ADAPT identifies an ESCRT complex composition that discriminates VCaP from LNCaP prostate cancer cell exosomes.

Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC-MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.

Toy Buying Today: Considerations, Information Seeking, and Thoughts about Manufacturer Suggested Age.

Children frequently play with toys purchased by their parents, yet little is known about the how these toys are selected by parents. Mothers with children from 0-12 years of age filled out a survey about their toy purchasing habits. Results revealed that the most common place mothers sought out information about toys was online, and mothers most frequently found the manufacturers' suggested age only somewhat accurate. Further analyses demonstrated that mothers who bought their toys from different sources (i.e., online, stores, secondhand) considered dissimilar items before purchasing a toy for their child and obtained information in distinct ways. In contrast, regardless of where mothers purchased toys, they regarded the manufacturers' suggested age in the same way. This study is one of the few to examine parental toy selection patterns, providing useful knowledge for understanding how to reach parents and provide them information they desire when selecting toys for their children.

Increasing phylogenetic stochasticity at high elevations on summits across a remote North American wilderness.

PREMISE: At the intersection of ecology and evolutionary biology, community phylogenetics can provide insights into overarching biodiversity patterns, particularly in remote and understudied ecosystems. To understand community assembly of the high alpine flora in the Sawtooth National Forest, USA, we analyzed phylogenetic structure within and between nine summit communities. METHODS: We used high-throughput sequencing to supplement existing data and infer a nearly completely sampled community phylogeny of the alpine vascular flora. We calculated mean nearest taxon distance (MNTD) and mean pairwise distance (MPD) to quantify phylogenetic divergence within summits, and assessed whether maximum elevation explains phylogenetic structure. To evaluate similarities between summits, we quantified phylogenetic turnover, taking into consideration microhabitats (talus vs. meadows). RESULTS: We found different patterns of community phylogenetic structure within the six most species-rich orders, but across all vascular plants phylogenetic structure was largely not different from random. There was a significant negative correlation between elevation and tree-wide phylogenetic diversity (MPD) within summits: overdispersion degraded as elevation increased. Between summits, we found high phylogenetic turnover driven by greater niche heterogeneity on summits with alpine meadows. CONCLUSIONS: Our results provide further evidence that stochastic processes may also play an important role in the assembly of vascular plant communities in high alpine habitats at regional scales. However, order-specific patterns suggest that adaptations are still important for assembly of specific sectors of the plant tree of life. Further studies quantifying functional diversity will be important in disentangling the interplay of eco-evolutionary processes that likely shape broad community phylogenetic patterns in extreme environments.

Children's Utilization of Toys is Moderated by Age-Appropriateness, Toy Category, and Child Age.

Play during childhood is essential to growth and learning. Little is known about whether categories of toys moderate play behaviors at different ages, or how children interact with toys that are simple, appropriate, or complex for their developmental level. Two hundred and forty-three children between the ages of 1 and 8 years, divided into four age groups, played with toys that were targeted to their age group as well as toys aimed at one age group younger and older. Toys fell into nine different categories. Whether children fully utilized each toy was evaluated. Analyses examined how children's utilization of toys was affected by the age-appropriateness of the toy, the category of toy, and the child's age. Considering all age groups and toys, children were less likely to fully utilize toys targeted toward older children than age-appropriate toys, but this effect was moderated by the toy category and the child's age.

Comparison of the complete genome sequences of Bifidobacterium animalis subsp. lactis DSM 10140 and Bl-04.

Bifidobacteria are important members of the human gut flora, especially in infants. Comparative genomic analysis of two Bifidobacterium animalis subsp. lactis strains revealed evolution by internal deletion of consecutive spacer-repeat units within a novel clustered regularly interspaced short palindromic repeat locus, which represented the largest differential content between the two genomes. Additionally, 47 single nucleotide polymorphisms were identified, consisting primarily of nonsynonymous mutations, indicating positive selection and/or recent divergence. A particular nonsynonymous mutation in a putative glucose transporter was linked to a negative phenotypic effect on the ability of the variant to catabolize glucose, consistent with a modification in the predicted protein transmembrane topology. Comparative genome sequence analysis of three Bifidobacterium species provided a core genome set of 1,117 orthologs complemented by a pan-genome of 2,445 genes. The genome sequences of the intestinal bacterium B. animalis subsp. lactis provide insights into rapid genome evolution and the genetic basis for adaptation to the human gut environment, notably with regard to catabolism of dietary carbohydrates, resistance to bile and acid, and interaction with the intestinal epithelium. The high degree of genome conservation observed between the two strains in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies and explains the inability to differentiate the strains by standard techniques such as pulsed-field gel electrophoresis.

