RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise.

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.

Counter-ion effect on surfactant-DNA gel particles as controlled DNA delivery systems

The ability to entrap drugs within vehicles and subsequently release them has led to new treatments for a number of diseases. Based on an associative phase separation and interfacial diffusion approach, we developed a way to prepare DNA gel particles without adding any kind of cross-linker or organic solvent. Among the various agents studied, cationic surfactants offered particularly efficient control for encapsulation and DNA release from these DNA gel particles. The driving force for this strong association is the electrostatic interaction between the two components, as induced by the entropic increase due to the release of the respective counter-ions. However, little is known about the influence of the respective counter-ions on this surfactant-DNA interaction. Here we examined the effect of different counter-ions on the formation and properties of the DNA gel particles by mixing DNA (either single- (ssDNA) or double-stranded (dsDNA)) with the single chain surfactant dodecyltrimethylammonium (DTA). In particular, we used as counter-ions of this surfactant the hydrogen sulfate and trifluoromethane sulfonate anions and the two halides, chloride and bromide. Effects on the morphology of the particles obtained, the encapsulation of DNA and its release, as well as the haemocompatibility of these particles, are presented, using the counter-ion structure and the DNA conformation as controlling parameters. Analysis of the data indicates that the degree of counter-ion dissociation from the surfactant micelles and the polar/hydrophobic character of the counter-ion are important parameters in the final properties of the particles. The stronger interaction with amphiphiles for ssDNA than for dsDNA suggests the important role of hydrophobic interactions in DNA.

DnaSP, DNA polymorphism analyses by the coalescent and other methods

DnaSP is a software package for the analysis of DNA polymorphism data. Present version introduces several new modules and features which, among other options allow: (1) handling big data sets (~5 Mb per sequence); (2) conducting a large number of coalescent-based tests by Monte Carlo computer simulations; (3) extensive analyses of the genetic differentiation and gene flow among populations; (4) analysing the evolutionary pattern of preferred and unpreferred codons; (5) generating graphical outputs for an easy visualization of results. Availability: The software package, including complete documentation and examples, is freely available to academic users from: http://www.ub.es/dnasp

Cooperative kinking at distant sites in mechanically stressed DNA.

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.

Indicators of therapeutic effect in FIT-06, a Phase II trial of a DNA vaccine, GTU(®)-Multi-HIVB, in untreated HIV-1 infected subjects.

BACKGROUND: Combination highly active antiretroviral therapy (HAART) has significantly decreased HIV-1 related morbidity and mortality globally transforming HIV into a controllable condition. HAART has a number of limitations though, including limited access in resource constrained countries, which have driven the search for simpler, affordable HIV-1 treatment modalities. Therapeutic HIV-1 vaccines aim to provide immunological support to slow disease progression and decrease transmission. We evaluated the safety, immunogenicity and clinical effect of a novel recombinant plasmid DNA therapeutic HIV-1 vaccine, GTU(®)-multi-HIVB, containing 6 different genes derived from an HIV-1 subtype B isolate. METHODS: 63 untreated, healthy, HIV-1 infected, adults between 18 and 40 years were enrolled in a single-blinded, placebo-controlled Phase II trial in South Africa. Subjects were HIV-1 subtype C infected, had never received antiretrovirals, with CD4 ≥ 350 cells/mm(3) and pHIV-RNA ≥ 50 copies/mL at screening. Subjects were allocated to vaccine or placebo groups in a 2:1 ratio either administered intradermally (ID) (0.5mg/dose) or intramuscularly (IM) (1mg/dose) at 0, 4 and 12 weeks boosted at 76 and 80 weeks with 1mg/dose (ID) and 2mg/dose (IM), respectively. Safety was assessed by adverse event monitoring and immunogenicity by HIV-1-specific CD4+ and CD8+ T-cells using intracellular cytokine staining (ICS), pHIV-RNA and CD4 counts. RESULTS: Vaccine was safe and well tolerated with no vaccine related serious adverse events. Significant declines in log pHIV-RNA (p=0.012) and increases in CD4+ T cell counts (p=0.066) were observed in the vaccine group compared to placebo, more pronounced after IM administration and in some HLA haplotypes (B*5703) maintained for 17 months after the final immunisation. CONCLUSIONS: The GTU(®)-multi-HIVB plasmid recombinant DNA therapeutic HIV-1 vaccine is safe, well tolerated and favourably affects pHIV-RNA and CD4 counts in untreated HIV-1 infected individuals after IM administration in subjects with HLA B*57, B*8101 and B*5801 haplotypes.

