Targeted genome enrichment for efficient purification of endosymbiont DNA from host DNA

Wolbachia endosymbionts are widespread in arthropods and are generally considered reproductive parasites, inducing various phenotypes including cytoplasmic incompatibility, parthenogenesis, feminization and male killing, which serve to promote their spread through populations. In contrast, Wolbachia infecting filarial nematodes that cause human diseases, including elephantiasis and river blindness, are obligate mutualists. DNA purification methods for efficient genomic sequencing of these unculturable bacteria have proven difficult using a variety of techniques. To efficiently capture endosymbiont DNA for studies that examine the biology of symbiosis, we devised a parallel strategy to an earlier array-based method by creating a set of SureSelect? (Agilent) 120-mer target enrichment RNA oligonucleotides (“baits”) for solution hybrid selection. These were designed from Wolbachia complete and partial genome sequences in GenBank and were tiled across each genomic sequence with 60 bp overlap. Baits were filtered for homology against host genomes containing Wolbachia using BLAT and sequences with significant host homology were removed from the bait pool. Filarial parasite Brugia malayi DNA was used as a test case, as the complete sequence of both Wolbachia and its host are known. DNA eluted from capture was size selected and sequencing samples were prepared using the NEBNext® Sample Preparation Kit. One-third of a 50 nt paired-end sequencing lane on the HiSeq? 2000 (Illumina) yielded 53 million reads and the entirety of the Wolbachia genome was captured. We then used the baits to isolate more than 97.1 % of the genome of a distantly related Wolbachia strain from the crustacean Armadillidium vulgare, demonstrating that the method can be used to enrich target DNA from unculturable microbes over large evolutionary distances.     

Steps Toward Developing an EEG Biofeedback Treatment for Chronic Pain

Chronic pain, usually refractory to analgesics, is a significant problem for many individuals with spinal cord injury (SCI). Preliminary studies suggest that electroencephalography (EEG) biofeedback (also known as neurofeedback, NF) has the potential to help patients with otherwise refractory chronic pain. However, there remain many unanswered questions about the effects and mechanisms of this treatment. We studied 13 individuals with SCI and chronic pain with NF. Ten of the 13 individuals completed 4 sessions each of three different neurofeedback protocols assigned in random order for a total of 12 NF sessions. All three protocols had similar immediate effects on pain intensity. In addition, the participants reported modest pre- to post-treatment decreases in worst pain and pain unpleasantness following completion of the 12 NF sessions. These improvements were maintained at 3-month follow-up. The majority of the participants felt they benefited from and were satisfied with the treatment. No significant effects on measures of other outcome domains (sleep quality, pain interference and fatigue) were observed, although there was a non-significant trend for an increase in fatigue. Finally, pre- to post-treatment changes in EEG bandwidth activity, consistent with the training protocols, were observed in θ and α but not β frequencies. The findings provide preliminary support for the potential efficacy of NF for the treatment of SCI-related pain, and suggest that further clinical studies are warranted.     

The portable UNIX programming system (PUPS) and CANTOR: a computational environment for dynamical representation and analysis of complex neurobiological data.

Many problems in analytical biology, such as the classification of organisms, the modelling of macromolecules, or the structural analysis of metabolic or neural networks, involve complex relational data. Here, we describe a software environment, the portable UNIX programming system (PUPS), which has been developed to allow efficient computational representation and analysis of such data. The system can also be used as a general development tool for database and classification applications. As the complexity of analytical biology problems may lead to computation times of several days or weeks even on powerful computer hardware, the PUPS environment gives support for persistent computations by providing mechanisms for dynamic interaction and homeostatic protection of processes. Biological objects and their interrelations are also represented in a homeostatic way in PUPS. Object relationships are maintained and updated by the objects themselves, thus providing a flexible, scalable and current data representation. Based on the PUPS environment, we have developed an optimization package, CANTOR, which can be applied to a wide range of relational data and which has been employed in different analyses of neuroanatomical connectivity. The CANTOR package makes use of the PUPS system features by modifying candidate arrangements of objects within the system's database. This restructuring is carried out via optimization algorithms that are based on user-defined cost functions, thus providing flexible and powerful tools for the structural analysis of the database content. The use of stochastic optimization also enables the CANTOR system to deal effectively with incomplete and inconsistent data. Prototypical forms of PUPS and CANTOR have been coded and used successfully in the analysis of anatomical and functional mammalian brain connectivity, involving complex and inconsistent experimental data. In addition, PUPS has been used for solving multivariate engineering optimization problems and to implement the digital identification system (DAISY), a system for the automated classification of biological objects. PUPS is implemented in ANSI-C under the POSIX.1 standard and is to a great extent architecture- and operating-system independent. The software is supported by systems libraries that allow multi-threading (the concurrent processing of several database operations), as well as the distribution of the dynamic data objects and library operations over clusters of computers. These attributes make the system easily scalable, and in principle allow the representation and analysis of arbitrarily large sets of relational data. PUPS and CANTOR are freely distributed (http://www.pups.org.uk) as open-source software under the GNU license agreement.     

Beauvericin and divalent cations: crystal structure of the barium complex.

The molecular structure of the 2:2 complex of the cyclohexadepsipeptide antibiotic beauvericin with barium picrate has been determined by X-ray crystallography. The structure serves to confirm previous observations on the bimolecular behavior of beauvericin and of the ions transported by beauvercin. The intimate involvement of the anions in the coordination of the barium also explains observations that the cation specificity of beauvericin in membrane transport depends on the species of anions present.     

