Amino Acid Sulfinate Salts as Alkyl Radical Precursors.

A general approach to the synthesis of amino acid sulfinate salts from commercially available α-chiral hydroxylated amino acids is reported. These reagents are shown to be valuable precursors to alkyl radicals under mild photochemical oxidation conditions. The photochemically generated amino acid radicals engage readily with alkyl and aryl disulfide radical traps to afford a diverse suite of modified amino acids.

Transcriptomic changes associated with maternal care in the brain of mouthbrooding cichlid Astatotilapia burtoni reflect adaptation to self-induced metabolic stress.

Parental care in Astatotilapia burtoni entails females protecting eggs and developing fry in a specialized buccal cavity in the mouth. During this mouthbrooding behavior, which can last 2-3 weeks, mothers undergo voluntary fasting accompanied by loss of body mass and major metabolic changes. Following release of fry, females resume normal feeding behavior and quickly recover body mass as they become reproductively active once again. In order to investigate the molecular underpinnings of such dramatic behavioral and metabolic changes, we sequenced whole-brain transcriptomes from females at four time points throughout their reproductive cycle: 2 days after the start of mouthbrooding, 14 days after the start of mouthbrooding, 2 days after the release of fry and 14 days after the release of fry. Differential expression analysis and clustering of expression profiles revealed a number of neuropeptides and hormones, including the strong candidate gene neurotensin, suggesting that molecular mechanisms underlying parental behaviors may be common across vertebrates despite de novo evolution of parental care in these lineages. In addition, oxygen transport pathways were found to be dramatically downregulated, particularly later in the mouthbrooding stage, while certain neuroprotective pathways were upregulated, possibly to mitigate negative consequences of metabolic depression brought about by fasting. Our results offer new insights into the evolution of parental behavior as well as revealing candidate genes that would be of interest for the study of hypoxic ischemia and eating disorders.

Correspondence of aCGH and long-read genome assembly for detection of copy number differences: A proof-of-concept with cichlid genomes.

Copy number variation is an important source of genetic variation, yet data are often lacking due to technical limitations for detection given the current genome assemblies. Our goal is to demonstrate the extent to which an array-based platform (aCGH) can identify genomic loci that are collapsed in genome assemblies that were built with short-read technology. Taking advantage of two cichlid species for which genome assemblies based on Illumina and PacBio are available, we show that inter-species aCGH log2 hybridization ratios correlate more strongly with inferred copy number differences based on PacBio-built genome assemblies than based on Illumina-built genome assemblies. With regard to inter-species copy number differences of specific genes identified by each platform, the set identified by aCGH intersects to a greater extent with the set identified by PacBio than with the set identified by Illumina. Gene function, according to Gene Ontology analysis, did not substantially differ among platforms, and platforms converged on functions associated with adaptive phenotypes. The results of the current study further demonstrate that aCGH is an effective platform for identifying copy number variable sequences, particularly those collapsed in short read genome assemblies.

Novel resin tissue array system reduces sample preparation time, labour and reagent costs in bone tissue histology.

Resin histology plays an essential role in the analysis of hard tissues, such as bone and teeth, as well as in the context of metallic implant analysis. However, the techniques of resin embedding, followed by ground sectioning, are very costly due to significantly increased reagent cost and labour time when compared to the conventional paraffin histology approach. In the present study, a novel resin array system was developed to increase the affordability of a project analysing rat femur tissues containing metallic or polymeric implants. The resin array system enabled the simultaneous embedding of the femur samples in groups of eight samples compared to the conventional resin method where samples are processed individually. The ground sections produced with the resin array system allowed uniform ROI selection, ground section thickness, staining consistency, and histological analysis with Goldner's trichrome stain, offering a substantial opportunity for reproducible immunohistochemistry which is unable to be achieved when processing samples embedded individually. The application of this novel resin array system significantly reduced resource usage when compared to doing the same analysis on individual samples. A reduction of approximately 40% was achieved for both total labour time and total reagent cost through the use of the array system compared with individual embedding. This novel resin array system has widespread applicability to many bone, hard tissue, and metallic implant studies, offering substantial conservation of research funds and increased accessibility to advanced techniques for commercial partners due to more cost-effective sample preparation and more accurate, reproducible data.

