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.

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.

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.

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.

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.