Citrulline-modified phage display: a novel high-throughput discovery approach for the identification of citrulline-containing ligands

Phage display is a high-throughput technology used to identify ligands for a given target. A drawback of the approach is the absence of PTMs in phage-displayed peptides. The applicability of phage display could be broadened considerably by the implementation of PTMs in this system. The aim of this study was to investigate the possible application of citrullination, a PTM of an arginine into a citrulline amino acid, in filamentous (M13) and lytic (T7) phage display. After in vitro citrullination of T7 and M13 phages, citrullination was confirmed and the infectivity of both citrullinated and non-citrullinated phage was compared by titer determination. We demonstrated the successful in vitro citrullination of T7 and M13 phage-displayed peptides. This in vitro modification did not affect the viability or infectivity of the T7 virions, a necessary prerequisite for the implementation of this approach in T7 phage display. For M13 phage, however, the infecting phage titer decreased five-fold upon citrullination, limiting the use of this modification in M13 phage display. In conclusion, in vitro citrullination can be applied in T7 phage display giving rise to a high-throughput and sensitive approach to identify citrulline-containing ligands by the use of the strengths of phage display technology.     

Recycling polarity

Three current papers in Cell and in this issue of Developmental Cell highlight the role of the exocyst in recycling of membrane proteins from endosomes to the plasma membrane in asymmetric cell division and polarized epithelial cells.     

Non-destructive sampling of maternal DNA from the external shell of bird eggs

The use of non-destructive sampling methods to collect genetic material from wildlife allows researchers to minimize disturbance. Most avian studies employ capturing and handling of young and parents to draw blood for DNA analysis. In some cases adult female birds are difficult to catch, so maternal genotyping has required collection of contour feathers from nests, or destructive sampling of eggs. Many species do not leave contour feathers in the nest, and destructive sampling has been unreliable due to contamination with embryonic DNA. Alternative field sampling techniques for collection of maternal DNA from birds are therefore desirable. Here we demonstrate that avian maternal DNA can be isolated in a non-invasive and non-destructive way from the external surface of eggs. We used cotton swabs to collect maternal DNA from the external shells of herring gull (Larus argentatus) and Caspian tern (Sterna caspia) eggs. DNA was then amplified by the polymerase chain reaction (PCR) for microsatellite genotyping. We verified that the DNA samples were maternal by comparing microsatellite profiles to those obtained from adults and chicks from the same nests. In 100% of Caspian tern (n=16) and herring gull families (n=12), the egg swabs that amplified matched the maternal microsatellite genotype. In a screening of many nests of both species, we successfully amplified microsatellite markers from 101/115 (88%) egg swabs. Swabs from eggs with blood stains on the shell were more likely to amplify successfully than those from clean eggs. The advantages of this new method include increased parentage assignment/exclusion power, and increased availability of maternal DNA for genotyping of species that do not deposit contour feathers in nests.     

Decrease in density of INa is in the common final pathway to heart block in murine hearts overexpressing calcineurin

Overexpression of calcineurin in transgenic mouse heart results in massive cardiac hypertrophy followed by sudden death. Sudden deaths are caused by abrupt transitions from sinus rhythm to heart block (asystole) in calcineurin-overexpressing (CN) mice. Preliminary studies showed decreased maximum change in potential over time (dV/dt(max)) of phase 0 of the action potential. Accordingly, the hypothesis was tested that decreased activity of the sodium channel contributes to heart block. Profound decreases in activity of sodium currents (I(Na)) paralleled the changes in action potential characteristics. Progressive age-dependent decreases were observed such that at 42-50 days of life little sodium channel function existed. However, this was not paralleled by decreased protein expression as assessed by immunocytochemistry or by Western blot. Since calcineurin can interact with the ryanodine receptor, we assessed whether chronic in vitro treatment with BAPTA-AM, thapsigargin, and ryanodine could rescue the decrease of I(Na). All of these treatments rescued I(Na) to levels indistinguishable from wild type. The nonspecific PKC inhibitor bisindolylmaleimide I also rescued the decrease of I(Na). To assess whether decreased sodium channel activity contributes to sudden death in vivo, the response to encainide (20 mg/kg) was assessed: 6 of 10 young CN mice died because of asystole, whereas 0 of 10 wild-type mice died (P < 0.01). Moreover, encainide produced exaggerated prolongation of the QRS width in sinus beats before the heart block. Catecholamine tone appears necessary to support life in older CN mice because propranolol (1 mg/kg) triggered asystolic death in five of six CN mice. We conclude that decrease in sodium channel activity is in the common final pathway to asystole in CN mice.     

Neuropsychological and behavioural correlates of CSF biomarkers in dementia

To improve clinical, neuropsychological and behavioural characterisation of the cerebrospinal fluid (CSF) biomarkers beta-amyloid((1-42)) protein (Abeta42), protein tau (tau) and tau phosphorylated at threonine 181 (P-tau181) across diagnostic dementia categories, a prospective study was set up. Patients with probable Alzheimer's disease (AD) (n=201), AD with cerebrovascular disease (CVD) (AD+CVD) (n=33), frontotemporal dementia (FTD) (n=27), dementia with Lewy bodies (DLB) (n=22) and healthy controls (n=148) were included. All patients underwent neuropsychological examination and behavioural assessment by means of a battery of behavioural assessment scales. CSF was obtained by lumbar puncture and levels of Abeta42, tau and P-tau181 were determined with commercially available ELISA kits. Negative correlations between CSF Abeta42 levels and aggressiveness (Spearman: r=-0.223; p=0.002) and positive correlations with age at inclusion (r=0.195; p=0.006), age at onset (r=0.205; p=0.003) and MMSE scores (r=0.198; p=0.005) were found in AD. In AD+CVD, CSF Abeta42 levels were correlated with MMSE (r=0.482; p=0.006), Hierarchic Dementia Scale (r=0.503; p=0.017) and Boston Naming Test (r=0.516; p=0.012) scores. In controls, age was positively correlated with CSF tau (r=0.465; p<0.001) and P-tau181 levels (r=0.312; p<0.001). CSF tau and P-tau181 levels correlated significantly in all groups, whereas CSF Abeta42 correlated with tau and P-tau181 levels in healthy controls only. Negative correlations between CSF Abeta42 levels and aggressiveness were found in AD patients. CSF Abeta42 seems to be a stage marker for AD (+/-CVD) given the positive correlations with neuropsychological test results suggesting that CSF Abeta42 might be of help for monitoring disease progression. Different correlations between age and CSF biomarker levels were obtained in healthy controls compared to AD patients, indicating that AD-induced pathophysiological processes change age-dependent regulation of CSF biomarker levels.