Myoviridae phage PDX kills enteroaggregative Escherichia coli without human microbiome dysbiosis.

Introduction. Bacteriophage therapy can be developed to target emerging diarrhoeal pathogens, but doing so in the absence of microbiome disruption, which occurs with antibiotic treatment, has not been established.Aim. Identify a therapeutic bacteriophage that kills diarrhoeagenic enteroaggregative Escherichia coli (EAEC) while leaving the human microbiome intact.Methodology. Phages from wastewater in Portland, OR, USA were screened for bacteriolytic activity by overlay assay. One isolated phage, PDX, was classified by electron microscopy and genome sequencing. A mouse model of infection determined whether the phage was therapeutic against EAEC. 16S metagenomic analysis of anaerobic cultures determined whether a normal human microbiome was altered by treatment.Results. Escherichia virus PDX, a member of the strictly lytic family Myoviridae, killed a case-associated EAEC isolate from a child in rural Tennessee in a dose-dependent manner, and killed EAEC isolates from Columbian children. A single dose of PDX (multiplicity of infection: 100) 1 day post-infection reduced EAEC recovered from mouse faeces. PDX also killed EAEC when cultured anaerobically in the presence of human faecal bacteria. While the addition of EAEC reduced the ß-diversity of the human microbiota, that of the cultures with either faeces alone, faeces with EAEC and PDX, or with just PDX phage was not different statistically.Conclusion. PDX killed EAEC isolate EN1E-0007 in vivo and in vitro, while not altering the diversity of normal human microbiota in anaerobic culture, and thus could be part of an effective therapy for children in developing countries and those suffering from EAEC-mediated traveller's diarrhoea without causing dysbiosis.

Traction load, tong position, and head support significantly influence cervical spine loading during traction.

Some cervical dislocation injuries may be acutely treated with traction via Gardner-Wells tongs, which are attached to the skull via two pins. While a variety of techniques have been proposed and utilized in the literature and clinical practice to use the tongs, these techniques have not been methodically studied to confirm how they transmit loads to the cervical spine. The current study investigated the mechanical effect of different traction techniques in a laboratory setting. A 50th male Hybrid anthropomorphic test device was used as a human surrogate to represent an average male in height and weight was modified to represent a patient with a unilateral facet dislocation injury. Electronic sensors at the atlanto-occipital joint recorded the loading delivered to the superior cervical spine by traction loading. Combinations of the following variables were evaluated as traction loads were progressively increased to one-third of body weight: tong pin position in the skull (anterior-posterior and superior-inferior to the recommended neutral position), traction cable angle in the sagittal plane (elevated, horizontal, declined), and presence or absence of an occipital support. Analysis of the cervical axial traction loads showed that the only significant predictor of cervical tension was the magnitude of the traction load. Anterior-posterior changes in the pin positions in the skull significantly influenced the cervical flexion-extension moment and anterior-posterior (AP) shear. The data show that a combined cervical tension, flexion moment, and anterior shear force can be achieved with posteriorly biased pins and a bolster behind the head. Increasing the angle of traction cable increased the cervical flexion moment and anterior shear force. The following variables should be carefully considered when applying cervical traction since they significantly affect cervical loading: magnitude of the hanging traction load, anterior-posterior pin position, use of an occipital bolster, and traction load angle.

The Genomic Substrate for Adaptive Radiation: Copy Number Variation across 12 Tribes of African Cichlid Species.

