Escherichia coli rimM and yjeQ null strains accumulate immature 30S subunits of similar structure and protein complement

Assembly of the Escherichia coli 30S ribosomal subunits proceeds through multiple parallel pathways. The protein factors RimM, YjeQ, RbfA, and Era work in conjunction to assist at the late stages of the maturation process of the small subunit. However, it is unclear how the functional interplay between these factors occurs in the context of multiple parallel pathways. To understand how these factors work together, we have characterized the immature 30S subunits that accumulate in ΔrimM cells and compared them with immature 30S subunits from a ΔyjeQ strain. The cryo-EM maps obtained from these particles showed that the densities representing helices 44 and 45 in the rRNA were partially missing, suggesting mobility of these motifs. These 30S subunits were also partially depleted in all tertiary ribosomal proteins, particularly those binding in the head domain. Using image classification, we identified four subpopulations of ΔrimM immature 30S subunits differing in the amount of missing density for helices 44 and 45, as well as the amount of density existing in these maps for the underrepresented proteins. The structural defects found in these immature subunits resembled those of the 30S subunits that accumulate in the ΔyjeQ strain. These findings are consistent with an “early convergency model” in which multiple parallel assembly pathways of the 30S subunit converge into a late assembly intermediate, as opposed to the mature state. Functionally related factors will bind to this intermediate to catalyze the last steps of maturation leading to the mature 30S subunit.     

Characterization of Novel CSF Tau and ptau Biomarkers for Alzheimer’s Disease

Cerebral spinal fluid (CSF) Aβ42, tau and p181tau are widely accepted biomarkers of Alzheimer’s disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases.     

Remote Source Document Verification in Two National Clinical Trials Networks: A Pilot Study

Objective

Barriers to executing large-scale randomized controlled trials include costs, complexity, and regulatory requirements. We hypothesized that source document verification (SDV) via remote electronic monitoring is feasible.

Methods

Five hospitals from two NIH sponsored networks provided remote electronic access to study monitors. We evaluated pre-visit remote SDV compared to traditional on-site SDV using a randomized convenience sample of all study subjects due for a monitoring visit. The number of data values verified and the time to perform remote and on-site SDV was collected.

Results

Thirty-two study subjects were randomized to either remote SDV (N=16) or traditional on-site SDV (N=16). Technical capabilities, remote access policies and regulatory requirements varied widely across sites. In the adult network, only 14 of 2965 data values (0.47%) could not be located remotely. In the traditional on-site SDV arm, 3 of 2608 data values (0.12%) required coordinator help. In the pediatric network, all 198 data values in the remote SDV arm and all 183 data values in the on-site SDV arm were located. Although not statistically significant there was a consistent trend for more time consumed per data value (minutes +/- SD): Adult 0.50 +/- 0.17 min vs. 0.39 +/- 0.10 min (two-tailed t-test p=0.11); Pediatric 0.99 +/- 1.07 min vs. 0.56 +/- 0.61 min (p=0.37) and time per case report form: Adult: 4.60 +/- 1.42 min vs. 3.60 +/- 0.96 min (p=0.10); Pediatric: 11.64 +/- 7.54 min vs. 6.07 +/- 3.18 min (p=0.10) using remote SDV.

Conclusions

Because each site had different policies, requirements, and technologies, a common approach to assimilating monitors into the access management system could not be implemented. Despite substantial technology differences, more than 99% of data values were successfully monitored remotely. This pilot study demonstrates the feasibility of remote monitoring and the need to develop consistent access policies for remote study monitoring.     

Controlling Hierarchy in Solution‐processed Polymer Solar Cells Based on Crosslinked P3HT

Understanding and controlling the morphology of donor/acceptor blends is critical for the development of solution processable organic solar cells. By crosslinking a poly(3‐n‐hexylthiophene‐2,5‐diyl) (P3HT) film we have been able to spin‐coat [6,6]‐phenyl‐C₆₁‐butyric acid methyl ester (PCBM) onto the film to form a structure that is close to a bilayer, thus creating an ideal platform for investigating interdiffusion in this model system. Neutron reflectometry (NR) demonstrates that without any thermal treatment a smaller amount of PCBM percolates throughout the crosslinked P3HT when compared to a non‐crosslinked P3HT film. Using time‐resolved NR we also show thermal annealing increases the rate of diffusion, resulting in a near‐uniform distribution of PCBM throughout the polymer film. XPS measurements confirm the presence of both P3HT and PCBM at the annealed film's surface indicating that the two components are intermixed. Photovoltaic devices fabricated using this bilayer approach and suitable annealing conditions yielded comparable power conversion efficiencies to bulk heterojunction devices made from the same materials. The crosslinking procedure has also enabled the formation of patterned P3HT films by photolithography. Pillars with feature sizes down to 2 μm were produced and after subsequent deposition of PCBM and thermal annealing devices with efficiencies of up to 1.4% were produced.     

Soluble Guanylate Cyclase α1–Deficient Mice: A Novel Murine Model for Primary Open Angle Glaucoma

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α₁ subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α₁–deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α₁ and β₁ subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.     

