Ubicación de la secuencia repetitiva PFCOL692 en fragmentos genómicos de Plasmodíum falciparum

In this paper we established the subtelomeric localization of the repetitive sequence PFCOL692 of Plasmodium falciparum by the characterization of four clones from a hEMBL41 PFCOL692 genomic library, which contains 15-23Kbp long inserts of parasite genomic DNA. Characterization was made by restriction analysis and the PFCOL692 localization in the genome was explored using specific probes for telomeric (pTB4.1) and subtelomeric regions (pRep20) of P falciparum chromosomes. We proposed a possible organization for the parasite chromosomes ends, where the copies of PFCOL692 are clusters in the subtelomeric region and their localization is conserved with respect to the pRep20 sequence. In addition, we established that PFCOL692 is not located next to the telomere.Las secuencias repetitivas son componentes estructurales de los genomas eucariotes. Se desconoce la función de la mayoría de ellas, pero, al parecer, no son simplemente ADN egoísta sino que intervienen en procesos de recombinación y regulación génica. En este estudio se estableció la localización subtelomérica dela secuencia repetitiva PFCOL692 en los cromosomas de Plasmodium falciparum, a través de la caracterización de cuatro clones de la genoteca ?EMBL4/PFCOL692, que contenían insertos entre 15 y 23Kb de ADN genómico del parásito; esta caracterización se hizo mediante análisis de restricción y la posible ubicación de PFCOL692 en el genoma se exploró utilizando sondas específicas para las regiones telomérica (pTB4.1) y subtelomérica (pRep20) de los cromosomas de P. falciparum. El análisis de los mapas de restricción obtenidos permitió plantear una posible ubicación de PFCOL692 en el extremo de los cromosomas del parásito, donde las copias de esta secuencia se encuentran agrupadas en un segmento del subtelómero y con una posición conservada con respecto a la secuencia pRep20 Se sugiere, además, que PFCOL692 no se encuentra en el limite entre el subtelómero y el telómero

Preliminary study of the enzyme ubiquitin carboxylterminal hydrolase 14 (UBP6) in Giardia intestinalis: structural bioinformatic analysis and transcriptional profile during encystation

This paper presents a combined approachwith two aims. The first is to analyze thereported sequence of the enzyme ubiquitincarboxyl-terminal hydrolase 14 of Giardiaintestinalis (UBP6) through computationalmethods to find components related withits hypothetical function. The second isto determine if the protein-coding gene isexpressed in G. intestinalis and, if such isthe case, also determine its transcriptionpattern along the life cycle of the parasite. Itwas established that the protein belongs tothe family of Cys-dependent deubiquitinasesand more specifically to ubiquitin specificproteases (USPs). Moreover, the catalyticcenter with the complete triad as well astypical features of the USP motif were alsoidentified. Since the computational findingssuggest that the enzyme could be functional,reverse transcription coupled to PCR wasused as a first approach to establish if in factthe coding gene is expressed in the parasite.Interestingly, it was found not only thatthe gene is expressed, but also that thereis a transcription variation along the lifecycle of the parasite. These two findings arethe starting point for further studies sincethey tentatively suggest that this enzymecould be involved in the protein turnoverthat occurs during parasite encystation.Although preliminary, this study is the firstreport concerning the study of a specificdeubiquitinating enzyme in the parasite G.intestinalis

Cost analysis of centralized viral load testing for antiretroviral therapy monitoring in Nicaragua, a low-HIV prevalence, low-resource setting

<p>Abstract</p> <p>Background</p> <p>HIV viral load testing as a component of antiretroviral therapy monitoring is costly. Understanding the full costs and the major sources of inefficiency associated with viral load testing is critical for optimizing the systems and technologies that support the testing process. The objective of our study was to estimate the costs associated with viral load testing performed for antiretroviral therapy monitoring to both patients and the public healthcare system in a low-HIV prevalence, low-resource country.</p> <p>Methods</p> <p>A detailed cost analysis was performed to understand the costs involved in each step of performing a viral load test in Nicaragua, from initial specimen collection to communication of the test results to each patient's healthcare provider. Data were compiled and cross referenced from multiple information sources: laboratory records, regional surveillance centre records, and scheduled interviews with the key healthcare providers responsible for HIV patient care in five regions of the country.</p> <p>Results</p> <p>The total average cost of performing a viral load test in Nicaragua varied by region, ranging from US$99.01 to US$124.58, the majority of which was at the laboratory level: $88.73 to$97.15 per specimen, depending on batch size. The average cost to clinics at which specimens were collected ranged from $3.31 to$20.92, depending on the region. The average cost per patient for transportation, food, lodging and lost income ranged from $3.70 to$14.93.</p> <p>Conclusions</p> <p>The quantitative viral load test remains the single most expensive component of the process. For the patient, the distance of his or her residence from the specimen collection site is a large determinant of cost. Importantly, the efficiency of results reporting has a large impact on the cost per result delivered to the clinician and utility of the result for patient monitoring. Detailed cost analysis can identify opportunities for removing barriers to effective antiretroviral therapy monitoring programmes in limited-resource countries with low HIV prevalence.</p

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Transcription of meiotic-like-pathway genes in Giardia intestinalis

The reproductive mechanism of Giardia intestinalis, considered one of the earliest divergent eukaryotes, has not been fully defined yet. Some evidence supports the hypothesis that Giardia is an exclusively asexual organism with a clonal population structure. However, the high genetic variability, the variation in ploidy during its life cycle, the low heterozygosity and the existence of genes involved in the meiotic-like recombination pathway in the parasite's genome cast doubt on exclusively asexual nature of Giardia. In this work, semiquantitative RT-PCR analysis was used to assess the transcription pattern of three meiosis-like-specific genes involved in homologues recombination: dmc1, hop1 and spo11. The mRNAs were amplified during the parasite's differentiation processes, encystation and excystation, and expression was found at each stage of its life cycle. A semiquantitative assessment also suggests that expression of some of the genes is regulated during encystation process