The initial sequencing of five cichlid genomes revealed an accumulation of genetic variation, including extensive copy number variation in cichlid lineages particularly those that have undergone dramatic evolutionary radiation. Gene duplication has the potential to generate substantial molecular substrate for the origin of evolutionary novelty. We use array-based comparative heterologous genomic hybridization to identify copy number variation events (CNVEs) for 168 samples representing 53 cichlid species including the 5 species for which full genome sequence is available. We identify an average of 50-100 CNVEs per individual. For those species represented by multiple samples, we identify 150-200 total CNVEs suggesting a substantial amount of intraspecific variation. For these species, only ∼10% of the detected CNVEs are fixed. Hierarchical clustering of species according to CNVE data recapitulates phylogenetic relationships fairly well at both the tribe and radiation level. Although CNVEs are detected on all linkage groups, they tend to cluster in "hotspots" and are likely to contain and be flanked by transposable elements. Furthermore, we show that CNVEs impact functional categories of genes with potential roles in adaptive phenotypes that could reasonably promote divergence and speciation in the cichlid clade. These data contribute to a more complete understanding of the molecular basis for adaptive natural selection, speciation, and evolutionary radiation.

Dynamic genomic architecture of mutualistic cooperation in a wild population of Mesorhizobium.

Research on mutualism seeks to explain how cooperation can be maintained when uncooperative mutants co-occur with cooperative kin. Gains and losses of the gene modules required for cooperation punctuate symbiont phylogenies and drive lifestyle transitions between cooperative symbionts and uncooperative free-living lineages over evolutionary time. Yet whether uncooperative symbionts commonly evolve from within cooperative symbiont populations or from within distantly related lineages with antagonistic or free-living lifestyles (i.e., third-party mutualism exploiters or parasites), remains controversial. We use genomic data to show that genotypes that differ in the presence or absence of large islands of symbiosis genes are common within a single wild recombining population of Mesorhizobium symbionts isolated from host tissues and are an important source of standing heritable variation in cooperation in this population. In a focal population of Mesorhizobium, uncooperative variants that lack a symbiosis island segregate at 16% frequency in nodules, and genome size and symbiosis gene number are positively correlated with cooperation. This finding contrasts with the genomic architecture of variation in cooperation in other symbiont populations isolated from host tissues in which the islands of genes underlying cooperation are ubiquitous and variation in cooperation is primarily driven by allelic substitution and individual gene gain and loss events. Our study demonstrates that uncooperative mutants within mutualist populations can comprise a significant component of genetic variation in nature, providing biological rationale for models and experiments that seek to explain the maintenance of mutualism in the face of non-cooperators.

Rapid establishment of a flowering cline in Medicago polymorpha after invasion of North America.

To establish and spread in a new location, an invasive species must be able to carry out its life cycle in novel environmental conditions. A key trait underlying fitness is the shift from vegetative to reproductive growth through floral development. In this study, we used a common garden experiment and genotyping-by-sequencing to test whether the latitudinal flowering cline of the North American invasive plant Medicago polymorpha was translocated from its European native range through multiple introductions, or whether the cline rapidly established due to evolution following a genetic bottleneck. Analysis of flowering time in 736 common garden plants showed a latitudinal flowering time cline in both the native and invaded ranges where genotypes from lower latitudes flowered earlier. Genotyping-by-sequencing of 9,658 SNPs in 446 individuals revealed two major subpopulations of M. polymorpha in the native range, only one of which is present in the invaded range. Additionally, native range populations have higher genetic diversity than invaded range populations, suggesting that a genetic bottleneck occurred during invasion. All invaded range individuals are closely related to plants collected from native range populations in Portugal and southern Spain, and population assignment tests assigned invaded range individuals to this same narrow source region. Taken together, our results suggest that latitudinal clinal variation in flowering time has rapidly evolved across the invaded range despite a genetic bottleneck following introduction.

The Effect of Booster Seat Use on Pediatric Injuries in Motor Vehicle Frontal Crashes.

