Development of a Cloning Strategy to Enable the Isolation of Novel Membrane Proteins

The aim of the project was to develop a cloning strategy for the isolation of novel membrane proteins. The scheme evolved from the surface antigen screening system developed by Seed and Aruffo (1987). The system was adapted to enable the cloning of cDNAs coding for membrane proteins to which no antibodies exist. This was achieved by taking a previously cloned membrane protein gene (CD2), deleting its transmembrane (Tm) and cytosolic regions, and replacing them with the polylinker and stuffer sequences of CDM8. cDNAs were then sub-cloned into this expression vector to generate a fusion library. which was then screened for surface expression of the CD2 protein

Spatial Variation in Erosion Rates in Mars Equatorial Regions Inferred from Ejecta Retention of 1-3 Km Diameter Craters

The modification of impact craters has long been used to infer the geomorphic forcing on Mars [1], as well as estimate the spatial and temporal variability of this erosion and gradation [e.g., 2]. Here, we studied the population of small primary craters (1-3 km) to understand differences in ejecta retention across equatorial Mars. Specifically, we evaluated whether craters in our study population had observable ejecta deposits (defined on the basis of distinct tone or texture with respect to their surroundings).This is a proxy for the resurfacing rate because only relatively fresh craters retain their ejecta deposits. More broadly, this is part of a larger project we are undertaking [3] to examine crater morphometry and other characteristics from CTX-derived digital terrain models (DTMs), augmented by qualitative observations

5-Aminolevulinic acid-mediated fluorescence diagnosis of colon cancer: A histopathological comparison of fluorescent and non-fluorescent tumours

Background: 5-Aminolevulinic acid (5-ALA) selectively accumulates in cancer cells and is metabolised in the mitochondria to the fluorophore protoporphyrin IX. The GLiSten trial evaluated 5-ALA as a fluorescent probe for intraoperative detection of colon cancer and lymph node metastases. Only 13 of 40 cases showed fluorescence, suggesting a fundamental difference between fluorescent and non-fluorescent cancers. The aim of this study was to investigate whether differences in fluorescence were due to tumour cellularity, in particular T cell infiltration, which may be of prognostic significance. Method: Primary tumour tissue was available from 30 patients. The density of tumour cells, vascularity and stromal compartment size were quantified using digitally scanned tissue sections stained with haematoxylin and eosin. A set of 300 random points was superimposed onto each tumour image. The structure indicated by each point was then categorised as tumour, stroma, vessel or other. The proportions of tumour and vessel points gave the tumour cell density and vessel density respectively. The relative size of the stromal compartment was given by the tumour to stroma ratio. A tissue section was also stained for the T cell marker CD3 by immunohistochemistry. Percentage staining was quantified in three high-density fields using the Nuance imaging system. Results: We were unable to detect any difference between fluorescent and non-fluorescent cancers in terms of tumour cell density (difference in means 3.7%; P = 0.452), vessel density (difference in means 0.17%; P = 0.684), tumour-stroma ratio (difference in mean ratios 0.12; P = 0.934), or T cell count (difference in means 0.92%; P = 0.726). Furthermore, comparisons of the distributions of each variable demonstrated substantial overlap between the fluorescent and non-fluorescent cohorts. Conclusion: The results suggest that tumour and microenvironment structure do not differ between cancers that fluoresce with 5-ALA and those that do not. We therefore propose that the cellular metabolism of 5-ALA is a more likely explanation for differential fluorescence

System-L amino acid transporters play a key role in pancreatic b-cell signalling and function

The branched-chain amino acids (BCAA) leucine, isoleucine and valine, are essential amino acids that play a critical role in cellular signalling and metabolism. They acutely stimulate insulin secretion and activate the regulatory serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1), a kinase that promotes increased β-cell mass and function. The effects of BCAA on cellular function are dependent on their active transport into mammalian cells via amino acid transporters and thus the expression and activity of these transporters likely influences β-cell signalling and function. In this report we show that the System-L transporters are required for BCAA uptake into clonal β-cell lines and pancreatic islets and that these are essential for signalling to mTORC1. Further investigation revealed that the System-L transporter LAT1 is abundantly expressed in islets and that knock-down of LAT1 using siRNA inhibits mTORC1 signalling, leucine-stimulated insulin secretion and islet cell proliferation. In summary, we show that the System-L transporter LAT1 is required for regulating β-cell signaling and function in islets and thus may be a novel pharmacological/nutritional target for the treatment and prevention of type-2 diabetes

pVAC-Seq: A genome-guided in silico approach to identifying tumor neoantigens

Cancer immunotherapy has gained significant momentum from recent clinical successes of checkpoint blockade inhibition. Massively parallel sequence analysis suggests a connection between mutational load and response to this class of therapy. Methods to identify which tumor-specific mutant peptides (neoantigens) can elicit anti-tumor T cell immunity are needed to improve predictions of checkpoint therapy response and to identify targets for vaccines and adoptive T cell therapies. Here, we present a flexible, streamlined computational workflow for identification of personalized Variant Antigens by Cancer Sequencing (pVAC-Seq) that integrates tumor mutation and expression data (DNA- and RNA-Seq). pVAC-Seq is available a

