Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate

Purpose The retinal pigment epithelium (RPE) and underlying Bruch’s membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell–substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch’s membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this “aged” substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. Methods Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. Results Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. Conclusions This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch”s membrane could play a significant role in age-related dysfunction of the RPE.

Vegetation responses to rapid climatic changes during the last deglaciation 13,500-8,000 years ago on southwest Andoya, arctic Norway

The late-glacial vegetation development in northern Norway in response to climate changes during the Allerod, Younger Dryas (YD), and the transition to the Holocene is poorly known. Here we present a high-resolution record of floral and vegetation changes at lake Lusvatnet, south-west Andoya, between 13500 and 8000 cal b.p. Plant macrofossil and pollen analyses were done on the same sediment core and the proxy records follow each other very closely. The core has also been analyzed using an ITRAX XRF scanner in order to check the sediment sequence for disturbances or hiatuses. The core has a good radiocarbon-based chronology. The Saksunarvatn tephra fits very well chronostratigraphically. During both the Allerod and the Younger Dryas time-periods arctic vegetation prevailed, dominated by Salix polaris associated with many typically arctic herbs such as Saxifraga cespitosa, Saxifraga rivularis and Oxyria digyna. Both periods were cold and dry. Between 12450 and 12250 cal b.p. during the Younger Dryas chronozone, the assemblage changed, particularly in the increased abundance of Papaver sect. Scapiflora and other high-Arctic herbs, suggesting the development of polar desert vegetation mainly as a response to increased aridity. After 11520 cal b.p. a gradually warmer and more oceanic climate initiated a succession to dwarf-shrub vegetation and the establishment of Betula woodland after 1,000 years at c. 10520 cal b.p. The overall late-glacial aridity contrasts with oceanic conditions in southern Norway and is probably related to sea-ice extent.

Production, characterisation and applications of monoclonal antibodies to two novel porcine bocaviruses from swine in Northern Ireland

The production, preliminary characterisation and applications of monoclonal antibodies (mAbs) against two novel swine bocaviruses isolated in cell culture from swine in Northern Ireland are described. Of the 17 stable final clones produced, four were characterised. All were of the IgG2a isotype and showed no cross-reactivity with either bocavirus strain. Partial neutralisation was observed with PBoV4 mAbs and homologous virus. The two mAbs selected for use in antigen-detecting ELISAs were successful in highlighting those fractions containing infectious virus within sucrose gradients. This is the first report of the production of specific reagents that will prove useful in the study of the biology of these viruses and swine bocavirus-associated diseases.

Keeping it real! Enhancing realism in standardised patient OSCE stations

Background: Objective structured clinical examinations (OSCEs) are a
commonly used method of assessing clinical competency in healthcare education. They can providean opportunity to observe candidates interacting with patients.
There are many challenges in using real patients in OSCEs, and increasingly standardised patients are being used as a preference. However, by using standardised patients there is a risk of making the encounter arti?cial and removed from actual clinical practice.
Context: Efforts made in terms of cognitive, auditory, visual, tactile, psychological and emotional cues can minimise the differences between a simulated
and real clinical scenario. However, a number of factors, including feasibility, cost and usability, need to be considered if such techniques are to be practicable
within an OSCE framework.
Innovation: This article describes a series of techniques that have been used in our institution to enhance the realism of a standardised patient encounter in an
OSCE. Efforts in preparing standardised patient roles, and how they portray these roles, will be considered. A wide variety of equipment can also be used in
combination with a patient and the surrounding environment, which can further enhance the authenticity of the simulated scenario.
Implications: By enhancing the realism in simulated patient OSCE encounters, there is potential to trigger more authentic conscious responses from candidates and implicit reactions that the candidates themselves may be less
aware of. Furthermore, using such techniques may allow faculty members to select scenarios that were previously not thought possible in an OSCE

SNP in the genome-wide association study hotspot on chromosome 9p21 confers susceptibility to diabetic nephropathy in type 1 diabetes

