sequences of different subtypes (n=58) were downloaded from the HIV-1 GenBank. Subtyping of the clinical strains was initially inferred with a maximum likelihood phylogenetic tree constructed with the HKY model and 1000 bootstrap datasets with PAUP 4.0Beta. The gene segments were analysed for recombinant screening with RIP3·0. To confirm consistent results, REGA HIV-1 Subtyping Tool (version 2·0) was also used. Phylogenetic analysis revealed that all five samples analysed were HIV-1 subtype B group M, which were distributed in two haplotypes. Hap 1 (n=4) was classified as X4 and Hap 2 (n=1) as R5 virus (which use CCR4 and CCR5 as a co-receptor, respectively). All samples showed the crown of the V3 loop with the aminoacid sequence glycine-proline-glycine-arginine. There was no phylogenetic clustering or differences between the samples according to sex, age, or method of acquisition of HIV infection. InterpretationThe genetic diversity of HIV has important implications for multiple aspects of the pandemic, including diagnostic and laboratory tests, susceptibility to ART, transmission capability, and disease progression. This study contributes to our understanding of the epidemiology of HIV infection in Mexico. We are now analysing more clinical samples collected from the same public hospitals, and the nucleotide sequences have been deposited in GenBank under accession numbers KF356165-67 and KF366441-42. FundingThis research was supported by Consejo Nacional de Ciencia y Tecnologia de Mexico (CONACyT) and Universidad Autonoma de Baja California.

Monitoring microbiological changes in drinking water systems using a fast and reproducible flow cytometric method

Flow cytometry (FCM) is a rapid, cultivation-independent tool to assess and evaluate bacteriological quality and biological stability of water. Here we demonstrate that a stringent, reproducible staining protocol combined with fixed FCM operational and gating settings is essential for reliable quantification of bacteria and detection of changes in aquatic bacterial communities. Triplicate measurements of diverse water samples with this protocol typically showed relative standard deviation values and 95% confidence interval values below 2.5% on all the main FCM parameters. We propose a straightforward and instrument-independent method for the characterization of water samples based on the combination of bacterial cell concentration and fluorescence distribution. Analysis of the fluorescence distribution (or so-called fluorescence fingerprint) was accomplished firstly through a direct comparison of the raw FCM data and subsequently simplified by quantifying the percentage of large and brightly fluorescent high nucleic acid (HNA) content bacteria in each sample. Our approach enables fast differentiation of dissimilar bacterial communities (less than 15 min from sampling to final result), and allows accurate detection of even small changes in aquatic environments (detection above 3% change). Demonstrative studies on (a) indigenous bacterial growth in water, (b) contamination of drinking water with wastewater, (c) household drinking water stagnation and (d) mixing of two drinking water types, univocally showed that this FCM approach enables detection and quantification of relevant bacterial water quality changes with high sensitivity. This approach has the potential to be used as a new tool for application in the drinking water field, e.g. for rapid screening of the microbial water quality and stability during water treatment and distribution in networks and premise plumbing.