BACKGROUND: Motor vehicle crashes are a significant source of pediatric mortality and morbidity. Studies indicate that booster seats significantly improve seat belt fit for children who have not attained a height of 145 cm (4' 9"). This study examined injuries occurring in booster age children up to age 12, as the majority of children do not attain 145 cm until this age. The purpose of the study was to identify differences in injuries due to the type of restraint used, with attention to musculoskeletal injuries. METHODS: Vehicle and occupant data were obtained from a publically available statistical sample of tow-away crashes. Frontal crashes over an 8-year period were examined. A data set of cases was created involving children ages 5 to 12 years who were unrestrained, restrained using the vehicle's lap and shoulder belt, and restrained using a booster seat with the vehicle's lap and shoulder seat belt. Injury severity, frequency, and patterns of distribution were compared. RESULTS: Unrestrained children experienced moderate to severe injuries 3.8 to 19 times more frequently than children using restraints. There were more injuries to the head and face in unrestrained versus restrained children, but the head and face was the most frequently injured region for all groups. There were no serious cervical spine injuries reported for any group. Lower extremity fractures were not observed in booster seat users but occurred at similar rates in both unrestrained and seat belt restrained children. These fractures occurred in older children who were involved in more severe crashes. CONCLUSIONS: Unrestrained children were more likely to experience moderate and severe injuries than restrained children. The data sample suggests that booster use may reduce the risk of extremity fracture, as there were no extremity fractures in children restrained with booster seats. CLINICAL RELEVANCE: This work provides evidence for the efficacy of booster use for preventing orthopaedic injury in children. This evidence can be used to inform parents and establish recommendations for best practices in transporting children.

Co-invading symbiotic mutualists of Medicago polymorpha retain high ancestral diversity and contain diverse accessory genomes.

Exotic, invasive plants and animals can wreak havoc on ecosystems by displacing natives and altering environmental conditions. However, much less is known about the identities or evolutionary dynamics of the symbiotic microbes that accompany invasive species. Most leguminous plants rely upon symbiotic rhizobium bacteria to fix nitrogen and are incapable of colonizing areas devoid of compatible rhizobia. We compare the genomes of symbiotic rhizobia in a portion of the legume's invaded range with those of the rhizobium symbionts from across the legume's native range. We show that in an area of California the legume Medicago polymorpha has invaded, its Ensifer medicae symbionts: (i) exhibit genome-wide patterns of relatedness that together with historical evidence support host-symbiont co-invasion from Europe into California, (ii) exhibit population genomic patterns consistent with the introduction of the majority of deep diversity from the native range, rather than a genetic bottleneck during colonization of California and (iii) harbor a large set of accessory genes uniquely enriched in binding functions, which could play a role in habitat invasion. Examining microbial symbiont genome dynamics during biological invasions is critical for assessing host-symbiont co-invasions whereby microbial symbiont range expansion underlies plant and animal invasions.

Anchored pseudo-de novo assembly of human genomes identifies extensive sequence variation from unmapped sequence reads.

The human genome reference (HGR) completion marked the genomics era beginning, yet despite its utility universal application is limited by the small number of individuals used in its development. This is highlighted by the presence of high-quality sequence reads failing to map within the HGR. Sequences failing to map generally represent 2-5 % of total reads, which may harbor regions that would enhance our understanding of population variation, evolution, and disease. Alternatively, complete de novo assemblies can be created, but these effectively ignore the groundwork of the HGR. In an effort to find a middle ground, we developed a bioinformatic pipeline that maps paired-end reads to the HGR as separate single reads, exports unmappable reads, de novo assembles these reads per individual and then combines assemblies into a secondary reference assembly used for comparative analysis. Using 45 diverse 1000 Genomes Project individuals, we identified 351,361 contigs covering 195.5 Mb of sequence unincorporated in GRCh38. 30,879 contigs are represented in multiple individuals with ~40 % showing high sequence complexity. Genomic coordinates were generated for 99.9 %, with 52.5 % exhibiting high-quality mapping scores. Comparative genomic analyses with archaic humans and primates revealed significant sequence alignments and comparisons with model organism RefSeq gene datasets identified novel human genes. If incorporated, these sequences will expand the HGR, but more importantly our data highlight that with this method low coverage (~10-20×) next-generation sequencing can still be used to identify novel unmapped sequences to explore biological functions contributing to human phenotypic variation, disease and functionality for personal genomic medicine.