AIMS/HYPOTHESIS: Parental type 2 diabetes mellitus increases the risk of diabetic nephropathy in offspring with type 1 diabetes mellitus. Several single nucleotide polymorphisms (SNPs) that predispose to type 2 diabetes mellitus have recently been identified. It is, however, not known whether such SNPs also confer susceptibility to diabetic nephropathy in patients with type 1 diabetes mellitus. METHODS: We genotyped nine SNPs associated with type 2 diabetes mellitus in genome-wide association studies in the Finnish population, and tested for their association with diabetic nephropathy as well as with severe retinopathy and cardiovascular disease in 2,963 patients with type 1 diabetes mellitus. Replication of significant SNPs was sought in 2,980 patients from three other cohorts. RESULTS: In the discovery cohort, rs10811661 near gene CDKN2A/B was associated with diabetic nephropathy. The association remained after robust Bonferroni correction for the total number of tests performed in this study (OR 1.33 [95% CI 1.14, 1.56], p?=?0.00045, p (36tests)?=?0.016). In the meta-analysis, the combined result for diabetic nephropathy was significant, with a fixed effects p value of 0.011 (OR 1.15 [95% CI 1.02, 1.29]). The association was particularly strong when patients with end-stage renal disease were compared with controls (OR 1.35 [95% CI 1.13, 1.60], p?=?0.00038). The same SNP was also associated with severe retinopathy (OR 1.37 [95% CI 1.10, 1.69] p?=?0.0040), but the association did not remain after Bonferroni correction (p (36tests)?=?0.14). None of the other selected SNPs was associated with nephropathy, severe retinopathy or cardiovascular disease. CONCLUSIONS/INTERPRETATION: A SNP predisposing to type 2 diabetes mellitus, rs10811661 near CDKN2A/B, is associated with diabetic nephropathy in patients with type 1 diabetes mellitus.

Modelling the cycling of persistent organic pollutants (POPs) in the North Sea system: Fluxes, loading, seasonality, trends

The fate and cycling of two selected POPs is investigated for the North Sea system with an improved version of a fate and transport ocean model (FANTOM). The model uses atmospheric data from the EMEP MSC East POP model (Gusev et al., 2009), giving reasonable concentrations and seasonal distributions for the entire region, as opposed to the three observation stations that Ilyina et al. (2006) were limited to. Other model improvements include changes in the calculation of POP exchange between the water column and sediment.

We chose to simulate the fate of two POPs with very different properties, ?-HCH and PCB 153. Since the fate and cycling of POPs are strongly affected by hydrodynamic processes, a high resolution version of the Hamburg Shelf Ocean Model (HAMSOM) was developed and utilised. Simulations were made for the period 1996–2005. Both models were validated by comparing results with available data, which showed that the simulations were of very satisfactory quality.

Model results show that the North Sea is a net sink for ?-HCH and a net source to the atmosphere of PCB 153. Total masses of ?-HCH and PCB 153 in 2005 are reduced to 30% and 50%, respectively, of 1996 values.

Storms resuspending bottom sediments into the water column mobilise POPs into the atmosphere and have the potential to deliver substantial loads of these POPs into Europe.

Expression of the SEPT9_i4 isoform confers resistance to microtubule-interacting drugs

BACKGROUND: The evolutionarily conserved septin family of genes encode GTP binding proteins involved in a variety of cellular functions including cytokinesis, apoptosis, membrane dynamics and vesicle trafficking. Septin proteins can form hetero-oligomeric complexes and interact with other proteins including actin and tubulin. The human SEPT9 gene on chromosome 17q25.3 has a complex genomic architecture with 18 different transcripts that can encode 15 distinct polypeptides. Two distinct transcripts with unique 5' ends (SEPT9_v4 and SEPT9_v4*) encode the same protein. In tumours the ratio of these transcripts changes with elevated levels of SEPT9_v4* mRNA, a transcript that is translated with enhanced efficiency leading to increased SEPT9_i4 protein.

METHODS: We have examined the effect of over-expression of SEPT9_i4 on the dynamics of microtubule polymer mass in cultured cells.

RESULTS: We show that the microtubule network in SEPT9_i4 over-expressing cells resists disruption by paclitaxel or cold incubation but also repolymerises tubulin more slowly after microtubule depolymerisation. Finally we show that SEPT9_i4 over-expressing cells have enhanced survival in the presence of clinically relevant microtubule acting drugs but not after treatment with DNAinteracting agents.

CONCLUSIONS: Given that SEPT9 over-expression is seen in diverse tumours and in particular ovarian and breast cancer, such data indicate that SEPT9_v4 expression may be clinically relevant and contribute to some forms of drug resistance.

A comparative analysis of the health and wellbeing of cancer survivors to the general population

Background
The population of people surviving cancer is continually increasing and currently cancer survivors represent approximately 3.7% of the American population and 3% of the UK population. There is limited and inconclusive empirical evidence regarding the long-term health and well-being of cancer survivors.