Odanacatib for the treatment of postmenopausal osteoporosis : Results of the LOFT multicentre, randomised, double-blind, placebo-controlled trial and LOFT Extension study

Background Odanacatib, a cathepsin K inhibitor, reduces bone resorption while maintaining bone formation. Previous work has shown that odanacatib increases bone mineral density in postmenopausal women with low bone mass. We aimed to investigate the efficacy and safety of odanacatib to reduce fracture risk in postmenopausal women with osteoporosis. Methods The Long-term Odanacatib Fracture Trial (LOFT) was a multicentre, randomised, double-blind, placebo-controlled, event-driven study at 388 outpatient clinics in 40 countries. Eligible participants were women aged at least 65 years who were postmenopausal for 5 years or more, with a femoral neck or total hip bone mineral density T-score between −2·5 and −4·0 if no previous radiographic vertebral fracture, or between −1·5 and −4·0 with a previous vertebral fracture. Women with a previous hip fracture, more than one vertebral fracture, or a T-score of less than −4·0 at the total hip or femoral neck were not eligible unless they were unable or unwilling to use approved osteoporosis treatment. Participants were randomly assigned (1:1) to either oral odanacatib (50 mg once per week) or matching placebo. Randomisation was done using an interactive voice recognition system after stratification for previous radiographic vertebral fracture, and treatment was masked to study participants, investigators and their staff, and sponsor personnel. If the study completed before 5 years of double-blind treatment, consenting participants could enrol in a double-blind extension study (LOFT Extension), continuing their original treatment assignment for up to 5 years from randomisation. Primary endpoints were incidence of vertebral fractures as assessed using radiographs collected at baseline, 6 and 12 months, yearly, and at final study visit in participants for whom evaluable radiograph images were available at baseline and at least one other timepoint, and hip and non-vertebral fractures adjudicated as being a result of osteoporosis as assessed by clinical history and radiograph. Safety was assessed in participants who received at least one dose of study drug. The adjudicated cardiovascular safety endpoints were a composite of cardiovascular death, myocardial infarction, or stroke, and new-onset atrial fibrillation or flutter. Individual cardiovascular endpoints and death were also assessed. LOFT and LOFT Extension are registered with ClinicalTrials.gov (number NCT00529373) and the European Clinical Trials Database (EudraCT number 2007-002693-66). Findings Between Sept 14, 2007, and Nov 17, 2009, we randomly assigned 16 071 evaluable patients to treatment: 8043 to odanacatib and 8028 to placebo. After a median follow-up of 36·5 months (IQR 34·43–40·15) 4297 women assigned to odanacatib and 3960 assigned to placebo enrolled in LOFT Extension (total median follow-up 47·6 months, IQR 35·45–60·06). In LOFT, cumulative incidence of primary outcomes for odanacatib versus placebo were: radiographic vertebral fractures 3·7% (251/6770) versus 7·8% (542/6910), hazard ratio (HR) 0·46, 95% CI 0·40–0·53; hip fractures 0·8% (65/8043) versus 1·6% (125/8028), 0·53, 0·39–0·71; non-vertebral fractures 5·1% (412/8043) versus 6·7% (541/8028), 0·77, 0·68–0·87; all p<0·0001. Combined results from LOFT plus LOFT Extension for cumulative incidence of primary outcomes for odanacatib versus placebo were: radiographic vertebral fractures 4·9% (341/6909) versus 9·6% (675/7011), HR 0·48, 95% CI 0·42–0·55; hip fractures 1·1% (86/8043) versus 2·0% (162/8028), 0·52, 0·40–0·67; non-vertebral fractures 6·4% (512/8043) versus 8·4% (675/8028), 0·74, 0·66–0·83; all p<0·0001. In LOFT, the composite cardiovascular endpoint of cardiovascular death, myocardial infarction, or stroke occurred in 273 (3·4%) of 8043 patients in the odanacatib group versus 245 (3·1%) of 8028 in the placebo group (HR 1·12, 95% CI 0·95–1·34; p=0·18). New-onset atrial fibrillation or flutter occurred in 112 (1·4%) of 8043 patients in the odanacatib group versus 96 (1·2%) of 8028 in the placebo group (HR 1·18, 0·90–1·55; p=0·24). Odanacatib was associated with an increased risk of stroke (1·7% [136/8043] vs 1·3% [104/8028], HR 1·32, 1·02–1·70; p=0·034), but not myocardial infarction (0·7% [60/8043] vs 0·9% [74/8028], HR 0·82, 0·58–1·15; p=0·26). The HR for all-cause mortality was 1·13 (5·0% [401/8043] vs 4·4% [356/8028], 0·98–1·30; p=0·10). When data from LOFT Extension were included, the composite of cardiovascular death, myocardial infarction, or stroke occurred in significantly more patients in the odanacatib group than in the placebo group (401 [5·0%] of 8043 vs 343 [4·3%] of 8028, HR 1·17, 1·02–1·36; p=0·029, as did stroke (2·3% [187/8043] vs 1·7% [137/8028], HR 1·37, 1·10–1·71; p=0·0051). Interpretation Odanacatib reduced the risk of fracture, but was associated with an increased risk of cardiovascular events, specifically stroke, in postmenopausal women with osteoporosis. Based on the overall balance between benefit and risk, the study's sponsor decided that they would no longer pursue development of odanacatib for treatment of osteoporosis