Performance of Ultra-Deep Pyrosequencing in Analysis of HIV-1 pol Gene Variation

INTRODUCTION: Ultra-deep pyrosequencing (UDPS) has been used to detect minority variants within HIV-1 populations. Some aspects of the quality and reproducibility of UDPS have been previously evaluated, but comprehensive studies are still needed. PRINCIPAL FINDING: In this study the UDPS technology (FLX platform) was evaluated by analyzing a 120 base pair fragment of the HIV-1 pol gene from plasma samples from two patients and artificial mixtures of molecular clones. UDPS was performed using an optimized experimental protocol and an in-house data cleaning strategy. Nine samples and mixtures were analyzed and the average number of reads per sample was 19,404 (range 8,858-26,846). The two patient plasma samples were analyzed twice and quantification of viral variants was found to be highly repeatable for variants representing >0.27% of the virus population, whereas some variants representing 0.11-0.27% were detected in only one of the two UDPS runs. Bland-Altman analysis showed that a repeated measurement would have a 95% likelihood to lie approximately within ±0.5 log(10) of the initial estimate. A similar level of agreement was observed for variant frequency estimates in forward vs. reverse sequencing direction, but here the agreement was higher for common variants than for rare variants. UDPS following PCR amplification with alternative primers indicated that some variants may be incorrectly quantified due to primer-related selective amplification. Finally, the in vitro recombination rate during PCR was evaluated using artificial mixtures of clones and was found to be low. The most abundant in vitro recombinant represented 0.25% of all UDPS reads. CONCLUSION: This study demonstrates that this UDPS protocol results in low experimental noise and high repeatability, which is relevant for future research and clinical use of the UDPS technology. The low rate of in vitro recombination suggests that this UDPS system can be used to study genetic variants and mutational linkage

Age of Child, More than HPV Type, Is Associated with Clinical Course in Recurrent Respiratory Papillomatosis

Background: RRP is a devastating disease in which papillomas in the airway cause hoarseness and breathing difficulty. The disease is caused by human papillomavirus (HPV), 6 or 11 and is very variable. Patients undergo multiple surgeries to maintain a patent airway and in order to communicate vocally. Several small studies have been published in which most have noted that HPV 11 is associated with a more aggressive course. Methodology/Principal Findings: Papilloma biopsies were taken from patients undergoing surgical treatment of RRP and were subjected to HPV typing. 118 patients with juvenile-onset RRP with a least 1 year of clinical data and infected with a single HPV type were analyzed. HPV 11 was encountered in 40% of the patients. By our definition, most of the patients in the sample (81%) had run an aggressive course. The odds of a patient with HPV 11 running an aggressive course were 3.9 times higher that that of patients with HPV 6 (Fisher's exact p=0.017). However, clinical course was more closely associated with age of the patient (at diagnosis and at the time of the current surgery) than with HPV type. Patients with HPV 11 were diagnosed at a younger age (2.4y) than were those with HPV 6 (3.4y) (p=0.014). Both by multiple linear regression and by multiple logistics regression HPV type was only weakly associated with metrics of disease course when simultaneously accounting for age. Conclusions/Significance Abstract: The course of RRP is variable and a quarter of the variability can be accounted for by the age of the patient. HPV 11 is more closely associated with a younger age at diagnosis than it is associated with an aggressive clinical course. These data suggest that there are factors other than HPV type and age of the patient that determine disease course. © 2008 Buchinsky et al

Interleukin-12p40 Modulates Human Metapneumovirus-Induced Pulmonary Disease in an Acute Mouse Model of Infection

The mechanisms that regulate the host immune response induced by human metapneumovirus (hMPV), a newly-recognized member of the Paramyxoviridae family, are largely unknown. Cytokines play an important role in modulating inflammatory responses during viral infections. IL-12p40, a known important mediator in limiting lung inflammation, is induced by hMPV and its production is sustained after the resolution phase of infection suggesting that this cytokine plays a role in the immune response against hMPV. In this work, we demonstrated that in mice deficient in IL-12p40, hMPV infection induced an exacerbated pulmonary inflammatory response and mucus production, altered cytokine response, and decreased lung function. However, hMPV infection in these mice does not have an effect on viral replication. These results identify an important regulatory role of IL-12p40 in hMPV infection

Percepção de estudantes de psicologia sobre a informática em sua formação profissional

