Application of a subtraction hybridization technique involving photoactivatable biotin and organic extraction to solution hybridization analysis of genomic DNA

We have adapted a subtraction hybridization technique involving photoactivatable biotin, streptavidin binding, and organic extraction for solution hybridization analysis of mammalian genomic DNA. By combining maximal hybridization conditions of high salt, dextran sulfate, and formamide with successive hybridization steps and sequence enrichment by agarose gel electrophoresis, up to 97% of tracer DNA can be reproducibly driven to hybridize with photobiotinylated driver DNA. We demonstrate that the fractionation of hybridized from unhybridized sequences by this technique differs from hydroxyapatite chromatography with respect to the handling of nondenatured tracer, foldback sequences, and tracer-tracer hybrids. Strategies are presented to control the contribution of these species to the final subtracted product thereby making this technology a useful adjunct to solution hybridization approaches such as deletion cloning.     

A map of 22 loci on human chromosome 22.

We constructed a genetic linkage map of the entire long arm of human chromosome 22 with 30 polymorphic markers, defining 22 loci. The map consists of a continuous linkage group 110 cM long, when male and female recombination fractions are combined; average distance between the loci is 5.2 cM. All loci were placed on the map with high support against alternative orders (odds in excess of 1000:1). The order of loci presented in our map is in full agreement with that of the previous linkage maps of chromosome 22 and with the physical assignment of markers. Two markers included in this map, KI-831 (D22S212) and pEFZ31 (D22S32), allowed us to better define the region of the (11;22) translocation breakpoint specific for Ewing sarcoma. Ten additional polymorphic markers were placed on the 22-loci map with odds lower than 1000:1 against alternative locations. In total, we have introduced 29 new markers on the linkage map of chromosome 22.     

Performance evaluation of Baermann techniques: The quest for developing a microscopy reference standard for the diagnosis of Strongyloides stercoralis

BackgroundSoil-transmitted helminths (STH) are common in low and middle income countries where there is lack of access to clean water and sanitation. Effective diagnosis and treatment are essential for the control of STH infections. However, among STH parasites, Strongyloides stercoralis is the most neglected species, both in diagnostics and control strategies. Diagnostic methods cover different approaches, each with different sensitivities and specificities, such as serology, molecular techniques and microscopy based techniques. Of the later, the Baermann technique is the most commonly used procedure. In the literature, several ways have been described to perform the Baermann method, which illustrates the overall lack of a ‘(gold) reference standard’ method for the diagnosis of S. stercoralis infection. In this study we have evaluated the performance of three Baermann techniques in order to improve the reference standard for the microscopic diagnosis of S. stercoralis infection thereby facilitating individual case detection, mapping of the disease and proper evaluation of treatment responses.Methods/Principal findingsA community based cross sectional study was conducted at Zenzelima, Bahir Dar Zuria Ethiopia. A total of 437 stool samples were collected and analyzed by the following procedures: conventional Baermann (CB), modified Baermann (MB), and modified Baermann with charcoal pre-incubation (MBCI). The diagnostic sensitivity and Negative Predictive Value (NPV) of each technique was calculated using the combination of all the three techniques as a composite reference standard. Our result indicated that larvae of S. stercoralis were detected in 151 (34.6%) stool samples. The prevalence of S. stercoralis infection based on the three diagnostic methods was 9.6%, 8.0%, and 31.3% by CB, MB, and MBCI respectively. The sensitivity and NPV for CB, MB, and MBCI were 26.7% and 70.8%, 22.1% and 69.6%, and 87.0% and 93.2%, respectively. The MBCI showed significant difference (P- value = <0.001) in the sensitivity and NPV values when compared with CB and MB values. The agreement between CB, MB, and MBCI with the composite reference standard was 31.8%, 26.7%, 89.6%, respectively.Conclusion/SignificanceOur results suggest the superior performance of MBCI. It is relatively easy to implement, simple to perform and comparatively cheaper. The CB is by far the commonly used method in routine diagnostic although this technique significantly underestimates the true burden of the disease and thereby contributing to the exclusion of S. stercoralis from the control strategies. Therefore, MBCI is recommended as a routine microscopy-based diagnostic test for S. stercoralis infection, particularly in settings where molecular procedures are not available.     

