Harmonization is more important than experience—results of the first Nordic–Baltic diatom intercalibration exercise 2007 (stream monitoring)

The goal of this study was a harmonization of diatom identification and counting among diatomists from the Scandinavian and Baltic countries to improve the comparison of diatom studies in this geographical area. An analysis of the results of 25 diatomists following the European standard EN 14407 during an intercalibration exercise showed that a high similarity was achieved by harmonization and not because of a long experience with diatoms. Sources of error were wrong calibration scales, overlooking of small taxa, especially small Navicula s.l., misidentifications (Eunotia rhomboidea was mistaken for Eunotia incisa) and unclear separation between certain taxa in the identification literature. The latter was discussed during a workshop with focus on the Achnanthes minutissima group, the separation of Fragilaria capucina var. gracilis from F. capucina var. rumpens, and Nitzschia palea var. palea from N. palea var. debilis. The exercise showed also that the Swedish standard diatom method tested here worked fine with acceptable error for the indices IPS (Indice de Polluo-sensibilité Spécifique) and ACID (ACidity Index for Diatoms) when diatomists with a low similarity (Bray–Curtis <60%) with the auditor in at least one of the samples are excluded.     

Changes in the quaternary structure of amelogenin when adsorbed onto surfaces

Amelogenin is a unique protein that self‐assembles into spherical aggregates called “nanospheres” and is believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin onto substrates is of great interest because protein‐surface interactions are critical to its function. We report studies of the adsorption of amelogenin onto self‐assembled monolayers containing COOH end group functionality as well as single crystal fluoroapatite, a biologically relevant surface. We found that although our solutions contained only nanospheres of narrow size distribution, smaller structures such as dimers or trimers were observed on the hydrophilic surfaces. This suggests that amelogenin can adsorb onto surfaces as small structures that “shed” or disassemble from the nanospheres that are present in solution. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 103–107, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Health Extension Workers Improve Tuberculosis Case Detection and Treatment Success in Southern Ethiopia: A Community Randomized Trial

Background

One of the main strategies to control tuberculosis (TB) is to find and treat people with active disease. Unfortunately, the case detection rates remain low in many countries. Thus, we need interventions to find and treat sufficient number of patients to control TB. We investigated whether involving health extension workers (HEWs: trained community health workers) in TB control improved smear-positive case detection and treatment success rates in southern Ethiopia.

Methodology/Principal Finding

We carried out a community-randomized trial in southern Ethiopia from September 2006 to April 2008. Fifty-one kebeles (with a total population of 296, 811) were randomly allocated to intervention and control groups. We trained HEWs in the intervention kebeles on how to identify suspects, collect sputum, and provide directly observed treatment. The HEWs in the intervention kebeles advised people with productive cough of 2 weeks or more duration to attend the health posts. Two hundred and thirty smear-positive patients were identified from the intervention and 88 patients from the control kebeles. The mean case detection rate was higher in the intervention than in the control kebeles (122.2% vs 69.4%, p<0.001). In addition, more females patients were identified in the intervention kebeles (149.0 vs 91.6, p<0.001). The mean treatment success rate was higher in the intervention than in the control kebeles (89.3% vs 83.1%, p = 0.012) and more for females patients (89.8% vs 81.3%, p = 0.05).

Conclusions/Significance

The involvement of HEWs in sputum collection and treatment improved smear-positive case detection and treatment success rate, possibly because of an improved service access. This could be applied in settings with low health service coverage and a shortage of health workers.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00803322","term_id":"NCT00803322"}}NCT00803322     

The efficiency of mapping of quantitative trait loci using cofactor analysis in half-sib design

This simulation study was designed to study the power and type I error rate in QTL mapping using cofactor analysis in half-sib designs. A number of scenarios were simulated with different power to identify QTL by varying family size, heritability, QTL effect and map density, and three threshold levels for cofactor were considered. Generally cofactor analysis did not increase the power of QTL mapping in a half-sib design, but increased the type I error rate. The exception was with small family size where the number of correctly identified QTL increased by 13% when heritability was high and 21% when heritability was low. However, in the same scenarios the number of false positives increased by 49% and 45% respectively. With a liberal threshold level of 10% for cofactor combined with a low heritability, the number of correctly identified QTL increased by 14% but there was a 41% increase in the number of false positives. Also, the power of QTL mapping did not increase with cofactor analysis in scenarios with unequal QTL effect, sparse marker density and large QTL effect (25% of the genetic variance), but the type I error rate tended to increase. A priori, cofactor analysis was expected to have higher power than individual chromosome analysis especially in experiments with lower power to detect QTL. Our study shows that cofactor analysis increased the number of false positives in all scenarios with low heritability and the increase was up to 50% in low power experiments and with lower thresholds for cofactors.     

