Single Molecule Analysis of Replicated DNA Reveals the Usage of Multiple KSHV Genome Regions for Latent Replication

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences

Borrelia valaisiana resist complement-mediated killing independently of the recruitment of immune regulators and inactivation of complement components

Spirochetes belonging to the Borrelia (B.) burgdorferi sensu lato complex differ in their resistance to complement-mediated killing, particularly in regard to human serum. In the present study, we elucidate the serum and complement susceptibility of B. valaisiana, a genospecies with the potential to cause Lyme disease in Europe as well as in Asia. Among the investigated isolates, growth of ZWU3 Ny3 was not affected while growth of VS116 and Bv9 was strongly inhibited in the presence of 50% human serum. Analyzing complement activation, complement components C3, C4 and C6 were deposited on the surface of isolates VS116 and Bv9, and similarly the membrane attack complex was formed on their surface. In contrast, no surface-deposited components and no aberrations in cell morphology were detected for serum-resistant ZWU3 Ny3. While further investigating the protective role of bound complement regulators in mediating complement resistance, we discovered that none of the B. valaisiana isolates analyzed bound complement regulators Factor H, Factor H-like protein 1, C4b binding protein or C1 esterase inhibitor. In addition, B. valaisiana also lacked intrinsic proteolytic activity to degrade complement components C3, C3b, C4, C4b, and C5. Taken together, these findings suggest that certain B. valaisiana isolates differ in their capability to resist complement-mediating killing by human serum. The molecular mechanism utilized by B. valaisiana to inhibit bacteriolysis appears not to involve binding of the key host complement regulators of the alternative, classical, and lectin pathways as already known for serum-resistant Lyme disease or relapsing fever borreliae

Structural issues affecting mixed methods studies in health research: a qualitative study

<p>Abstract</p> <p>Background</p> <p>Health researchers undertake studies which combine qualitative and quantitative methods. Little attention has been paid to the structural issues affecting this mixed methods approach. We explored the facilitators and barriers to undertaking mixed methods studies in health research.</p> <p>Methods</p> <p>Face-to-face semi-structured interviews with 20 researchers experienced in mixed methods research in health in the United Kingdom.</p> <p>Results</p> <p>Structural facilitators for undertaking mixed methods studies included a perception that funding bodies promoted this approach, and the multidisciplinary constituency of some university departments. Structural barriers to exploiting the potential of these studies included a lack of education and training in mixed methods research, and a lack of templates for reporting mixed methods articles in peer-reviewed journals. The 'hierarchy of evidence' relating to effectiveness studies in health care research, with the randomised controlled trial as the gold standard, appeared to pervade the health research infrastructure. Thus integration of data and findings from qualitative and quantitative components of mixed methods studies, and dissemination of integrated outputs, tended to occur through serendipity and effort, further highlighting the presence of structural constraints. Researchers are agents who may also support current structures - journal reviewers and editors, and directors of postgraduate training courses - and thus have the ability to improve the structural support for exploiting the potential of mixed methods research.</p> <p>Conclusion</p> <p>The environment for health research in the UK appears to be conducive to mixed methods research but not to exploiting the potential of this approach. Structural change, as well as change in researcher behaviour, will be necessary if researchers are to fully exploit the potential of using mixed methods research.</p

Synergistic ecoclimate teleconnections from forest loss in different regions structure global ecological responses

ABSTRACT: Forest loss in hotspots around the world impacts not only local climate where loss occurs, but also influences climate and vegetation in remote parts of the globe through ecoclimate teleconnections. The magnitude and mechanism of remote impacts likely depends on the location and distribution of forest loss hotspots, but the nature of these dependencies has not been investigated. We use global climate model simulations to estimate the distribution of ecologically-relevant climate changes resulting from forest loss in two hotspot regions: western North America (wNA), which is experiencing accelerated dieoff, and the Amazon basin, which is subject to high rates of deforestation. The remote climatic and ecological net effects of simultaneous forest loss in both regions differed from the combined effects of loss from the two regions simulated separately, as evident in three impacted areas. Eastern South American Gross Primary Productivity (GPP) increased due to changes in seasonal rainfall associated with Amazon forest loss and changes in temperature related to wNA forest loss. Eurasia’s GPP declined with wNA forest loss due to cooling temperatures increasing soil ice volume. Southeastern North American productivity increased with simultaneous forest loss, but declined with only wNA forest loss due to changes in VPD. Our results illustrate the need for a new generation of local-to-global scale analyses to identify potential ecoclimate teleconnections, their underlying mechanisms, and most importantly, their synergistic interactions, to predict the responses to increasing forest loss under future land use change and climate change

