Analysis of Mice with Single and Multiple Copies of Transgenes Reveals a Novel Arrangement for the λ5-VpreB1 Locus Control Region

The murine λ5-V_preB1 locus encodes two proteins that form part of the pre-B-cell receptor and play a key role in B-lymphocyte development. We have identified a locus control region (LCR) which is responsible for coordinate activation of both genes in pre-B cells. Analysis of mice with single and multiple copies of transgenes shows a clear difference in the expression behavior of the genes depending on the transgene copy number. While expression of both λ5 and V_preB1 in single- and two-copy integrations requires the presence of a set of DNase I hypersensitive sites located 3′ of the λ5 gene, small fragments containing the genes have LCR activity when arranged in multiple-copy tandem arrays, indicating that additional components of the LCR are located within or close to the genes. The complete LCR is capable of driving efficient copy-dependent expression of a λ5 gene in pre-B cells even when it is integrated into centomeric γ-satellite DNA. The finding that activation of expression of the locus by positively acting factors is fully dominant over the silencing effect of heterochromatin has implications for models for chromatin-mediated gene silencing during B-cell development.     

Foraging environment determines the genetic architecture and evolutionary potential of trophic morphology in cichlid fishes

Phenotypic plasticity allows organisms to change their phenotype in response to shifts in the environment. While a central topic in current discussions of evolutionary potential, a comprehensive understanding of the genetic underpinnings of plasticity is lacking in systems undergoing adaptive diversification. Here, we investigate the genetic basis of phenotypic plasticity in a textbook adaptive radiation, Lake Malawi cichlid fishes. Specifically, we crossed two divergent species to generate an F₃ hybrid mapping population. At early juvenile stages, hybrid families were split and reared in alternate foraging environments that mimicked benthic/scraping or limnetic/sucking modes of feeding. These alternate treatments produced a variation in morphology that was broadly similar to the major axis of divergence among Malawi cichlids, providing support for the flexible stem theory of adaptive radiation. Next, we found that the genetic architecture of several morphological traits was highly sensitive to the environment. In particular, of 22 significant quantitative trait loci (QTL), only one was shared between the environments. In addition, we identified QTL acting across environments with alternate alleles being differentially sensitive to the environment. Thus, our data suggest that while plasticity is largely determined by loci specific to a given environment, it may also be influenced by loci operating across environments. Finally, our mapping data provide evidence for the evolution of plasticity via genetic assimilation at an important regulatory locus, ptch1. In all, our data address long‐standing discussions about the genetic basis and evolution of plasticity. They also underscore the importance of the environment in affecting developmental outcomes, genetic architectures, morphological diversity and evolutionary potential.     

Bommies away! Logistics and early effects of repositioning 400 tonnes of displaced coral colonies following cyclone impacts on the Great Barrier Reef

For over 40 years, management of the Great Barrier Reef Marine Park (GBRMP) in Australia has focused on limiting human‐use impacts to facilitate natural resilience and recovery. Compounding acute disturbances and chronic stressors have resulted in degradation of coral reef habitats in many areas of the Marine Park. Given current trends and predictions of escalating climate‐driven disturbances, it is increasingly evident that effective management of the GBRMP requires adaptive and novel approaches to protect and restore coral reef health. Here, we provide an overview of the logistical requirements and early‐stage ecological benefits of repositioning 400 tonnes of moderately sized (1–3 m diameter) Porites spp. coral colonies (bommies) that were displaced by cyclone‐generated swells that impacted reefs in the Whitsunday Islands during March 2017. An ecological survey conducted 16 months after the bommie repositioning revealed that several genera of hard coral had settled onto the bommies and that a range of reef fish species were associating with the restored habitat. Early findings suggest that the repositioning of the displaced bommies has assisted in restoring reef habitat structure and settlement habitat for juvenile corals, while improving natural aesthetics, vessel access and tourist experiences at Manta Ray Bay.     