Genome-wide DNA polymorphism analyses using VariScan

BACKGROUND: DNA sequence polymorphisms analysis can provide valuable information on the evolutionary forces shaping nucleotide variation, and provides an insight into the functional significance of genomic regions. The recent ongoing genome projects will radically improve our capabilities to detect specific genomic regions shaped by natural selection. Current available methods and software, however, are unsatisfactory for such genome-wide analysis. RESULTS: We have developed methods for the analysis of DNA sequence polymorphisms at the genome-wide scale. These methods, which have been tested on a coalescent-simulated and actual data files from mouse and human, have been implemented in the VariScan software package version 2.0. Additionally, we have also incorporated a graphical-user interface. The main features of this software are: i) exhaustive population-genetic analyses including those based on the coalescent theory; ii) analysis adapted to the shallow data generated by the high-throughput genome projects; iii) use of genome annotations to conduct a comprehensive analyses separately for different functional regions; iv) identification of relevant genomic regions by the sliding-window and wavelet-multiresolution approaches; v) visualization of the results integrated with current genome annotations in commonly available genome browsers. CONCLUSION: VariScan is a powerful and flexible suite of software for the analysis of DNA polymorphisms. The current version implements new algorithms, methods, and capabilities, providing an important tool for an exhaustive exploratory analysis of genome-wide DNA polymorphism data.

Task 4: Testing Iowa Portland Cement Concrete Mixtures for the AASHTO Mechanistic-Empirical Pavement Design Procedure, May 2008

The present research project was designed to identify the typical Iowa material input values that are required by the Mechanistic-Empirical Pavement Design Guide (MEPDG) for the Level 3 concrete pavement design. It was also designed to investigate the existing equations that might be used to predict Iowa pavement concrete for the Level 2 pavement design. In this project, over 20,000 data were collected from the Iowa Department of Transportation (DOT) and other sources. These data, most of which were concrete compressive strength, slump, air content, and unit weight data, were synthesized and their statistical parameters (such as the mean values and standard variations) were analyzed. Based on the analyses, the typical input values of Iowa pavement concrete, such as 28-day compressive strength (f’c), splitting tensile strength (fsp), elastic modulus (Ec), and modulus of rupture (MOR), were evaluated. The study indicates that the 28-day MOR of Iowa concrete is 646 + 51 psi, very close to the MEPDG default value (650 psi). The 28-day Ec of Iowa concrete (based only on two available data of the Iowa Curling and Warping project) is 4.82 + 0.28x106 psi, which is quite different from the MEPDG default value (3.93 x106 psi); therefore, the researchers recommend re-evaluating after more Iowa test data become available. The drying shrinkage (εc) of a typical Iowa concrete (C-3WR-C20 mix) was tested at Concrete Technology Laboratory (CTL). The test results show that the ultimate shrinkage of the concrete is about 454 microstrain and the time for the concrete to reach 50% of ultimate shrinkage is at 32 days; both of these values are very close to the MEPDG default values. The comparison of the Iowa test data and the MEPDG default values, as well as the recommendations on the input values to be used in MEPDG for Iowa PCC pavement design, are summarized in Table 20 of this report. The available equations for predicting the above-mentioned concrete properties were also assembled. The validity of these equations for Iowa concrete materials was examined. Multiple-parameters nonlinear regression analyses, along with the artificial neural network (ANN) method, were employed to investigate the relationships among Iowa concrete material properties and to modify the existing equations so as to be suitable for Iowa concrete materials. However, due to lack of necessary data sets, the relationships between Iowa concrete properties were established based on the limited data from CP Tech Center’s projects and ISU classes only. The researchers suggest that the resulting relationships be used by Iowa pavement design engineers as references only. The present study furthermore indicates that appropriately documenting concrete properties, including flexural strength, elastic modulus, and information on concrete mix design, is essential for updating the typical Iowa material input values and providing rational prediction equations for concrete pavement design in the future.