The effect of laparoscopic adjustable gastric bands on esophageal motility and the gastroesophageal junction: analysis using high-resolution video manometry.

BACKGROUND: Laparoscopic adjustable gastric bands (LAGB) are a safe and effective treatment for obesity. Conflicting data exist concerning their effect on the esophagus, gastroesophageal junction, and mechanism of action. These patients will increasingly require accurate assessment of their esophageal function. METHODS: Twenty LAGB patients underwent high-resolution video manometry with the LAGB empty, 20% under, 20% over, and at their optimal volume. Twenty obese controls were also studied. Effects on esophageal motility, the lower esophageal sphincter (LES), and the gastroesophageal junction were measured. Transit during liquid and semisolid swallows was assessed. RESULTS: The intraluminal pressure at the level of LAGB was a mean of 26.9 (19.8) mm Hg. This pressure varied depending on the volume within the LAGB and was separate to and distal to the lower esophageal sphincter LES. The LES was attenuated compared to controls (10 vs 18 mm Hg; p < 0.01) although relaxed normally. Esophageal motility was well preserved at optimal volume compared to 20% overfilled, with 77% normal swallows vs 51%, p = 0.008. Repetitive esophageal contractions were observed in 40% of swallows at optimal volume compared to 16% in controls, p = 0.024. In comparison to controls, the transit of liquid, 21 vs 8 s (p < 0.001), and semisolids, 50 vs 16 s (p < 0.001), was delayed. CONCLUSIONS: In LAGB patients, the LES is attenuated, although relaxes normally. Esophageal motility is preserved, although disrupted by overfilling the band. In the optimally adjusted LAGB, a delay in transit of liquids and semisolids through the esophagus and band is produced, along with an increase in repeated esophageal contractions.

Translocation of a Cell Surface Spliceosomal Complex Induces Alternative Splicing Events and Lymphoma Cell Necrosis.

Spliceosomal dysregulation dramatically affects many cellular processes, notably signal transduction, metabolism, and proliferation, and has led to the concept of targeting intracellular spliceosomal proteins to combat cancer. Here we show that a subset of lymphoma cells displays a spliceosomal complex on their surface, which we term surface spliceosomal complex (SSC). The SSC consists of at least 13 core components and was discovered as the binding target of the non-Hodgkin's lymphoma-specific aptamer C10.36. The aptamer triggers SSC internalization, causing global changes in alternative splicing patterns that eventually lead to necrotic cell death. Our study reveals an exceptional spatial arrangement of a spliceosomal complex and defines it not only as a potential target of anti-cancer drugs, but also suggests that its localization plays a fundamental role in cell survival.

Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus.

Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes that provide acquired immunity against foreign genetic elements. Here, we characterize a novel Streptococcus thermophilus locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several S. thermophilus strains. We show that both CRISPR repeats and cas genes are locus specific and functionally coupled. A total of 124 strains were studied, and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique), and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%), and S. thermophilus chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers, cas genes, CRISPR motif, locus architecture, and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers after exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that the activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the coevolution of host and viral genomes.

Primers for Castilleja and their utility across Orobanchaceae: II. Single-copy nuclear loci.

PREMISE OF THE STUDY: We developed primers targeting nuclear loci in Castilleja with the goal of reconstructing the evolutionary history of this challenging clade. These primers were tested across other major clades in Orobanchaceae to assess their broader utility. METHODS AND RESULTS: We assembled low-coverage genomes for three taxa in Castilleja and developed primer combinations for the single-copy conserved ortholog set (COSII) and the pentatricopeptide repeat (PPR) gene family. These primer combinations were designed to take advantage of the Fluidigm microfluidic PCR platform and are well suited for high-throughput sequencing applications. Eighty-seven primers were designed for Castilleja, and 27 were found to have broader utility in Orobanchaceae. CONCLUSIONS: These results demonstrate the utility of these primers, not only across Castilleja, but for other lineages within Orobanchaceae as well. This expanded molecular toolkit will be an asset to future phylogenetic studies in Castilleja and throughout Orobanchaceae.