Methods
Two hundred eighty-nine cancer survivors and 262 matched-age and sex patients from the same group of General (primary care) Practices completed postal questionnaires measuring health and well-being, health service utilisation and satisfaction and health care needs.

Main Results
Cancer survivors reported poorer health and well-being and health service utilisation than the general population. Despite this poorer health, the majority of cancer survivors reported satisfaction with services and almost two-thirds of the survivors did not report any needs.

Conclusions
The majority of cancer survivors do not appear to require additional support services. There is, however, a subgroup of survivors who warrant specialist support, particularly survivors who are older, experience late effects and have had adjuvant treatments. Future research should focus on developing methods that could be used in routine clinical practice to identify ‘at risk’ or vulnerable patients and to provide appropriate and timely support.

Development of an objective structured clinical examination (OSCE) to assess formulation and extemporaneous dispensing skills in MPharm undergraduates

Background: The use of Objective Structured Clinical Examination (OSCE) in Pharmacy has been explored; however this is the first attempt in Queen’s University School of Pharmacy, Belfast to assess students via this method in a module where chemistry is the main discipline.

Aims: To devise an OSCE to assess undergraduate ability to check extemporaneously dispensed products for clinical and formulation errors. This activity also aims to consider whether it is a viable method of assessment in such a science-based class, from a staff and student perspective.

Method: Students rotated around a number of stations, performing a check of the product, corresponding prescription and formulation record sheet detailing the theory behind the formulation. They were assessed on their ability to spot intentional mistakes at each one.

Results: Of the 79 students questioned, 95% indicated that OSCE made them aware of the importance of the clinical check carried out by the pharmacist. Nearly all of the undergraduates (72 out of 79) felt that OSCE made them aware of the type of mistakes that students make in class. Most (5 out of 7) of the academic team members strongly agreed that it made students aware of ‘point of dispensing’ checks carried out by pharmacists, in addition to helping them to prepare for their exam.

Conclusion: OSCE assesses both scientific and formulation skills, and has increased the diversity of assessment of this module, bringing with it many additional benefits for the undergraduates since it measures their ability to exercise professional judgement in a time- constrained environment and, in this way, mirrors the conditions many pharmacists work within.

Multi-epoch high-resolution spectroscopy of SN2011fe - Linking the progenitor to its environment

This study attempts to establish a link between the reasonably well known nature of the progenitor of SN2011fe and its surrounding environment. This is done with the aim of enabling the identification of similar systems in the vast majority of the cases, when distance and epoch of discovery do not allow a direct approach. To study the circumstellar environment of SN2011fe we have obtained high-resolution spectroscopy of SN2011fe on 12 epochs, from 8 to 86 days after the estimated date of explosion, targeting in particular at the time evolution of CaII and NaI. Three main absorption systems are identified from CaII and NaI, one associated to the Milky Way, one probably arising within a high-velocity cloud, and one most likely associated to the halo of M101. The Galactic and host galaxy reddening, deduced from the integrated equivalent widths (EW) of the NaI lines are E(B-V)=0.011+/-0.002 and E(B-V)=0.014+/-0.002 mag, respectively. The host galaxy absorption is dominated by a component detected at the same velocity measured from the 21-cm HI line at the projected SN position (~180 km/s). During the ~3 months covered by our observations, its EW changed by 15.6+/-6.5 mA. This small variation is shown to be compatible with the geometric effects produced by therapid SN photosphere expansion coupled to the patchy fractal structure of the ISM. The observed behavior is fully consistent with ISM properties similar to those derived for our own Galaxy, with evidences for structures on scales

Laboratory tests to study the influence of rock stress confinement on the performances of TBM discs in tunnels

To clarify some aspects of rock destruction with a disc acting on a high confined tunnel face, a series of tests were carried out to examine fracture mechanisms under an indenter that simulates the tunnel boring machine (TBM) tool action, in the presence of an adjacent groove, when a state of stress (lateral confinement) is imposed on a rock sample. These tests proved the importance of carefully establishing the optimal distance of grooves produced by discs acting on a confined surface, and the value (as a mere order of magnitude) of the increase of the thrust to produce the initiation of chip formation, as long as the confinement pressure becomes greater. © University of Science and Technology Beijing and Springer-Verlag Berlin Heidelberg 2011.