Pseudo-De Novo Assembly and Analysis of Unmapped Genome Sequence Reads in Wild Zebrafish Reveal Novel Gene Content.

Zebrafish represents the third vertebrate with an officially completed genome, yet it remains incomplete with additions and corrections continuing with the current release, GRCz10, having 13% of zebrafish cDNA sequences unmapped. This disparity may result from population differences, given that the genome reference was generated from clonal individuals with limited genetic diversity. This is supported by the recent analysis of a single wild zebrafish, which identified over 5.2 million SNPs and 1.6 million in/dels in the previous genome build, zv9. Re-examination of this sequence data set indicated that 13.8% of quality sequence reads failed to align to GRCz10. Using a novel bioinformatics de novo assembly pipeline on these unmappable reads, we identified 1,514,491 novel contigs covering ∼224 Mb of genomic sequence. Among these, 1083 contigs were found to contain a potential gene coding sequence. RNA-seq data comparison confirmed that 362 contigs contained a transcribed DNA sequence, suggesting that a large amount of functional genomic sequence remains unannotated in the zebrafish reference genome. By utilizing the bioinformatics pipeline developed in this study, the zebrafish genome will be bolstered as a model for human disease research. Adaptation of the pipeline described here also offers a cost-efficient and effective method to identify and map novel genetic content across any genome and will ultimately aid in the completion of additional genomes for a broad range of species.

Saccular Transcriptome Profiles of the Seasonal Breeding Plainfin Midshipman Fish (Porichthys notatus), a Teleost with Divergent Sexual Phenotypes.

Acoustic communication is essential for the reproductive success of the plainfin midshipman fish (Porichthys notatus). During the breeding season, type I males use acoustic cues to advertise nest location to potential mates, creating an audible signal that attracts reproductive females. Type II (sneaker) males also likely use this social acoustic signal to find breeding pairs from which to steal fertilizations. Estrogen-induced changes in the auditory system of breeding females are thought to enhance neural encoding of the advertisement call, and recent anatomical data suggest the saccule (the main auditory end organ) as one possible target for this seasonal modulation. Here we describe saccular transcriptomes from all three sexual phenotypes (females, type I and II males) collected during the breeding season as a first step in understanding the mechanisms underlying sexual phenotype-specific and seasonal differences in auditory function. We used RNA-Seq on the Ion Torrent platform to create a combined transcriptome dataset containing over 79,000 assembled transcripts representing almost 9,000 unique annotated genes. These identified genes include several with known inner ear function and multiple steroid hormone receptors. Transcripts most closely matched to published genomes of nile tilapia and large yellow croaker, inconsistent with the phylogenetic relationship between these species but consistent with the importance of acoustic communication in their life-history strategies. We then compared the RNA-Seq results from the saccules of reproductive females with a separate transcriptome from the non-reproductive female phenotype and found over 700 differentially expressed transcripts, including members of the Wnt and Notch signaling pathways that mediate cell proliferation and hair cell addition in the inner ear. These data constitute a valuable resource for furthering our understanding of the molecular basis for peripheral auditory function as well as a range of future midshipman and cross-species comparative studies of the auditory periphery.

Comparative Analysis of the Shared Sex-Determination Region (SDR) among Salmonid Fishes.

Salmonids present an excellent model for studying evolution of young sex-chromosomes. Within the genus, Oncorhynchus, at least six independent sex-chromosome pairs have evolved, many unique to individual species. This variation results from the movement of the sex-determining gene, sdY, throughout the salmonid genome. While sdY is known to define sexual differentiation in salmonids, the mechanism of its movement throughout the genome has remained elusive due to high frequencies of repetitive elements, rDNA sequences, and transposons surrounding the sex-determining regions (SDR). Despite these difficulties, bacterial artificial chromosome (BAC) library clones from both rainbow trout and Atlantic salmon containing the sdY region have been reported. Here, we report the sequences for these BACs as well as the extended sequence for the known SDR in Chinook gained through genome walking methods. Comparative analysis allowed us to study the overlapping SDRs from three unique salmonid Y chromosomes to define the specific content, size, and variation present between the species. We found approximately 4.1 kb of orthologous sequence common to all three species, which contains the genetic content necessary for masculinization. The regions contain transposable elements that may be responsible for the translocations of the SDR throughout salmonid genomes and we examine potential mechanistic roles of each one.

