Utilization of a deoxynucleoside diphosphate substrate by HIV reverse transcriptase

Background: Deoxynucleoside triphosphates (dNTPs) are the normal substrates for DNA sysnthesis is catalyzed by polymerases such as HIV-1 reverse transcriptase (RT). However, substantial amounts of deoxynucleoside diphosphates (dNDPs) are also present in the cell. Use of dNDPs in HIV-1 DNA sysnthesis could have significant implications for the efficacy of nucleoside RT inhibitors such as AZT which are first line therapeutics fro treatment of HIV infection. Our earlier work on HIV-1 reverse transcriptase (RT) suggested that the interaction between the γ phosphate of the incoming dNTP and RT residue K65 in the active site is not essential for dNTP insertion, implying that this polymerase may be able to insert dNPs in addition to dNTPs. Methodology/Principal Findings: We examined the ability of recombinant wild type (wt) and mutant RTs with substitutions at residue K65 to utilize a dNDP substrate in primer extension reactions. We found that wild type HIV-1 RT indeed catalyzes incorporation of dNDP substrates whereas RT with mutations of residue K645 were unable to catalyze this reaction. Wild type HIV-1 RT also catalyzed the reverse reaction, inorganic phosphate-dependent phosphorolysis. Nucleotide-mediated phosphorolytic removal of chain-terminating 3′-terminal nucleoside inhibitors such as AZT forms the basis of HIV-1 resistance to such drugs, and this removal is enhanced by thymidine analog mutations (TAMs). We found that both wt and TAM-containing RTs were able to catalyze Pi-mediated phosphorolysis of 3′-terminal AZT at physiological levels of Pi with an efficacy similar to that for ATP-dependent AZT-excision. Conclusion: We have identified two new catalytic function of HIV-1 RT, the use of dNDPs as substrates for DNA synthesis, and the use of Pi as substrate for phosphorolytic removal of primer 3′-terminal nucleotides. The ability to insert dNDPs has been documented for only one other DNA polymerase The RB69 DNA polymerase and the reverse reaction employing inorganic phosphate has not been documented for any DNA polymerase. Importantly, our results show that Pi-mediated phosphorolysis can contribute to AZT resistance and indicates that factors that influence HIV resistance to AZT are more complex than previously appreciated. © 2008 Garforth et al

MACI - a new era?

Full thickness articular cartilage defects have limited regenerative potential and are a significant source of pain and loss of knee function. Numerous treatment options exist, each with their own advantages and drawbacks. The goal of this review is to provide an overview of the problem of cartilage injury, a brief description of current treatment options and outcomes, and a discussion of the current principles and technique of Matrix-induced Autologous Chondrocyte Implantation (MACI). While early results of MACI have been promising, there is currently insufficient comparative and long-term outcome data to demonstrate superiority of this technique over other methods for cartilage repair

Techniques for measuring aerosol attenuation using the Central Laser Facility at the Pierre Auger Observatory

The Pierre Auger Observatory in Malargüe, Argentina, is designed to study the properties of ultra-high energy cosmic rays with energies above 10(18) eV. It is a hybrid facility that employs a Fluorescence Detector to perform nearly calorimetric measurements of Extensive Air Shower energies. To obtain reliable calorimetric information from the FD, the atmospheric conditions at the observatory need to be continuously monitored during data acquisition. In particular, light attenuation due to aerosols is an important atmospheric correction. The aerosol concentration is highly variable, so that the aerosol attenuation needs to be evaluated hourly. We use light from the Central Laser Facility, located near the center of the observatory site, having an optical signature comparable to that of the highest energy showers detected by the FD. This paper presents two procedures developed to retrieve the aerosol attenuation of fluorescence light from CLF laser shots. Cross checks between the two methods demonstrate that results from both analyses are compatible, and that the uncertainties are well understood. The measurements of the aerosol attenuation provided by the two procedures are currently used at the Pierre Auger Observatory to reconstruct air shower data

The rapid atmospheric monitoring system of the Pierre Auger Observatory

The Pierre Auger Observatory is a facility built to detect air showers produced by cosmic rays above 10(17) eV. During clear nights with a low illuminated moon fraction, the UV fluorescence light produced by air showers is recorded by optical telescopes at the Observatory. To correct the observations for variations in atmospheric conditions, atmospheric monitoring is performed at regular intervals ranging from several minutes (for cloud identification) to several hours (for aerosol conditions) to several days (for vertical profiles of temperature, pressure, and humidity). In 2009, the monitoring program was upgraded to allow for additional targeted measurements of atmospheric conditions shortly after the detection of air showers of special interest, e. g., showers produced by very high-energy cosmic rays or showers with atypical longitudinal profiles. The former events are of particular importance for the determination of the energy scale of the Observatory, and the latter are characteristic of unusual air shower physics or exotic primary particle types. The purpose of targeted (or 'rapid') monitoring is to improve the resolution of the atmospheric measurements for such events. In this paper, we report on the implementation of the rapid monitoring program and its current status. The rapid monitoring data have been analyzed and applied to the reconstruction of air showers of high interest, and indicate that the air fluorescence measurements affected by clouds and aerosols are effectively corrected using measurements from the regular atmospheric monitoring program. We find that the rapid monitoring program has potential for supporting dedicated physics analyses beyond the standard event reconstruction