Pathophysiology of laparoscopic adjustable gastric bands: analysis and classification using high-resolution video manometry and a stress barium protocol.

BACKGROUND: Symmetrical pouch dilatation has become the most common problem following laparoscopic adjustable gastric banding (LAGB). Although, in a significant number of symptomatic patients, no explanation for the underlying problem is identified with a contrast swallow. There is a need for a better understanding of the pathophysiology of LAGBs and more sensitive diagnostic tests. METHODS: LAGB patients with adverse symptoms or poor weight loss (symptomatic patients), in whom a contrast swallow had not shown an abnormality, underwent high-resolution video manometry. This incorporated a semi-solid, stress barium, swallow protocol. Outcomes were categorized based on anatomical appearance, transit through the LAGB, and esophageal motility. Cohorts of successful (>50% excess weight loss with no adverse symptoms) and pre-operative patients were used as controls. RESULTS: One hundred twenty-three symptomatic patients participated along with 30 successful and 56 pre-operative patients. Five pathophysiological patterns were defined: transhiatal enlargement (n = 40), sub-diaphragmatic enlargement (n = 39), no abnormality (n = 30), aperistaltic esophagus (n = 7), and intermittent gastric prolapse (n = 3). Esophageal motility disorders were more common in symptomatic and pre-operative patients than in successful patients (p = 0.01). Differences between successful and symptomatic patients were identified in terms of the length of the high-pressure zone above the LAGB (p < 0.005), peristaltic velocity (p < 0.005), frequency of previous surgery(p = 0.01), and lower esophageal sphincter tone (p = 0.05). CONCLUSIONS: Video manometry identified abnormalities in three quarters of symptomatic patients where conventional contrast swallow had not been diagnostic. Five primary patterns of pathophysiology were defined. These were used to develop a seven category, clinical, classification system based on the anatomical appearance at stress barium. This system stratifies the spectrum of symmetrical pouch dilatation and can be used to logically guide treatment.

Effects of gastric band adjustments on intraluminal pressure.

BACKGROUND: Understanding of the effects of adjustments to laparoscopic adjustable gastric band (LAGB) volume is limited. Changes in intraluminal pressure may be important and explain patients reporting a tighter LAGB after saline is removed and an identical volume replaced. METHODS: Using high-resolution manometry, changes in the basal intraluminal pressure at the level of the LAGB in response to sequential, small alterations in LAGB volume were recorded. All fluid was removed from the LAGB and replaced, pressures and motility were reassessed. RESULTS: Sixteen patients (four males, age 45.4+/-13.2 years) participated. A linear increase (r2=0.87+/-0.12) in intraluminal pressure was observed after a threshold volume was reached. The threshold volume varied considerably (1.0 to 5.8 ml). The gradient of the linear increase was 21.2+/- 8.7 mmHg/ml. The mean basal intraluminal pressure at the level of the LAGB was initially 19.1+/-8.9 mmHg and increased to 37.0+/-20.4 mmHg (p=0.001) after removing and replacing the same volume of saline. There was an increase in distal esophageal peristaltic pressure (123.5+/- 34.7 vs. 157.4+/-52.6 mmHg, p=0.003) and a decrease in the proportion of normal swallows (0.85+/-0.22 vs. 0.53+/- 0.47, p=0.02). Nine patients also developed adverse symptoms. CONCLUSIONS: Intraluminal pressure at the level of the LAGB is an objective measure of the restriction produced by LAGBs. The addition of fluid to the LAGB results in a linear increase in intraluminal pressure once a threshold volume is reached. The removal and replacement of the same volume of saline from the LAGB may temporarily increase intraluminal pressure.

CRISPR provides acquired resistance against viruses in prokaryotes.

Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.