Impact of Moderate to Severe Renal Impairment on Mortality and Appropriate Shocks in Patients with Implantable Cardioverter Defibrillators

Background. Due to underrepresentation of patients with chronic kidney disease (CKD) in large Implantable-Cardioverter Defibrillator (ICD) clinical trials, the impact of ICD remains uncertain in this population. Methods. Consecutive patients who received ICD at Creighton university medical center between years 2000–2004 were included in a retrospective cohort after excluding those on maintenance dialysis. Based on baseline Glomerular filtration rate (GFR), patients were classified as severe CKD: GFR < 30 mL/min; moderate CKD: GFR: 30–59 mL/min; and mild or no CKD: GFR ≥ 60 mL/min. The impact of GFR on appropriate shocks and survival was assessed using Kaplan-Meier method and Generalized Linear Models (GLM) with log-link function. Results. There were 509 patients with a mean follow-up of 3.0 + 1.3 years. Mortality risk was inversely proportional to the estimated GFR: 2 fold higher risk with GFR between 30–59 mL/min and 5 fold higher risk with GFR < 30 mL/min. One hundred and seventy-seven patients received appropriate shock(s); appropriate shock-free survival was lower in patients with severe CKD (GFR < 30) compared to mild or no CKD group (2.8 versus 4.2 yrs). Conclusion. Even moderate renal dysfunction increases all cause mortality in CKD patients with ICD. Severe but not moderate CKD is an independent predictor for time to first appropriate shock

catena-Poly[(diaqua­strontium)-bis­(μ-2-methyl-3,5-dinitro­benzoato)]

The title compound, [Sr(C8H5N2O6)2(H2O)2]n, essentially consists of a one-dimensional polymeric network with Sr2O2 rings extending along the [100] direction. The range of Sr—O bond lengths is 2.4822 (13)–2.8113 (13) Å. C—H⋯O and O—H⋯O hydrogen-bonding inter­actions stabilize the mol­ecules in the form of a two-dimensional polymeric network parallel to (001). One of the nitro groups is disordered over three sets of sites with the occupancy ratio of 0.46:0.32:0.22

Metformin reduces airway glucose permeability and hyperglycaemia-induced Staphylococcus aureus load independently of effects on blood glucose

Background Diabetes is a risk factor for respiratory infection, and hyperglycaemia is associated with increased glucose in airway surface liquid and risk of Staphylococcus aureus infection. Objectives To investigate whether elevation of basolateral/blood glucose concentration promotes airway Staphylococcus aureus growth and whether pretreatment with the antidiabetic drug metformin affects this relationship. Methods Human airway epithelial cells grown at air–liquid interface (±18 h pre-treatment, 30 μM–1 mM metformin) were inoculated with 5×105 colony-forming units (CFU)/cm2 S aureus 8325-4 or JE2 or Pseudomonas aeruginosa PA01 on the apical surface and incubated for 7 h. Wild-type C57BL/6 or db/db (leptin receptor-deficient) mice, 6–10 weeks old, were treated with intraperitoneal phosphate-buffered saline or 40 mg/kg metformin for 2 days before intranasal inoculation with 1×107 CFU S aureus. Mice were culled 24 h after infection and bronchoalveolar lavage fluid collected. Results Apical S aureus growth increased with basolateral glucose concentration in an in vitro airway epithelia–bacteria co-culture model. S aureus reduced transepithelial electrical resistance (RT) and increased paracellular glucose flux. Metformin inhibited the glucose-induced growth of S aureus, increased RT and decreased glucose flux. Diabetic (db/db) mice infected with S aureus exhibited a higher bacterial load in their airways than control mice after 2 days and metformin treatment reversed this effect. Metformin did not decrease blood glucose but reduced paracellular flux across ex vivo murine tracheas. Conclusions Hyperglycaemia promotes respiratory S aureus infection, and metformin modifies glucose flux across the airway epithelium to limit hyperglycaemia-induced bacterial growth. Metformin might, therefore, be of additional benefit in the prevention and treatment of respiratory infection