Merocyanine 540, a fluorescent probe sensitive to lipid packing

Binding of the lipophilic probe merocyanine 540 to artificial bilayers was assessed by measuring the enhancement of fluorescence which results when dye enters the hydrophobic environment of the membrane. Titration of a constant amount of dye with increasing amounts of vesicles revealed that much more dye binds to multilamellar and 1000-Å, unilamellar vesicles which are in the fluid-phase state than to comparable vesicles which are in the gel-phase state. Incorporation of cholesterol into fluid-phase vesicles at levels of greater than 20 mol% reduced dye binding, whereas cholesterol had no effect at any concentration when incorporated into gel-phase vesicles. Sonicated 200–300-Å unilamellar gel-phase vesicles, which because of their reduced radius of curvature resemble fluid-phase bilayers in their more widely spaced exterior leaflet lipids, bound more dye than 1000-Å unilameilar gel-phase vesicles constructed from the same lipid. These results suggest that merocyanine 540 is able to sense the degree of lipid packing of bilayers and inserts preferentially into bilayers whose lipids are more widely spaced.     

Exploratory analysis of quantitative histopathology of cervical intraepithelial neoplasia: objectivity, reproducibility, malignancy-associated changes, and human papillomavirus.

BACKGROUND: As part of a project to evaluate emerging optical technologies for cervical neoplasia, our group is performing quantitative histopathological analyses of biopsy specimens from 1,190 patients. Objectives in the interim analysis are (a) quantitatively assessing progression of the neoplastic process of cervical intraepithelial neoplasia (CIN)/squamous intraepithelial lesions (SIL), (b) detecting malignancy-associated changes (MACs), and (c) phenotypically measuring human papillomavirus (HPV) detected by DNA testing. METHODS: The diagnostic region of interest (ROI) from immediately adjacent sections were imaged, and the basal lamina and surface of the superficial layer were delimited. Nonoverlapping quantitatively stained nuclei were selected from 1,190 samples with histopathological characteristics of normal (929), koilocytosis (130), CIN 1 (40), CIN 2 (23), and CIN 3/carcinoma in situ (CIS) (68). A fully automatic procedure located and recorded the center of every nucleus in the region of interest (ROI). We used linear discriminant analysis to assess the changes between normal and CIN 3/CIS. RESULTS: Scores computed from the cell-by cell features and the clinical grade of CIN/SIL were highly correlated, as were those of the architectural features and the clinical grade of CIN/SIL. We found even higher correlations between a combination of cell-by-cell and architectural scores, and clinical grade. Using these scores, we found MACs in the normal biopsy specimens from patients with high-grade CIN/SIL. Furthermore, the same scores correlated with the molecular detection of HPV. CONCLUSIONS: Quantitative histopathology can be used in large clinical trials as an objective and reproducible measure of CIN/SIL. Detectable phenotypic changes correlate well with CIN/SIL neoplastic progression. It can also be used to infer the presence of CIN/SIL (MACs) and molecular changes associated with increased risk of cancer development (high-risk HPV).     

Closing the ‘phenotype gap’ in precision medicine: improving what we measure to understand complex disease mechanisms

The central concept underlying precision medicine is a mechanistic understanding of each disease and its response to therapy sufficient to direct a specific intervention. To execute on this vision requires parsing incompletely defined disease syndromes into discrete mechanistic subsets and developing interventions to precisely address each of these etiologically distinct entities. This will require substantial adjustment of traditional paradigms which have tended to aggregate high-level phenotypes with very different etiologies. In the current environment, where diagnoses are not mechanistic, drug development has become so expensive that it is now impractical to imagine the cost-effective creation of new interventions for many prevalent chronic conditions. The vision of precision medicine also argues for a much more seamless integration of research and development with clinical care, where shared taxonomies will enable every clinical interaction to inform our collective understanding of disease mechanisms and drug responses. Ideally, this would be executed in ways that drive real-time and real-world discovery, innovation, translation, and implementation. Only in oncology, where at least some of the biology is accessible through surgical excision of the diseased tissue or liquid biopsy, has “co-clinical” modeling proven feasible. In most common germline disorders, while genetics often reveal the causal mutations, there still remain substantial barriers to efficient disease modeling. Aggregation of similar disorders under single diagnostic labels has directly contributed to the paucity of etiologic and mechanistic understanding by directly reducing the resolution of any subsequent studies. Existing clinical phenotypes are typically anatomic, physiologic, or histologic, and result in a substantial mismatch in information content between the phenomes in humans or in animal ‘models’ and the variation in the genome. This lack of one-to-one mapping of discrete mechanisms between disease and animal models causes a failure of translation and is one form of ‘phenotype gap.’ In this review, we will focus on the origins of the phenotyping deficit and approaches that may be considered to bridge the gap, creating shared taxonomies between human diseases and relevant models, using cardiovascular examples.