Environmental influence on cyanobacteria abundance and microcystin toxin production in a shallow temperate lake.

The increasing frequency of harmful cyanobacterial blooms in freshwater systems is a commonly recognized problem due to detrimental effects on water quality. Vancouver Lake, a shallow, tidally influenced lake in the flood plain of the Columbia River within the city of Vancouver, WA, USA, has experienced numerous summertime cyanobacterial blooms, dominated by Aphanizomenon sp. and Anabaena sp. Cyanobacteria abundance and toxin (microcystin) levels have been monitored in this popular urban lake for several years; however, no previous studies have identified which cyanobacteria species produce toxins, nor analyzed how changes in environmental variables contribute to the fluctuations in toxic cyanobacteria populations. We used a suite of molecular techniques to analyze water samples from Vancouver Lake over two summer bloom cycles (2009 and 2010). Both intracellular and extracellular microcystin concentrations were measured using an ELISA kit. Intracellular microcystin concentrations exceeded WHO guidelines for recreational waters several times throughout the sampling period. PCR results demonstrated that Microcystis sp. was the sole microcystin-producing cyanobacteria species present in Vancouver Lake, although Microcystis sp. was rarely detected in microscopical counts. qPCR results indicated that the majority of the Microcystis sp. population contained the toxin-producing gene (mcyE), although Microcystis sp. abundance rarely exceeded 1 percent of overall cyanobacteria abundance. Non-metric multidimensional scaling (NMDS) revealed that PO4-P was the main environmental variable influencing the abundance of toxic and non-toxic cyanobacteria, as well as intracellular microcystin concentrations. Our study underscores the importance of using molecular genetic techniques, in addition to traditional microscopy, to assess the importance of less conspicuous species in the dynamics of harmful algal blooms.

Characterization of the OmyY1 Region on the Rainbow Trout Y Chromosome.

We characterized the male-specific region on the Y chromosome of rainbow trout, which contains both sdY (the sex-determining gene) and the male-specific genetic marker, OmyY1. Several clones containing the OmyY1 marker were screened from a BAC library from a YY clonal line and found to be part of an 800 kb BAC contig. Using fluorescence in situ hybridization (FISH), these clones were localized to the end of the short arm of the Y chromosome in rainbow trout, with an additional signal on the end of the X chromosome in many cells. We sequenced a minimum tiling path of these clones using Illumina and 454 pyrosequencing. The region is rich in transposons and rDNA, but also appears to contain several single-copy protein-coding genes. Most of these genes are also found on the X chromosome; and in several cases sex-specific SNPs in these genes were identified between the male (YY) and female (XX) homozygous clonal lines. Additional genes were identified by hybridization of the BACs to the cGRASP salmonid 4x44K oligo microarray. By BLASTn evaluations using hypothetical transcripts of OmyY1-linked candidate genes as query against several EST databases, we conclude at least 12 of these candidate genes are likely functional, and expressed.

A serendipitous discovery of isomotuporin-containing sponge populations of Theonella swinhoei.

An in-depth LCMS examination of 14 different collections of Indo-Pacific Theonella swinhoei sponges resulted in the discovery of four diastereomeric analogues of the cyclic pentapeptide motuporin. These motuporin analogues all contain a novel 2R configuration for the Adda amino acid. Additionally, one analogue has a unique nonoxygenated Adda amino acid. In all, 15 different compounds were observed by LCMS or isolated. The stereochemistries of the constituent amino acids were determined through a combination of the advanced Marfey technique and 1H NMR data.