Reliability of goniometric measurements in children with cerebral palsy: A comparative analysis of universal goniometer and electronic inclinometer. A pilot study

<p>Abstract</p> <p>Background</p> <p>Even though technological progress has provided us with more and more sophisticated equipment for making goniometric measurements, the most commonly used clinical tools are still the universal goniometer and, to a lesser extent, the inclinometer. There is, however, no published study so far that uses an inclinometer for measurements in children with cerebral palsy (CP). The objective of this study was two-fold: to independently assess the intra and inter-examiner reliability for measuring the hip abduction range of motion in children with CP using two different instruments, the universal two-axis goniometer and electronic inclinometer. A pool of 5 examiners with different levels of experience as paediatric physiotherapists participated. The study did not compare both instruments because the measurement procedure and the hip position were different for each.</p> <p>Methods</p> <p>A prospective, observational study of goniometery was carried out with 14 lower extremities of 7 children with spastic CP. The inclinometer study was carried out with 8 lower extremities of 4 children with spastic CP. This study was divided into two independent parts: a study of the reliability of the hip abduction range of motion measured with a universal goniometer (hip at 0° flexion) and with an electronic inclinometer (hip at 90° flexion). The Intraclass Correlation Coefficient (ICC) was calculated to analyse intra and inter-examiner agreement for each instrument.</p> <p>Results</p> <p>For the goniometer, the intra-examiner reliability was excellent (>0.80), while the inter-examiner reliability was low (0.375 and 0.475). For the inclinometer, both the intra-examiner (0.850-0.975) and inter-examiner reliability were excellent (0.965 and 0.979).</p> <p>Conclusions</p> <p>The inter-examiner reliability for goniometric measurement of hip abduction in children with CP was low, in keeping with other results found in previous publications. The inclinometer has proved to be a highly reliable tool for measuring the hip abduction range of motion in children with CP, which opens up new possibilities in this field, despite having some measurement limitations.</p

Sensory Processing of Motor Inaccuracy Depends on Previously Performed Movement and on Subsequent Motor Corrections: A Study of the Saccadic System

When goal-directed movements are inaccurate, two responses are generated by the brain: a fast motor correction toward the target and an adaptive motor recalibration developing progressively across subsequent trials. For the saccadic system, there is a clear dissociation between the fast motor correction (corrective saccade production) and the adaptive motor recalibration (primary saccade modification). Error signals used to trigger corrective saccades and to induce adaptation are based on post-saccadic visual feedback. The goal of this study was to determine if similar or different error signals are involved in saccadic adaptation and in corrective saccade generation. Saccadic accuracy was experimentally altered by systematically displacing the visual target during motor execution. Post-saccadic error signals were studied by manipulating visual information in two ways. First, the duration of the displaced target after primary saccade termination was set at 15, 50, 100 or 800 ms in different adaptation sessions. Second, in some sessions, the displaced target was followed by a visual mask that interfered with visual processing. Because they rely on different mechanisms, the adaptation of reactive saccades and the adaptation of voluntary saccades were both evaluated. We found that saccadic adaptation and corrective saccade production were both affected by the manipulations of post-saccadic visual information, but in different ways. This first finding suggests that different types of error signal processing are involved in the induction of these two motor corrections. Interestingly, voluntary saccades required a longer duration of post-saccadic target presentation to reach the same amount of adaptation as reactive saccades. Finally, the visual mask interfered with the production of corrective saccades only during the voluntary saccades adaptation task. These last observations suggest that post-saccadic perception depends on the previously performed action and that the differences between saccade categories of motor correction and adaptation occur at an early level of visual processing