     

Effect of Crystallization State on the Gel Properties of Oleogels Based on β-sitosterol

Oleogels based on three different oils (sunflower oil, solid coconut oil and liquid coconut oil) were formulated using β-sitosterol. In general, an observed increase in crystallinity was correlated with an increase in the gel storage modulus and hardness. Addition of lecithin promoted the formation of needle-like crystals of β-sitosterol with a corresponding increase in strain tolerance and oil-trapping capacity for oleogels produced with liquid oils. However, the incorporation of β-sitosterol crystals with or without lecithin into oleogels containing solid coconut oil reduced its strain tolerance by interrupting the formation of continual radiolitic crystal structures. The use of sunflower oil (long chain fatty acids) was more favourable to the packing and growth of gelator crystals and the formation of an elastic gel, in comparison to liquid coconut oil (short chain fatty acids). Overall, the type and physical state of oil influence the formation of oil crystal network, thus affecting its gel properties. These findings allow the better understanding of β-sitosterol-based oleogels, providing opportunity to design for the application as a fat-replacer and lowering solid fat content.

     

High-resolution array CGH increases heterogeneity tolerance in the analysis of clinical samples

Recent advances in array comparative genomic hybridization (array CGH) technology are revolutionizing our understanding of tumor genomes. Marker-based arrays enable rapid survey at megabase intervals, while tiling path arrays examine the entire genome in unprecedented detail. Tumor biopsies are typically small and contain infiltrating stromal cells, requiring tedious microdissection. Tissue heterogeneity is a major barrier to high-throughput profiling of tumor genomes and is also an important consideration for the introduction of array CGH to clinical settings. We propose that increasing array resolution will enhance detection sensitivity in mixed tissues and as a result significantly reduce microdissection requirements. In this study, we first simulated normal cell contamination to determine the heterogeneity tolerance of array CGH and then validated this detection sensitivity model on cancer specimens using the newly developed submegabase resolution tiling-set (SMRT) array, which spans the human genome with 32,433 overlapping BAC clones.     

Natural history of cervical intraepithelial neoplasia: a meta-analysis

OBJECTIVE: To determine the probabilities of transition of stages in the cervical cancer by conducting a meta-studies on the topic. STUDY DESIGN: We identified health states of interest in the natural history of cervical precancer, identified all possible papers that could meet selection criteria, developed relevance and acceptability criteria for inclusion, then thoroughly reviewed the selected studies. To determine the transition probability data we used a random effects model. We determined probabilities for 4 health state transitions. The 6-month mean predictive transition probability (95% confidence intervals with "prediction interval" in parentheses) for high grade squamous intraepithelial lesions (HSIL) to cancer was 0.0037 (0.00004, 0.03386), for low grade squamous intraepithelial lesions (LSIL) to HSIL was 0.0362 (0.00055, 0.23220), for HSIL to LSIL was 0.0282 (0.00027, 0.35782), and for LSIL to normal was 0.0740 (0.00119, 0.42672). CONCLUSION: The transition probabilities between cervical cancer health states for 6-month intervals are small; however, the cumulative risk of cervical cancer is significant. Markers to identify the cervical precursors that will lead to the transition to cervical cancer are needed.