Methodological Rigour and Transparency of Clinical Practice Guidelines Developed by Neurology Professional Societies in Croatia

Background

Clinical practice guidelines are systematically created documents that summarize knowledge and assist in delivering high-quality medicine by identifying evidence that supports best clinical care. They are produced not only by international professional groups but also by local professionals to address locally-relevant clinical practice. We evaluated the methodological rigour and transparency of guideline development in neurology formulated by professionals in a local medical community.

Methods

We analyzed clinical guidelines in neurology publicly available at the web-site of the Physicians’ Assembly in Croatia in 2012: 6 guidelines developed by Croatian authors and 1 adapted from the European Federation of Neurological Societies. The quality was assessed by 2 independent evaluators using the AGREE II instrument. We also conducted a search of the Cochrane Library to identify potential changes in recommendation from Cochrane systematic reviews included in guideline preparation.

Results

The methodological quality of the guidelines greatly varied across different domains. „Scope and Purpose” and „Clarity of Presentation“ domains received high scores (100% [95% confidence interval (CI) 98.5–100] and 97% [77.9–100], respectively), the lowest scores were in “Stakeholder Involvement“ (19% [15.5–34.6]) and “Editorial Independence” (0% [0–19.2]). Conclusions of 3 guidelines based on Cochrane systematic reviews were confirmed in updated versions and one update provided new information on the effectiveness of another antidepressant. Two Cochrane reviews used in guidelines were withdrawn and split into new reviews and their findings are now considered to be out of date.

Conclusion

Neurological guidelines used in Croatia differ in structure and their methodological quality. We recommend to national societies and professional groups to develop a more systematic and rigorous approach to the development of the guidelines, timely inclusion of best evidences and an effort to involve target users and patients in the guideline development procedures.     

Adverse responses of Cabera pusaria caterpillars to high dietary manganese concentration

The adaptation of insects to environmental changes can constitute a crucial factor in their development and activity. The response of Cabera pusaria L. (Lepidoptera: Geometridae) caterpillars to high manganese (Mn) concentrations in the diet was studied. Birch leaves were treated by dipping in MnCl₂·4H₂O solutions, thereby achieving Mn contents of 370 (T0), 695 (T1), 3 198 (T2), and 6 302 mg kg⁻¹ (T3). The reactions were determined by observing caterpillar mortality, development time, food consumption, and pupal weight. Manganese concentrations in larval excrement, pupae, and food were determined. Manganese in the diet at unnaturally increased concentrations caused great stress for caterpillars. All individuals in the treatment with the highest Mn concentration (T3) died during rearing and successful pupation occurred in only four individuals in T2. Even in the case of caterpillars from T1 (twofold higher than T0) a negative reaction (increased food consumption and prolonged development) was recorded. We also determined significantly increased Mn concentration in pupae from T1 (T2 and T3 were not included in this evaluation due to mortality) and excrement (T1‒T3) compared with T0 having a natural Mn concentration. Caterpillars were seen to eliminate negatively acting dietary Mn by its translocation to excrement. However, the highest mortality rate in T2 and T3 and negative reactions of individuals in T1 very likely demonstrate energy insufficiency and the high energy requirements of Mn elimination mechanisms.     

Evolution under pH stress and high population densities leads to increased density-dependent fitness in the protist Tetrahymena thermophila

Abiotic stress is a major force of selection that organisms are constantly facing. While the evolutionary effects of various stressors have been broadly studied, it is only more recently that the relevance of interactions between evolution and underlying ecological conditions, that is, eco-evolutionary feedbacks, have been highlighted. Here, we experimentally investigated how populations adapt to pH-stress under high population densities. Using the protist species Tetrahymena thermophila, we studied how four different genotypes evolved in response to stressfully low pH conditions and high population densities. We found that genotypes underwent evolutionary changes, some shifting up and others shifting down their intrinsic rates of increase (r₀). Overall, evolution at low pH led to the convergence of r₀ and intraspecific competitive ability (α) across the four genotypes. Given the strong correlation between r₀ and α, we argue that this convergence was a consequence of selection for increased density-dependent fitness at low pH under the experienced high density conditions. Increased density-dependent fitness was either attained through increase in r₀, or decrease of α, depending on the genetic background. In conclusion, we show that demography can influence the direction of evolution under abiotic stress.