Measuring fixed charge density of goat articular cartilage using indentation methods and biochemical analysis

An important indicator of osteoarthritis (OA) progression is the loss of proteoglycan (PG) aggregates from the cartilage tissue. Using the indentation creep test, two analytical methods, as previously developed by Lu et al. [Lu, X. L., Miller, C., Chen, F. H., Guo, X. E., Mow, V. C., 2007. The generalized triphasic correspondence principle for simultaneous determination of the mechanical properties and proteoglycan content of articular cartilage by indentation. Journal of Biomechanics 40, 2434-2441 (EPub).], for predicting the fixed charge density (FCD) of goat knee articular cartilage in the normal (control) and degenerated states were compared: (1) a "dual-stage" method to calculate FCD from the mechanical properties of the tissue when tested in isotonic and hypertonic solutions; and (2) a "single-stage" method to predict FCD (as in (1)) assuming an intrinsic Poisson's ratio of 0.05 in the hypertonic state. A biochemical analysis using 1,9-dimethylmethylene blue (DMMB) assay was conducted to directly measure PG content, and hence FCD. The association between the FCD and the aggregate modulus of the tissue was also explored. The mean (+/-S.D.) FCD values measured using the dual-stage method were the closest (control: 0.129+/-0.039, degenerated: 0.046+/-029) to the DMMB results (control: 0.125+/-0.034, degenerated: 0.057+/-0.024) as compared to those of the single-stage method (control: 0.147+/-0.035, degenerated: 0.063+/-0.026). The single-stage method was more reliable (r(2)=0.81) when compared to the dual-stage method (r(2)=0.79). A prediction of FCD from the aggregate modulus generated the least reliable FCD prediction (r(2)=0.68). Because both the dual- and single-stage methods provided reliable FCD estimates for normal and degenerated tissue, the less time-consuming single-stage method was concluded to be the ideal technique for predicting FCD and hence PG content of the tissue.     

Discovery and SAR of benzyl phenyl ethers as inhibitors of bacterial phenylalanyl-tRNA synthetase

A series of benzyl phenyl ethers (BPEs) is described that displays potent inhibition of bacterial phenylalanyl-tRNA synthetase. The synthesis, SAR, and select ADMET data are provided.     

Microbiological and Geochemical Heterogeneity in an In Situ Uranium Bioremediation Field Site

The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.     

Access to health care among status Aboriginal people with chronic kidney disease

Background

Ethnic disparities in access to health care and health outcomes are well documented. It is unclear whether similar differences exist between Aboriginal and non-Aboriginal people with chronic kidney disease in Canada. We determined whether access to care differed between status Aboriginal people (Aboriginal people registered under the federal Indian Act) and non-Aboriginal people with chronic kidney disease.

Methods

We identified 106 511 non-Aboriginal and 1182 Aboriginal patients with chronic kidney disease (estimated glomerular filtration rate less than 60 mL/min/1.73 m²). We compared outcomes, including hospital admissions, that may have been preventable with appropriate outpatient care (ambulatory-care–sensitive conditions) as well as use of specialist services, including visits to nephrologists and general internists.

Results

Aboriginal people were almost twice as likely as non-Aboriginal people to be admitted to hospital for an ambulatory-care–sensitive condition (rate ratio 1.77, 95% confidence interval [CI] 1.46–2.13). Aboriginal people with severe chronic kidney disease (estimated glomerular filtration rate < 30 mL/min/1.73 m²) were 43% less likely than non-Aboriginal people with severe chronic kidney disease to visit a nephrologist (hazard ratio 0.57, 95% CI 0.39–0.83). There was no difference in the likelihood of visiting a general internist (hazard ratio 1.00, 95% CI 0.83–1.21).

Interpretation

Increased rates of hospital admissions for ambulatory-care–sensitive conditions and a reduced likelihood of nephrology visits suggest potential inequities in care among status Aboriginal people with chronic kidney disease. The extent to which this may contribute to the higher rate of kidney failure in this population requires further exploration.Ethnic disparities in access to health care are well documented;^1,2 however, the majority of studies include black and Hispanic populations in the United States. The poorer health status and increased mortality among Aboriginal populations than among non-Aboriginal populations,^3,4 particularly among those with chronic medical conditions,^5,6 raise the question as to whether there is differential access to health care and management of chronic medical conditions in this population.The prevalence of end-stage renal disease, which commonly results from chronic kidney disease, is about twice as common among Aboriginal people as it is among non-Aboriginal people.^7,8 Given that the progression of chronic kidney disease can be delayed by appropriate therapeutic interventions^9,10 and that delayed referral to specialist care is associated with increased mortality,^11,12 issues such as access to health care may be particularly important in the Aboriginal population. Although previous studies have suggested that there is decreased access to primary and specialist care in the Aboriginal population,^13–15 these studies are limited by the inclusion of patients from a single geographically isolated region,¹³ the use of survey data,¹⁴ and the inability to differentiate between different types of specialists and reasons for the visit.¹⁵In addition to physician visits, admission to hospital for ambulatory-care–sensitive conditions (conditions that, if managed effectively in an outpatient setting, do not typically result in admission to hospital) has been used as a measure of access to appropriate outpatient care.^16,17 Thus, admission to hospital for an ambulatory-care–sensitive condition reflects a potentially preventable complication resulting from inadequate access to care. Our objective was to determine whether access to health care differs between status Aboriginal (Aboriginal people registered under the federal Indian Act) and non-Aboriginal people with chronic kidney disease. We assess differences in care by 2 measures: admission to hospital for an ambulatory-care–sensitive condition related to chronic kidney disease; and receipt of nephrology care for severe chronic kidney disease as recommended by clinical practice guidelines.¹⁸