The Rapid Atmospheric Monitoring System of the Pierre Auger Observatory

The Pierre Auger Observatory is a facility built to detect air showers produced by cosmic rays above 10^17 eV. During clear nights with a low illuminated moon fraction, the UV fluorescence light produced by air showers is recorded by optical telescopes at the Observatory. To correct the observations for variations in atmospheric conditions, atmospheric monitoring is performed at regular intervals ranging from several minutes (for cloud identification) to several hours (for aerosol conditions) to several days (for vertical profiles of temperature, pressure, and humidity). In 2009, the monitoring program was upgraded to allow for additional targeted measurements of atmospheric conditions shortly after the detection of air showers of special interest, e.g., showers produced by very high-energy cosmic rays or showers with atypical longitudinal profiles. The former events are of particular importance for the determination of the energy scale of the Observatory, and the latter are characteristic of unusual air shower physics or exotic primary particle types. The purpose of targeted (or "rapid") monitoring is to improve the resolution of the atmospheric measurements for such events. In this paper, we report on the implementation of the rapid monitoring program and its current status. The rapid monitoring data have been analyzed and applied to the reconstruction of air showers of high interest, and indicate that the air fluorescence measurements affected by clouds and aerosols are effectively corrected using measurements from the regular atmospheric monitoring program. We find that the rapid monitoring program has potential for supporting dedicated physics analyses beyond the standard event reconstruction

Trigger and Aperture of the Surface Detector Array of the Pierre Auger Observatory

The surface detector array of the Pierre Auger Observatory consists of 1600 water-Cherenkov detectors, for the study of extensive air showers (EAS) generated by ultra-high-energy cosmic rays. We describe the trigger hierarchy, from the identification of candidate showers at the level of a single detector, amongst a large background (mainly random single cosmic ray muons), up to the selection of real events and the rejection of random coincidences. Such trigger makes the surface detector array fully efficient for the detection of EAS with energy above $3\times 10^{18}$ eV, for all zenith angles between 0$^\circ$ and 60$^\circ$, independently of the position of the impact point and of the mass of the primary particle. In these range of energies and angles, the exposure of the surface array can be determined purely on the basis of the geometrical acceptance.Comment: 29 pages, 12 figure

Role of biomechanics in the understanding of normal, injured, and healing ligaments and tendons

Ligaments and tendons are soft connective tissues which serve essential roles for biomechanical function of the musculoskeletal system by stabilizing and guiding the motion of diarthrodial joints. Nevertheless, these tissues are frequently injured due to repetition and overuse as well as quick cutting motions that involve acceleration and deceleration. These injuries often upset this balance between mobility and stability of the joint which causes damage to other soft tissues manifested as pain and other morbidity, such as osteoarthritis

Ultrahigh energy neutrinos at the Pierre Auger observatory

The observation of ultrahigh energy neutrinos (UHEνs) has become a priority in experimental astroparticle physics. UHEνs can be detected with a variety of techniques. In particular, neutrinos can interact in the atmosphere (downward-going ν) or in the Earth crust (Earth-skimming ν), producing air showers that can be observed with arrays of detectors at the ground. With the surface detector array of the Pierre Auger Observatory we can detect these types of cascades. The distinguishing signature for neutrino events is the presence of very inclined showers produced close to the ground (i.e., after having traversed a large amount of atmosphere). In this work we review the procedure and criteria established to search for UHEνs in the data collected with the ground array of the Pierre Auger Observatory. This includes Earth-skimming as well as downward-going neutrinos. No neutrino candidates have been found, which allows us to place competitive limits to the diffuse flux of UHEνs in the EeV range and above.P. Abreu ... K. B. Barber ... J. A. Bellido ... R. W. Clay ... M. J. Cooper ... B. R. Dawson ... T. A. Harrison ... A. E. Herve ... V. C. Holmes ... J. Sorokin ... P. Wahrlich ... B. J. Whelan ... et al

Epigenetic investigation of variably X chromosome inactivated genes in monozygotic female twins discordant for primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an autoimmune chronic cholestatic liver disease with a strong genetic susceptibility due to the high concordance in monozygotic (MZ) twins and a striking female predominance. Women with PBC manifest an enhanced X monosomy rate in peripheral lymphocytes and we thus hypothesized an X chromosome epigenetic component to explain PBC female prevalence. While most genes on the female inactive X chromosome are silenced by promoter methylation following X chromosome inactivation (XCI), approximately 10% of X-linked genes exhibit variable escape from XCI in healthy females. This study was designed to test the hypothesis that susceptibility to PBC is modified by one or more X-linked gene with variable XCI status. Peripheral blood mRNA and DNA samples were obtained from a unique cohort of MZ twin sets discordant and concordant for PBC. Transcript levels of the 125 variable XCI status genes were determined by quantitative RT-PCR analysis and two genes (CLIC2 and PIN4) were identified as consistently downregulated in the affected twin of discordant pairs. Both CLIC2 and PIN4 demonstrated partial and variable methylation of CpG sites within 300 bp of the transcription start site that did not predict the XCI status. Promoter methylation of CLIC2 manifested no significant difference between samples and no significant correlation with transcript levels. PIN4 methylation showed a positive trend with transcription in all samples but no differential methylation was observed between discordant twins. A genetic polymorphism affecting the number of CpG sites in the PIN4 promoter did not impact methylation or transcript levels in a heterozygous twin pair and showed a similar frequency in independent series of unrelated PBC cases and controls. Our results suggest that epigenetic factors influencing PBC onset are more complex than methylation differences at X-linked promoters and variably inactivated X-linked genes may be characterized by partial promoter methylation and biallelic transcription