Neste artigo analisa-se uma pesquisa de natureza qualitativa, cujo objetivo era explorar a percepção de 45 alunos de psicologia de diferentes universidades da cidade de Santiago do Chile, sobre a incorporação da informática como apoio para a aprendizagem em sua formação profissional. A informação foi coletada através de entrevistas semiestruturadas e analisou-se mediante o processo de codificação da Teoría Fundamentada. Os resultados mostram como categorias principais, o conhecimento da informática, a valoração da informática, e os fatores que obstaculizam o uso desta. Da codificação seletiva se desprende uma alta valoração da informática como ferramenta econômica e cômoda, mas também se observa uma alta reticência dos alunos diante de sua utilização no trabalho profissional tradicional do psicólogo. Encontraram-se fatores pessoais e institucionais nos que se baseia a valoração, a motivação e o nível de conhecimento dos estudantes diante do uso de ferramentas informáticas.This paper analyses a qualitative research aimed to exploring the perception of 45 psychology students from different universities in Santiago de Chile, about incorporating information systems to support learning in their professional training. Data were collected through semi structured interviews and their analysis was based on Grounded Theory's coding process. Results show that the major categories are computer knowledge, appreciation of computers and factors that hinder their use. From the selective coding a positive evaluation emerges of information systems as an economic and useful tool. However, there is also a high reluctance on the part of the students regarding the use of such tools in the psychologist' traditional professional work. Personal and organizational factors were found as bases for students' assessment, motivation and level of knowledge facing the use of these tools.En este artículo se analiza una investigación de naturaleza cualitativa, cuyo objetivo era explorar la percepción de 45 alumnos de psicología de diferentes universidades de la ciudad de Santiago de Chile, sobre la incorporación de la informática como apoyo para el aprendizaje en su formación profesional. La información se recolectó a través de entrevistas semiestructuradas y se analizó mediante el proceso de codificación de la Teoría Fundamentada. Los resultados muestran como categorías principales, el conocimiento de la informática, la valoración de la informática, y los factores que obstaculizan el uso de ésta. De la codificación selectiva se desprende una alta valoración de la informática como herramienta económica y cómoda, pero también se observa una alta reticencia de los alumnos frente a su utilización en el trabajo profesional tradicional del psicólogo. Se encontraron factores personales e institucionales en los cuales se fundamenta la valoración, la motivación y el nivel de conocimiento de los estudiantes frente al uso de herramientas informáticas

Impact of climate change on larch budmoth cyclic outbreaks

Periodic outbreaks of the larch budmoth Zeiraphera diniana population (and the massive forest defoliation they engender) have been recorded in the Alps over the centuries and are known for their remarkable regularity. But these have been conspicuously absent since 1981. On the other hand, budmoth outbreaks have been historically unknown in the larches of the Carpathian Tatra mountains. To resolve this puzzle, we propose here a model which includes the influence of climate and explains both the 8–9 year periodicity in the budmoth cycle and the variations from this, as well as the absence of cycles. We successfully capture the observed trend of relative frequencies of outbreaks, reproducing the dominant periodicities seen. We contend that the apparent collapse of the cycle in 1981 is due to changing climatic conditions following a tipping point and propose the recurrence of the cycle with a changed periodicity of 40 years – the next outbreak could occur in 2021. Our model also predicts longer cycles

Downregulation of drug transport and metabolism in mice bearing extra-hepatic malignancies

There is increasing evidence of a systemic inflammatory response associated with malignancy, which may have an impact on both drug disposition and resistance to cytotoxic therapy. The impact of inflammation on drug disposition was studied in mice bearing a number of common tumour xenografts. C57BL/6 mice were inoculated with tumour xenografts. Hepatic expressions of Cyp3a and drug transporters were analysed at the mRNA, protein and functional levels (Cyp3a only). Circulating serum cytokines and the hepatic expression of acute phase proteins (APPs) were measured. Intratumoral levels of multidrug resistance genes were determined. Tumour xenografts elicited an inflammatory response that coincided with repression in hepatic Cyp3a11 activity and the expression of a number of hepatic drug transporters. With tumour growth, a progressive reduction in hepatic Cyp3a11 mRNA expression was seen. Conversely, an increase in the hepatic APP expression and circulating interleukin (IL)-6 levels was observed. Furthermore, a correlation was seen between increased intratumoral expression of the multidrug resistance gene, Mdr1a, and levels of circulating IL-6. Malignancy results in reduced hepatic drug disposition that correlates with an associated inflammatory response. Reduction of inflammation may improve the clinical outcome for patients receiving chemotherapeutic agents that undergo hepatic metabolism

Proteins of Leishmania (Viannia) shawi confer protection associated with Th1 immune response and memory generation

<p>Abstract</p> <p>Background</p> <p><it>Leishmania (Viannia) shawi </it>parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA) from <it>L. (V.) shawi </it>promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained.</p> <p>Methods</p> <p>F1 fraction was purified from L. (V.) shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 μg of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated.</p> <p>Results</p> <p>The F1 fraction induced a high degree of protection associated with an increase in IFN-γ, a decrease in IL-4, increased cell proliferation and activation of CD8⁺T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4⁺central memory T lymphocytes and activation of both CD4⁺and CD8⁺T cells. In addition, F1-immunized groups showed an increase in IgG2a levels.</p> <p>Conclusions</p> <p>The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis.</p