Rapid and repeatable host plant shifts drive reproductive isolation following a recent human-mediated introduction of the apple maggot fly,Rhagoletis pomonella

Ecological speciation via host-shifting is often invoked as a mechanism for insect diversification, but the relative importance of this process is poorly understood. The shift of Rhagoletis pomonella in the 1850s from the native downy hawthorn, Crataegus mollis, to introduced apple, Malus pumila, is a classic example of sympatric host race formation, a hypothesized early stage of ecological speciation. The accidental human-mediated introduction of R. pomonella into the Pacific Northwest (PNW) in the late 1970s allows us to investigate how novel ecological opportunities may trigger divergent adaptation and host race formation on a rapid timescale. Since the introduction, the fly has spread in the PNW, where in addition to apple, it now infests native black hawthorn, Crataegus douglasii, and introduced ornamental hawthorn, Crataegus monogyna. We use this “natural experiment” to test for genetic differentiation among apple, black, and ornamental hawthorn flies co-occurring at three sympatric sites. We report evidence that populations of all three host-associations are genetically differentiated at the local level, indicating that partial reproductive isolation has evolved in this novel habitat. Our results suggest that conditions suitable for initiating host-associated divergence may be common in nature, allowing for the rapid evolution of new host races when ecological opportunity arises.     

The new sequencer on the block: comparison of Life Technology’s Proton sequencer to an Illumina HiSeq for whole-exome sequencing

We assessed the performance of the new Life Technologies Proton sequencer by comparing whole-exome sequence data in a Centre d’Etude du Polymorphisme Humain trio (family 1463) to the Illumina HiSeq instrument. To simulate a typical user’s results, we utilized the standard capture, alignment and variant calling methods specific to each platform. We restricted data analysis to include the capture region common to both methods. The Proton produced high quality data at a comparable average depth and read length, and the Ion Reporter variant caller identified 96 % of single nucleotide polymorphisms (SNPs) detected by the HiSeq and GATK pipeline. However, only 40 % of small insertion and deletion variants (indels) were identified by both methods. Usage of the trio structure and segregation of platform-specific alleles supported this result. Further comparison of the trio data with Complete Genomics sequence data and Illumina SNP microarray genotypes documented high concordance and accurate SNP genotyping of both Proton and Illumina platforms. However, our study underscored the problem of accurate detection of indels for both the Proton and HiSeq platforms.     

Robust Fall Social Displays Predict Spring Reproductive Behavior in Brown‐Headed Cowbirds (Molothrus ater ater)

The ability to engage others in close proximity may be an essential component of social life and shapes the development of social skills. Variation in the willingness to initiate and sustain close interaction with conspecifics is known as sociability. The Brown‐headed Cowbird (Molothrus ater) uses an affiliative display called the head‐down to bring individuals into close proximity. During fall 2009, we manipulated a large flock of cowbirds in a fission–fusion perturbation and recorded the frequency of head‐downs and social approaches. During the fission–fusion perturbation, the rate of head‐downs remained both correlated and repeatable across perturbations. In spring 2010, we separated individuals into three aviaries, a high, intermediate, and low aviary, based on the frequency of head‐down displays they initiated during the previous fall 2009. When breeding, males in the high flock produced a higher number of songs within counter‐singing matches, and females laid more eggs in comparison with the other aviaries. These findings suggest that head‐down displays performed outside the breeding season may contribute to the development and maintenance of reproductive competence by providing intimate social interactions with others.     

Evaluation of the suchey–brooks method of age estimation in an Australian subpopulation using computed tomography of the pubic symphyseal surface

Despite the prominent use of the Suchey–Brooks (S–B) method of age estimation in forensic anthropological practice, it is subject to intrinsic limitations, with reports of differential interpopulation error rates between geographical locations. This study assessed the accuracy of the S–B method to a contemporary adult population in Queensland, Australia and provides robust age parameters calibrated for our population. Three‐dimensional surface reconstructions were generated from computed tomography scans of the pubic symphysis of male and female Caucasian individuals aged 15–70 years (n = 195) in Amira® and Rapidform®. Error was analyzed on the basis of bias, inaccuracy and percentage correct classification for left and right symphyseal surfaces. Application of transition analysis and Chi‐square statistics demonstrated 63.9 and 69.7% correct age classification associated with the left symphyseal surface of Australian males and females, respectively, using the S–B method. Using Bayesian statistics, probability density distributions for each S–B phase were calculated, providing refined age parameters for our population. Mean inaccuracies of 6.77 (±2.76) and 8.28 (±4.41) years were reported for the left surfaces of males and females, respectively; with positive biases for younger individuals (<55 years) and negative biases in older individuals. Significant sexual dimorphism in the application of the S–B method was observed; and asymmetry in phase classification of the pubic symphysis was a frequent phenomenon. These results recommend that the S–B method should be applied with caution in medico‐legal death investigations of Queensland skeletal remains and warrant further investigation of reliable age estimation techniques. Am J Phys Anthropol, 2013. © 2013 Wiley Periodicals, Inc.