Differential involvement of MEK kinase 1 (MEKK1) in the induction of apoptosis in response to microtubule-targeted drugs versus DNA damaging agents.

MEK kinase 1 (MEKK1) is a 196-kDa enzyme that is involved in the regulation of the c-Jun N-terminal kinase (JNK) pathway and apoptosis. In cells exposed to genotoxic agents including etoposide and cytosine arabinoside, MEKK1 is cleaved at Asp874 by caspases. The cleaved kinase domain of MEKK1, itself, stimulates caspase activity leading to apoptosis. Kinase-inactive MEKK1 expressed in HEK293 cells effectively blocks genotoxin-induced apoptosis. Treatment of cells with taxol, a microtubule stabilizing agent, did not induce MEKK1 cleavage in cells, and kinase-inactive MEKK1 expression failed to block taxol-induced apoptosis. MEKK1 became activated in HEK293 cells exposed to taxol, but in contrast to etoposide-treatment, taxol failed to increase JNK activity. Taxol treatment of cells, therefore, dissociates MEKK1 activation from the regulation of the JNK pathway. Overexpression of anti-apoptotic Bcl2 blocked MEKK1 and taxol-induced apoptosis but did not block the caspase-dependent cleavage of MEKK1 in response to etoposide. This indicates Bcl2 inhibition of apoptosis is, therefore, downstream of caspase-dependent MEKK1 cleavage. The results define the involvement of MEKK1 in the induction of apoptosis by genotoxins but not microtubule altering drugs.

Evaluation of Fly Ash in Water Reduced Paving Mixtures, June 1985

Fly ash was used to replace 15% of the cement in C3WR and C6WR concrete paving mixes containing ASTM C494 Type A water reducin9 admixtures. Two Class C ashes and one Class F ash from Iowa approved sources were examined in each mix. When Class C ashes were used they were substituted on the basis of 1 pound of ash added for each pound of cement deleted. When Class F was used it was substituted on the basis of 1.25 pounds of ash added for each pound of cement deleted. Compressive strengths of the water reduced mixes, with and without fly ash, were determined at 7, 28, and 56 days of age. In every case except one the mixes containing the fly ash exhibited higher strengths than the same concrete mix without the fly ash. An excellent correlation existed between the C3WR and C6WR mixes both with and without fly ash substitutions. The freeze-thaw durability of the concrete studied was not affected by presence or absence of fly ash. The data gathered suggests that the present Class C water reduced concrete paving mixes can be modified to allow the substitution of 15% of the cement with an approved fly ash.

Optimizing Cementious Content in Concrete Mixtures for Required Performance Final Report, January 2012

