546 research outputs found
A mutate-and-map protocol for inferring base pairs in structured RNA
Chemical mapping is a widespread technique for structural analysis of nucleic
acids in which a molecule's reactivity to different probes is quantified at
single-nucleotide resolution and used to constrain structural modeling. This
experimental framework has been extensively revisited in the past decade with
new strategies for high-throughput read-outs, chemical modification, and rapid
data analysis. Recently, we have coupled the technique to high-throughput
mutagenesis. Point mutations of a base-paired nucleotide can lead to exposure
of not only that nucleotide but also its interaction partner. Carrying out the
mutation and mapping for the entire system gives an experimental approximation
of the molecules contact map. Here, we give our in-house protocol for this
mutate-and-map strategy, based on 96-well capillary electrophoresis, and we
provide practical tips on interpreting the data to infer nucleic acid
structure.Comment: 22 pages, 5 figure
RNA secondary structure prediction from multi-aligned sequences
It has been well accepted that the RNA secondary structures of most
functional non-coding RNAs (ncRNAs) are closely related to their functions and
are conserved during evolution. Hence, prediction of conserved secondary
structures from evolutionarily related sequences is one important task in RNA
bioinformatics; the methods are useful not only to further functional analyses
of ncRNAs but also to improve the accuracy of secondary structure predictions
and to find novel functional RNAs from the genome. In this review, I focus on
common secondary structure prediction from a given aligned RNA sequence, in
which one secondary structure whose length is equal to that of the input
alignment is predicted. I systematically review and classify existing tools and
algorithms for the problem, by utilizing the information employed in the tools
and by adopting a unified viewpoint based on maximum expected gain (MEG)
estimators. I believe that this classification will allow a deeper
understanding of each tool and provide users with useful information for
selecting tools for common secondary structure predictions.Comment: A preprint of an invited review manuscript that will be published in
a chapter of the book `Methods in Molecular Biology'. Note that this version
of the manuscript may differ from the published versio
Design principles for riboswitch function
Scientific and technological advances that enable the tuning of integrated regulatory components to match network and system requirements are critical to reliably control the function of biological systems. RNA provides a promising building block for the construction of tunable regulatory components based on its rich regulatory capacity and our current understanding of the sequence–function relationship. One prominent example of RNA-based regulatory components is riboswitches, genetic elements that mediate ligand control of gene expression through diverse regulatory mechanisms. While characterization of natural and synthetic riboswitches has revealed that riboswitch function can be modulated through sequence alteration, no quantitative frameworks exist to investigate or guide riboswitch tuning. Here, we combined mathematical modeling and experimental approaches to investigate the relationship between riboswitch function and performance. Model results demonstrated that the competition between reversible and irreversible rate constants dictates performance for different regulatory mechanisms. We also found that practical system restrictions, such as an upper limit on ligand concentration, can significantly alter the requirements for riboswitch performance, necessitating alternative tuning strategies. Previous experimental data for natural and synthetic riboswitches as well as experiments conducted in this work support model predictions. From our results, we developed a set of general design principles for synthetic riboswitches. Our results also provide a foundation from which to investigate how natural riboswitches are tuned to meet systems-level regulatory demands
Symptom Dimensions in OCD: Item-Level Factor Analysis and Heritability Estimates
To reduce the phenotypic heterogeneity of obsessive-compulsive disorder (OCD) for genetic, clinical and translational studies, numerous factor analyses of the Yale-Brown Obsessive Compulsive Scale checklist (YBOCS-CL) have been conducted. Results of these analyses have been inconsistent, likely as a consequence of small sample sizes and variable methodologies. Furthermore, data concerning the heritability of the factors are limited. Item and category-level factor analyses of YBOCS-CL items from 1224 OCD subjects were followed by heritability analyses in 52 OCD-affected multigenerational families. Item-level analyses indicated that a five factor model: (1) taboo, (2) contamination/cleaning, (3) doubts, (4) superstitions/rituals, and (5) symmetry/hoarding provided the best fit, followed by a one-factor solution. All 5 factors as well as the one-factor solution were found to be heritable. Bivariate analyses indicated that the taboo and doubts factor, and the contamination and symmetry/hoarding factor share genetic influences. Contamination and symmetry/hoarding show shared genetic variance with symptom severity. Nearly all factors showed shared environmental variance with each other and with symptom severity. These results support the utility of both OCD diagnosis and symptom dimensions in genetic research and clinical contexts. Both shared and unique genetic influences underlie susceptibility to OCD and its symptom dimensions.Obsessive Compulsive FoundationTourette Syndrome AssociationAnxiety Disorders Association of AmericaAmerican Academy of Child and Adolescent Psychiatr
Neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-HSD type 4
<p>Abstract</p> <p>Background</p> <p>Steroids affect many tissues, including the brain. In the zebra finch, the estrogenic steroid estradiol (E<sub>2</sub>) is especially effective at promoting growth of the neural circuit specialized for song. In this species, only the males sing and they have a much larger and more interconnected song circuit than females. Thus, it was surprising that the gene for 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4), an enzyme that converts E<sub>2 </sub>to a less potent estrogen, had been mapped to the Z sex chromosome. As a consequence, it was likely that HSD17B4 was differentially expressed in males (ZZ) and females (ZW) because dosage compensation of Z chromosome genes is incomplete in birds. If a higher abundance of HSD17B4 mRNA in males than females was translated into functional enzyme in the brain, then contrary to expectation, males could produce less E<sub>2 </sub>in their brains than females.</p> <p>Results</p> <p>Here, we used molecular and biochemical techniques to confirm the HSD17B4 Z chromosome location in the zebra finch and to determine that HSD17B4 mRNA and activity were detectable in the early developing and adult brain. As expected, HSD17B4 mRNA expression levels were higher in males compared to females. This provides further evidence of the incomplete Z chromosome inactivation mechanisms in birds. We detected HSD17B4 mRNA in regions that suggested a role for this enzyme in the early organization and adult function of song nuclei. We did not, however, detect significant sex differences in HSD17B4 activity levels in the adult brain.</p> <p>Conclusions</p> <p>Our results demonstrate that the HSD17B4 gene is expressed and active in the zebra finch brain as an E<sub>2 </sub>metabolizing enzyme, but that dosage compensation of this Z-linked gene may occur via post-transcriptional mechanisms.</p
Role of previous hospitalization in clinically-significant MRSA infection among HIV-infected inpatients: results of a case-control study
<p>Abstract</p> <p>Background</p> <p>HIV-infected subjects have high incidence rates of <it>Staphylococcus aureus </it>infections, with both methicillin-susceptible and methicillin-resistant (MRSA) strains. Possible explanations could include the high burden of colonization, the behavioral risk factors, and the frequent exposures to health care facilities of HIV-infected patients. The purpose of the study was to assess the risk factors for clinically- significant methicillin-resistant <it>Staphylococcus aureus </it>(CS-MRSA) infections in HIV-infected patients admitted to Infectious Diseases Units.</p> <p>Methods</p> <p>From January 1, 2002 to December 31, 2005, we conducted a retrospective case-control (1:2) study. We identified all the cases of CS-MRSA infections in HIV-infected patients admitted to the National Institute for Infectious Diseases (INMI) "Lazzaro Spallanzani" in the 4-year study period. A conditional logistic regression model was used to identify risk factors for CS-MRSA infection.</p> <p>Results</p> <p>We found 27 CS-MRSA infections, i.e. 0.9 CS-MRSA infections per 100 HIV-infected individuals cared for in our Institute. At multivariate analysis, independent predictors of CS-MRSA infection were cumulative hospital stay, invasive procedures in the previous year, and low CD4 cell count. Particularly, the risk for CS-MRSA increased by 14% per an increase of 5 days hospitalization in the previous year. Finally, we identified a low frequency of community-acquired MRSA infections (only 1 of 27; 3.7%) among HIV-infected patients.</p> <p>Conclusion</p> <p>Clinicians should be aware of the risk for CS-MRSA infection in the clinical management of HIV-infected patients, especially in those patients with a low CD4 cell count, longer previous hospital stay, and previous invasive procedures.</p
Increased sensitivity for detecting malaria parasites in human umbilical cord blood using scaled-up DNA preparation
BACKGROUND: All mothers donating umbilical cord blood units to the NHS cord blood bank undergo an assessment for the likelihood of prior exposure to malaria infection. Those deemed at risk due to a history of travel to, or residence in, malaria endemic regions are screened serologically to detect anti-malaria antibodies. A positive result excludes the use of the cord blood for transplant therapy unless a risk assessment can ensure that malaria transmission is extremely unlikely. This paper details the screening of cord blood units from malaria serology positive mothers to detect malaria parasite DNA using a highly sensitive nested PCR. METHODS: Uninfected blood from a healthy volunteer was spiked with known quantities of malaria parasites and 5 millilitre and 200 microlitre aliquots were subjected to DNA extraction using QIAamp DNA maxi and DNA mini kits respectively. Nested PCR, to detect malarial SSU rRNA sequences, was performed on the purified DNA samples to determine the limit of detection for this assay with both extraction methodologies. Following assay validation, 54 cord blood units donated by mothers who were positive for anti-malaria antibodies were screened by this approach. RESULTS: When DNA was purified from 5 millilitres of blood it was possible to routinely detect as few as 50 malaria parasites per millilitre using nested PCR. This equates to a significant increase in the sensitivity of the current gold standard nucleic acid amplification technique used to detect malaria parasites (routinely performed from > 200 microlitre volumes of blood). None of the 54 donated cord blood units from serology positive mothers tested positive for malaria parasites using this scaled up DNA preparation method. CONCLUSION: Serological testing for malaria parasites may be overly conservative, leading to unnecessary rejection of cord blood donations that lack malaria parasites and which are, therefore, safe for use in stem cell therapy