Patients present earlier and survival has improved, but pre-ART attrition is high in a six-year HIV cohort data from Ethiopia

Background

Previous studies showed higher early mortality rates among patients treated with antiretroviral drugs in settings with limited resources. One of the reasons was late presentation of patients to care. With improved access to HIV services, we expect improvements in disease stage at presentation. Our objective was to assess the effect of improved availability of HIV services on patient presentation to care and subsequent pre-ART and on-ART outcomes.

Methodology and Principal Findings

At Arba Minch Hospital in Ethiopia, we reviewed baseline characteristics and outcomes of 2191 adult HIV patients. Nearly a half were in WHO stage III at presentation. About two-thirds of the patients (1428) started ART. Patients enrolled in the early phase (OR = 4.03, 95% CI 3.07–5.27), men (OR = 1.78, 95%CI 1.47–2.16), and those aged 45 years and above (OR = 2.04, 95%CI 1.48–2.82) were at higher risk of being in advanced clinical stage at presentation. The pre-treatment mortality rate was 13.1 per 100 PYO, ranging from 1.4 in the rapid scale-up phase to 25.9 per 100 PYO in the early phase. A quarter of the patients were lost to follow-up before starting treatment. Being in less advanced stage (HR = 1.9, 95% CI = 1.6, 2.2), being in the recent cohort (HR = 2.0, 95% CI = 1.6, 2.6), and rural residence (HR = 1.8, 95% CI = 1.5, 2.2) were independent predictors of pre-ART loss to follow-up. Of those who started ART, 13.4% were lost to follow-up and 15.4% died. The survival improved during the study. Patients with advanced disease, men and older people had higher death rates.

Conclusions and Significance

Patients started to present at earlier stages of their illness and death has decreased among adult HIV patients visiting Arba Minch Hospital. However, many patients were lost from pre-treatment follow-up. Early treatment start contributed to improved survival. Both pre-ART and on-ART patient retention mechanisms should be strengthened.     

Cost and Cost-Effectiveness of Smear-Positive Tuberculosis Treatment by Health Extension Workers in Southern Ethiopia: A Community Randomized Trial

by HEWs in the health posts and general health workers at health facility were compared along a community-randomized trial. Costs were analysed from societal perspective in 2007 in US $ using standard methods. We prospectively enrolled smear positive patients, and calculated cost-effectiveness as the cost per patient successfully treated. The total cost for each successfully treated smear-positive patient was higher in health facility ($158.9) compared with community ($61.7). Community-based treatment reduced the total, patient and caregiver cost by 61.2%, 68.1% and 79.8%, respectively. Involving HEWs added a total cost of $8.80 (14.3% of total cost) on health service per patient treated in the community.

Conclusions/Significance

Community-based treatment by HEWs costs only 39% of what treatment by general health workers costs for similar outcomes. Involving HEWs in TB treatment is a cost effective treatment alternative to the health service, to the patients and the family. There is an economic and public health reason to consider involving HEWs in TB treatment in Ethiopia. However, community-based treatment requires initial investment to start its implementation, training and supervision.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00803322","term_id":"NCT00803322"}}NCT00803322     

Recombinant purple acid phosphatase isoform 3 from sweet potato is an enzyme with a diiron metal center

