Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation

Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes

LRP1 influences trafficking of N-type calcium channels via interaction with the auxiliary α2δ-1 subunit.

Voltage-gated Ca(2+) (CaV) channels consist of a pore-forming α1 subunit, which determines the main functional and pharmacological attributes of the channel. The CaV1 and CaV2 channels are associated with auxiliary β- and α2δ-subunits. The molecular mechanisms involved in α2δ subunit trafficking, and the effect of α2δ subunits on trafficking calcium channel complexes remain poorly understood. Here we show that α2δ-1 is a ligand for the Low Density Lipoprotein (LDL) Receptor-related Protein-1 (LRP1), a multifunctional receptor which mediates trafficking of cargoes. This interaction with LRP1 is direct, and is modulated by the LRP chaperone, Receptor-Associated Protein (RAP). LRP1 regulates α2δ binding to gabapentin, and influences calcium channel trafficking and function. Whereas LRP1 alone reduces α2δ-1 trafficking to the cell-surface, the LRP1/RAP combination enhances mature glycosylation, proteolytic processing and cell-surface expression of α2δ-1, and also increase plasma-membrane expression and function of CaV2.2 when co-expressed with α2δ-1. Furthermore RAP alone produced a small increase in cell-surface expression of CaV2.2, α2δ-1 and the associated calcium currents. It is likely to be interacting with an endogenous member of the LDL receptor family to have these effects. Our findings now provide a key insight and new tools to investigate the trafficking of calcium channel α2δ subunits

Assessing the Consultation and Relational Empathy (CARE) Measure in sexual health nurses' consultations

Background: Increasingly healthcare policies emphasise the importance of person-centred, empathic care. Consequently, healthcare professionals are expected to demonstrate the ‘human’ aspects of care in training and in practice. The Consultation and Relational Empathy (CARE) Measure is a patient-rated measure of the interpersonal skills of healthcare practitioners. It has been widely validated for use by healthcare professionals in both primary and secondary care. This paper reports on the validity and reliability of the CARE Measure with sexual health nurses. Methods: Patient questionnaires were collected for 943 consultations with 20 sexual health nurses. Participating patients self-completed the questionnaire immediately after the encounter with the nurse. The questionnaire included the ten item CARE Measure, the Patient Enablement Index, and overall satisfaction instruments. Construct validity was assessed through Spearman’s correlation and principal component analysis. Internal consistence was assessed through Cronbach’s alpha and the inter-rater reliability through Generalisability Theory. Data were collected in 2013 in Scotland. Results: Female patients completed 68% of the questionnaires. The mean patient age was 28.8 years (standard deviation 9.8 years). Two of the 20 participating nurses withdrew from the study. Most patients (71.7%) regarded the CARE Measure items as very important to their consultation and the number of ‘not applicable’ and missing responses’ were low (2.6% and 0.1% respectively). The participating nurses had high CARE Measure scores; out of a maximum possible score of 50, the overall mean CARE measure score was 47.8 (standard deviation 4.4). The scores were moderately correlated with patient enablement (rho = 0.232, p = 0.001) and overall satisfaction (rho = 0.377, p = 0.001. Cronbach’s alpha showed the measure’s high internal consistency (Cronbach’s alpha coefficient = 0.95), but the inter-rater reliability could not be calculated due to the high achieved CARE Measure scores that varied little between nurses. Conclusions: Within this clinical context the CARE Measure has high perceived relevance and face validity. The findings support construct validity and some evidence of reliability. The high CARE Measure scores may have been due to sample bias. A future study which ensures a representative sample of patients on a larger group of nurses is required to determine whether the measure can discriminate between nurses

Increase in Tau Pathology in P290S Mapt Knock-In Mice Crossed with AppNL-G-F Mice

Alzheimer's Disease (AD) is characterized by the pathological assembly of Aβ peptide, which deposits into extracellular plaques, and tau, which accumulates in intraneuronal inclusions. To investigate the link between Aβ and tau pathologies, experimental models featuring both pathologies are needed. We developed a mouse model featuring both tau and Aβ pathologies by knocking the P290S mutation into murine Mapt and crossing these MaptP290S KI mice with the AppNL-G-F KI line. MaptP290S KI mice developed a small number of tau inclusions, which increased with age. The amount of tau pathology was significantly larger in AppNL-G-FxMaptP290S KI mice from 18-months of age onwards. Tau pathology was higher in limbic areas, including hippocampus, amygdala and piriform/entorhinal cortex. We also observed AT100-and Gallyas-Braak-silver-positive dystrophic neurites containing assembled filamentous tau, as visualized by in situ EM. Using a cell-based tau seeding assay, we showed that sarkosyl-insoluble brain extracts from both 18-month-old MaptP290S KI and AppNL-G-FxMaptP290S KI mice were seed-competent, with brain extracts from double KI mice seeding significantly more than those from the MaptP290S KI mice. Finally, we showed that AppNL-G-FxMaptP290S KI mice had neurodegeneration in the piriform cortex from 18-months of age. We suggest that AppNL-G-F x MaptP290S KI mice provide a good model for studying the interactions of aggregation-prone tau, Aβ, neuritic plaques, neurodegeneration and aging