This research investigated the effects of changing the cementitious content required at a given water-to-cement ratio (w/c) on workability, strength, and durability of a concrete mixture. An experimental program was conducted in which 64 concrete mixtures with w/c ranging between 0.35 and 0.50, cementitious content ranging from 400 to 700 per cubic yard (pcy), and containing four different supplementary cementitious material (SCM) combinations were tested. The fine-aggregate to total-aggregate ratio was fixed at 0.42 and the void content of combined aggregates was held constant for all the mixtures. Fresh (i.e., slump, unit weight, air content, and setting time) and hardened properties (i.e., compressive strength, chloride penetrability, and air permeability) were determined. The hypothesis behind this study is that when other parameters are kept constant, concrete properties such as strength, chloride penetration, and air permeability will not be improved significantly by increasing the cement after a minimum cement content is used. The study found that about 1.5 times more paste is required than voids between the aggregates to obtain a minimum workability. Below this value, water-reducing admixtures are of no benefit. Increasing paste thereafter increased workability. In addition, for a given w/c, increasing cementitious content does not significantly improve compressive strength once the critical minimum has been provided. The critical value is about twice the voids content of the aggregate system. Finally, for a given w/c, increasing paste content increases chloride penetrability and air permeability.

Optimizing Cementitious Content in Concrete Mixtures for Required Performance Final Report, January 2012

This research investigated the effects of changing the cementitious content required at a given water-to-cement ratio (w/c) on workability, strength, and durability of a concrete mixture. An experimental program was conducted in which 64 concrete mixtures with w/c ranging between 0.35 and 0.50, cementitious content ranging from 400 to 700 per cubic yard (pcy), and containing four different supplementary cementitious material (SCM) combinations were tested. The fine-aggregate to total-aggregate ratio was fixed at 0.42 and the void content of combined aggregates was held constant for all the mixtures. Fresh (i.e., slump, unit weight, air content, and setting time) and hardened properties (i.e., compressive strength, chloride penetrability, and air permeability) were determined. The hypothesis behind this study is that when other parameters are kept constant, concrete properties such as strength, chloride penetration, and air permeability will not be improved significantly by increasing the cement after a minimum cement content is used. The study found that about 1.5 times more paste is required than voids between the aggregates to obtain a minimum workability. Below this value, water-reducing admixtures are of no benefit. Increasing paste thereafter increased workability. In addition, for a given w/c, increasing cementitious content does not significantly improve compressive strength once the critical minimum has been provided. The critical value is about twice the voids content of the aggregate system. Finally, for a given w/c, increasing paste content increases chloride penetrability and air permeability.

Respiratory chain dysfunction associated with multiple mitochondrial DNA deletions in antiretroviral therapy-related lipodystrophy.

Highly-active antiretroviral therapy (HAART) can induce a characteristic lipodystrophy syndrome characterized by peripheral fat wasting and central adiposity, usually associated with hyperlipidaemia and insulin resistance [1,2]. Indirect data have led some authors to propose that mitochondrial dysfunction could play a role in this syndrome [3,4].To date, as recently outlined by Kakuda et al. [5] in this journal, HIV-infected patients developing lipodystrophy have not been studied for mitochondrial changes or respiratory chain capacity...

The inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune response.

The innate immune system recognizes nucleic acids during infection and tissue damage. Whereas viral RNA is detected by endosomal toll-like receptors (TLR3, TLR7, TLR8) and cytoplasmic RIG-I and MDA5, endosomal TLR9 and cytoplasmic DAI bind DNA, resulting in the activation of nuclear factor-kappaB and interferon regulatory factor transcription factors. However, viruses also trigger pro-inflammatory responses, which remain poorly defined. Here we show that internalized adenoviral DNA induces maturation of pro-interleukin-1beta in macrophages, which is dependent on NALP3 and ASC, components of the innate cytosolic molecular complex termed the inflammasome. Correspondingly, NALP3- and ASC-deficient mice display reduced innate inflammatory responses to adenovirus particles. Inflammasome activation also occurs as a result of transfected cytosolic bacterial, viral and mammalian (host) DNA, but in this case sensing is dependent on ASC but not NALP3. The DNA-sensing pro-inflammatory pathway functions independently of TLRs and interferon regulatory factors. Thus, in addition to viral and bacterial components or danger signals in general, inflammasomes sense potentially dangerous cytoplasmic DNA, strengthening their central role in innate immunity.