Mild Transient Hypercapnia as a Novel Fear Conditioning Stimulus Allowing Re-Exposure during Sleep

Introduction:Studies suggest that sleep plays a role in traumatic memories and that treatment of sleep disorders may help alleviate symptoms of posttraumatic stress disorder. Fear-conditioning paradigms in rodents are used to investigate causal mechanisms of fear acquisition and the relationship between sleep and posttraumatic behaviors. We developed a novel conditioning stimulus (CS) that evoked fear and was subsequently used to study re-exposure to the CS during sleep.Methods:Experiment 1 assessed physiological responses to a conditioned stimulus (mild transient hypercapnia, mtHC; 3.0% CO2; n = 17)+footshock for the purpose of establishing a novel CS in male FVB/J mice. Responses to the novel CS were compared to tone+footshock (n = 18) and control groups of tone alone (n = 17) and mild transient hypercapnia alone (n = 10). A second proof of principle experiment re-exposed animals during sleep to mild transient hypercapnia or air (control) to study sleep processes related to the CS.Results:Footshock elicited a response of acute tachycardia (30-40 bpm) and increased plasma epinephrine. When tone predicted footshock it elicited mild hypertension (1-2 mmHg) and a three-fold increase in plasma epinephrine. When mtHC predicted footshock it also induced mild hypertension, but additionally elicited a conditioned bradycardia and a smaller increase in plasma epinephrine. The overall mean 24 hour sleep-wake profile was unaffected immediately after fear conditioning.Discussion:Our study demonstrates the efficacy of mtHC as a conditioning stimulus that is perceptible but innocuous (relative to tone) and applicable during sleep. This novel model will allow future studies to explore sleep-dependent mechanisms underlying maladaptive fear responses, as well as elucidate the moderators of the relationship between fear responses and sleep. © 2013 McDowell et al

Using indirect methods to constrain symbiotic nitrogen fixation rates : a case study from an Amazonian rain forest

© The Authors 2009. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License. The definitive version was published in Biogeochemistry 99 (2010): 1-13, doi:10.1007/s10533-009-9392-y.Human activities have profoundly altered the global nitrogen (N) cycle. Increases in anthropogenic N have had multiple effects on the atmosphere, on terrestrial, freshwater and marine ecosystems, and even on human health. Unfortunately, methodological limitations challenge our ability to directly measure natural N inputs via biological N fixation (BNF)—the largest natural source of new N to ecosystems. This confounds efforts to quantify the extent of anthropogenic perturbation to the N cycle. To address this gap, we used a pair of indirect methods—analytical modeling and N balance—to generate independent estimates of BNF in a presumed hotspot of N fixation, a tropical rain forest site in central Rondônia in the Brazilian Amazon Basin. Our objectives were to attempt to constrain symbiotic N fixation rates in this site using indirect methods, and to assess strengths and weaknesses of this approach by looking for areas of convergence and disagreement between the estimates. This approach yielded two remarkably similar estimates of N fixation. However, when compared to a previously published bottom-up estimate, our analysis indicated much lower N inputs via symbiotic BNF in the Rondônia site than has been suggested for the tropics as a whole. This discrepancy may reflect errors associated with extrapolating bottom-up fluxes from plot-scale measures, those resulting from the indirect analyses, and/or the relatively low abundance of legumes at the Rondônia site. While indirect methods have some limitations, we suggest that until the technological challenges of directly measuring N fixation are overcome, integrated approaches that employ a combination of model-generated and empirically-derived data offer a promising way of constraining N inputs via BNF in natural ecosystems.We acknowledge and are grateful for financial support from the Andrew W. Mellon Foundation (C.C. and B.H.), the National Science Foundation (NSF DEB-0515744 to C.C. and A.T. and DEB-0315656 to C.N.), and the NASA LBA Program (NCC5-285 to C.N.)