Purple acid phosphatases (PAPs) from sweet potato (sp) have been classified on the basis of their primary structure and the dinuclear metal center into isoforms spPAP1 [Fe(III)-Zn(II)] and spPAP2 [Fe(III)-Mn(II)]; for spPAP3 only the cDNA is known. With the aim of unraveling the character of the dinuclear metal center we report here the characterization of this isoform at the protein level. We cloned spPAP3 cDNA in a baculovirus and overexpressed this enzyme in Sf9 insect cells. Preparation of recombinant spPAP3 in two steps afforded pure enzyme with yields of 4.5 mg.L(-1) culture medium. This enzyme is a dimeric, disulfide-linked PAP of 110 kDa, similar to known PAP isoforms from higher plants. Enzymatic studies and spectroscopic properties (max. absorption at 550-565 nm) indicated a diiron enzyme; quantitative and semiquantitative metal analysis using ICP-OES and TOF-SIMS, respectively, revealed the presence of only iron in purified spPAP3. Metal replacement in the second metal-binding site upon preparation of the semiapo-enzyme with Fe(II), Zn(II), or Mn(II) showed highest activities with Fe(II). The data show that recombinant spPAP3 has a diiron metal center. Site-directed mutagenesis was conducted to check catalytic efficiency at the atomic level. Tyr291 at the substrate-binding site in spPAP3 was mutated to His and Ala, the respective residues found in spPAP1 and spPAP2. Kinetic analysis showed that conversion of Tyr291 to His further optimized the performance of this protein as a diiron enzyme, whereas the Ala mutation weakened the catalytic efficiency regardless of the metal present in the second binding site.     

Glyoxysomal acetoacetyl-CoA thiolase and 3-oxoacyl-CoA thiolase from sunflower cotyledons

Following chromatography on hydroxyapatite, the elution profile of the thiolase activity of the glyoxysomal fraction from sunflower (Helianthus annuus L.) cotyledons exhibited two peaks when the enzyme activity was assayed with acetoacetyl-CoA as substrate. Only one of these two activity peaks was detectable when a long-chain thiolase substrate was used in the activity assay. The proteins (thiolase I and thiolase II) underlying the two activity peaks detected with acetoacetyl-CoA were of glyoxysomal origin. They were purified using glyoxysomal matrices as starting material, and biochemically characterized. Thiolase I is an acetoacetyl-CoA thiolase (EC 2.3.1.9) exhibiting activity only towards acetoacetyl-CoA (Km = 11 microM). Its contribution to the total glyoxysomal thiolytic activity towards acetoacetyl-CoA amounted to about 15%. Thiolase II is a 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The activity of the enzyme towards 3-oxoacyl-CoAs increased with increasing chain length of the substrate. Thiolase II exhibited a Km value of 27 microM with acetoacetyl-CoA as substrate. and Km values between 3 and 7 microM with substrates having a carbon chain length from 6 to 16 carbon atoms. The thiolase activity of the glyoxysomes towards acetoacetyl-CoA and 3-oxopalmitoyl-CoA exceeded the glyoxysomal butyryl-CoA and palmitoyl-CoA beta-oxidation rates, respectively, by about 10-fold at all substrate concentrations employed (1-15 microM).     

Spatial variation in the evolutionary potential and constraints of basal metabolic rate and body mass in a wild bird

An organism's energy budget is strongly related to resource consumption, performance, and fitness. Hence, understanding the evolution of key energetic traits, such as basal metabolic rate (BMR), in natural populations is central for understanding life-history evolution and ecological processes. Here we used quantitative genetic analyses to study evolutionary potential of BMR in two insular populations of the house sparrow (Passer domesticus). We obtained measurements of BMR and body mass (M_b) from 911 house sparrows on the islands of Leka and Vega along the coast of Norway. These two populations were the source populations for translocations to create an additional third, admixed ‘common garden’ population in 2012. With the use of a novel genetic group animal model concomitant with a genetically determined pedigree, we differentiate genetic and environmental sources of variation, thereby providing insight into the effects of spatial population structure on evolutionary potential. We found that the evolutionary potential of BMR was similar in the two source populations, whereas the Vega population had a somewhat higher evolutionary potential of M_b than the Leka population. BMR was genetically correlated with M_b in both populations, and the conditional evolutionary potential of BMR (independent of body mass) was 41% (Leka) and 53% (Vega) lower than unconditional estimates. Overall, our results show that there is potential for BMR to evolve independently of M_b, but that selection on BMR and/or M_b may have different evolutionary consequences in different populations of the same species.     

Plasmids with temperature-dependent copy number for amplification of cloned genes and their products

Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1. At 30°C these miniplasmids are present in 20–50 copies per cell of Escherichia coli, whereas at temperatures above 35°C the plasmids replicate without copy number control during 2–3 h. At the end of this period plasmid DNA amounts to about 75% of the total DNA. During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products. Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plasmid pKN410 has a single BamHI site and carries ampicillin resistance. The plasmids can therefore be used as cloning vectors. Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded β-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication. Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products. These plasmids have lost their mobilization capacity. Runaway replication is lethal to the host bacteria in rich media. These two properties contribute to the safe use of the plasmids as cloning vehicles.