Pilot, randomized, placebo-controlled clinical field study to evaluate the effectiveness of bupivacaine liposome injectable suspension for the provision of post-surgical analgesia in dogs undergoing stifle surgery
Abstract Background Local anesthetics are an important component of perioperative pain management, but the duration of action of available products is limited. We hypothesized that a single local infiltration of a novel bupivacaine liposome injectable suspension (AT-003) would provide clinically effective analgesia over a 72-h period. In a masked, randomized, placebo-controlled, multi-center pilot field study, dogs undergoing lateral retinacular suture placement for cranial cruciate insufficiency were randomly assigned to surgical site infiltration with AT-003 (5.3 mg/kg) or an equivalent volume of saline. Infiltration of the surgical site was done prior to closure. Primary outcome measure was the Glasgow Composite Measure Pain Scale (CMPS-SF) assessed prior to surgery and at 2, 4, 8, 12, 24, 30, 36, 48, 54, 60 and 72 h following surgery by trained individuals. Provision for rescue analgesia was employed. Repeated measures analysis of variance were utilized to test for possible differences between treatment groups and a success/failure analysis was also employed, based on the need for rescue analgesia. Results Forty-six dogs were enrolled and evaluated. For CMPS-SF scores there was a significant overall treatment effect (p = 0.0027) in favor of AT-003. There were significantly more successes in the AT-003 group compared to placebo over each time period (p = 0.0001 for 0–24 h, p = 0.0349 for 0–48 h, and p = 0.0240 for 0-72 h). No significant adverse events were seen. Conclusions AT-003 (bupivacaine liposome injectable suspension) provided measurable local analgesia over a 72-h period following post-stifle surgery surgical site tissue infiltration. Further work is indicated to develop this product for clinical use
Direct effects of diazepam on emotional processing in healthy volunteers
RATIONALE: Pharmacological agents used in the treatment of anxiety have been reported to decrease threat relevant processing in patients and healthy controls, suggesting a potentially relevant mechanism of action. However, the effects of the anxiolytic diazepam have typically been examined at sedative doses, which do not allow the direct actions on emotional processing to be fully separated from global effects of the drug on cognition and alertness. OBJECTIVES: The aim of this study was to investigate the effect of a lower, but still clinically effective, dose of diazepam on emotional processing in healthy volunteers. MATERIALS AND METHODS: Twenty-four participants were randomised to receive a single dose of diazepam (5 mg) or placebo. Sixty minutes later, participants completed a battery of psychological tests, including measures of non-emotional cognitive performance (reaction time and sustained attention) and emotional processing (affective modulation of the startle reflex, attentional dot probe, facial expression recognition, and emotional memory). Mood and subjective experience were also measured. RESULTS: Diazepam significantly modulated attentional vigilance to masked emotional faces and significantly decreased overall startle reactivity. Diazepam did not significantly affect mood, alertness, response times, facial expression recognition, or sustained attention. CONCLUSIONS: At non-sedating doses, diazepam produces effects on attentional vigilance and startle responsivity that are consistent with its anxiolytic action. This may be an underlying mechanism through which benzodiazepines exert their therapeutic effects in clinical anxiety
Clusters of galaxies : observational properties of the diffuse radio emission
Clusters of galaxies, as the largest virialized systems in the Universe, are
ideal laboratories to study the formation and evolution of cosmic
structures...(abridged)... Most of the detailed knowledge of galaxy clusters
has been obtained in recent years from the study of ICM through X-ray
Astronomy. At the same time, radio observations have proved that the ICM is
mixed with non-thermal components, i.e. highly relativistic particles and
large-scale magnetic fields, detected through their synchrotron emission. The
knowledge of the properties of these non-thermal ICM components has increased
significantly, owing to sensitive radio images and to the development of
theoretical models. Diffuse synchrotron radio emission in the central and
peripheral cluster regions has been found in many clusters. Moreover
large-scale magnetic fields appear to be present in all galaxy clusters, as
derived from Rotation Measure (RM) studies. Non-thermal components are linked
to the cluster X-ray properties, and to the cluster evolutionary stage, and are
crucial for a comprehensive physical description of the intracluster medium.
They play an important role in the cluster formation and evolution. We review
here the observational properties of diffuse non-thermal sources detected in
galaxy clusters: halos, relics and mini-halos. We discuss their classification
and properties. We report published results up to date and obtain and discuss
statistical properties. We present the properties of large-scale magnetic
fields in clusters and in even larger structures: filaments connecting galaxy
clusters. We summarize the current models of the origin of these cluster
components, and outline the improvements that are expected in this area from
future developments thanks to the new generation of radio telescopes.Comment: Accepted for the publication in The Astronomy and Astrophysics
Review. 58 pages, 26 figure
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