Long-term results after liver transplantation for primary hepatic epithelioid hemangioendothelioma

Background: Hepatic epithelioid hemangioendothelioma (PHEHE) is a multifocal, low-grade malignant neoplasia characterized by its epithelial-like appearance and vascular endothelial histogenesis. The outcome of 16 patients treated with orthotopic liver transplantation (OLT) is the subject of this report. Methods: A retrospective study of 16 patients with HEHE (7 men, 9 women) with ages ranging from 24 to 58 years (mean 37 ± 10.6 years). Follow-up intervals ranged from 1 to 15 years (median of 4.5 years). Results: Actual patient survival at 1, 3, and 5 years was 100, 87.5, and 71.3%, respectively. Disease-free survival at 1, 3, and 5 years was 81.3, 68.8, and 60.2%, respectively. The 90-day operative mortality was 0. Involvement of the hilar lymph nodes or vascular invasion did not affect survival. The 5-year survival of HEHE compares favorably with that of hepatocellular carcinoma at the same stage (stage 4A): 71.3 versus 9.8% (p=0.001) Conclusions: The long-term survival obtained in this series justifies OLT for these tumors even in the presence of limited extrahepatic disease. © 1995 The Society of Surgical Oncology, Inc

Slightly Non-Minimal Dark Matter in PAMELA and ATIC

We present a simple model in which dark matter couples to the standard model through a light scalar intermediary that is itself unstable. We find this model has several notable features, and allows a natural explanation for a surplus of positrons, but no surplus of anti-protons, as has been suggested by early data from PAMELA and ATIC. Moreover, this model yields a very small nucleon coupling, well below the direct detection limits. In this paper we explore the effect of this model in both the early universe and in the galaxy.Comment: 7 pages, 6 figures, v3: updated for new data, added discussion of Ferm

Data-Oriented Declarative Language for Optimizing Business Processes

There is a signifi cant number of declarative languages to describe business processes. They tend to be used when business processes need to be fl exible and adaptable, being not possible to use an imperative description. Declarative languages in business process have been traditionally used to describe the order of activities, specifi cally the order allowed or prohibited. Unfortunately, none of them is worried about a declarative description of exchanged data between the activities and how they can infl uence the model. In this paper, we analyse the data description capacity of a variety of declarative languages in business processes. Using this analysis, we have detected the necessity to include data exchanged aspects in the declarative descriptions. In order to solve the gap, we propose a Data-Oriented Optimization Declarative LanguagE, called DOODLE, which includes the process requirements referred to data description, and the possibility to include an optimization function about the process output data

Distributions of charged massive scalars and fermions from evaporating higher-dimensional black holes

A detailed numerical analysis is performed to obtain the Hawking spectrum for charged, massive brane scalars and fermions on the approximate background of a brane charged rotating higher-dimensional black hole constructed in arXiv:0907.5107. We formulate the problem in terms of a "spinor-like" first order system of differential wave equations not only for fermions, but for scalars as well and integrate it numerically. Flux spectra are presented for non-zero mass, charge and rotation, confirming and extending previous results based on analytic approximations. In particular we describe an inverted charge splitting at low energies, which is not present in four or five dimensions and increases with the number of extra dimensions. This provides another signature of the evaporation of higher-dimensional black holes in TeV scale gravity scenarios.Comment: 19 pages, 6 figures, minor typos corrected, 1 page added with a discussion on higher spins, added reference

On 4d rank-one N=3 superconformal field theories

We study the properties of 4d N=3 superconformal field theories whose rank is one, i.e. those that reduce to a single vector multiplet on their moduli space of vacua. We find that the moduli space can only be of the form C^3/Z_k for k=1,2,3,4,6, and that the supersymmetry automatically enhances to N=4 for k=1,2. In addition, we determine the central charges a and c in terms of k, and construct the associated 2d chiral algebras, which turn out to be exotic N=2 supersymmetric W-algebras.Comment: 24 page