Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

The MK5/PRAK kinase and Myc form a negative feedback loop that is disrupted during colorectal tumorigenesis

Expression of the Myc oncoprotein is downregulated in response to stress signals to allow cells to cease proliferation and escape apoptosis, but the mechanisms involved in this process are poorly understood. Cell cycle arrest in response to DNA damage requires downregulation of Myc via a p53-independent signaling pathway. Here we have used siRNA?screening of the human kinome to identify MAPKAPK5 (MK5, PRAK) as a negative regulator of Myc expression. MK5 regulates translation of Myc, since it is required for expression of miR-34b and miR-34c that bind to the 3'UTR of MYC. MK5 activates miR-34b/c expression via phosphorylation of FoxO3a, thereby promoting nuclear localization of FoxO3a and enabling it to induce miR-34b/c expression and arrest proliferation. Expression of MK5 in turn is directly activated by Myc, forming a negative feedback loop. MK5 is downregulated in colon carcinomas, arguing that this feedback loop is disrupted during colorectal tumorigenesis.     

DNA-based faecal dietary analysis: a comparison of qPCR and high throughput sequencing approaches

The genetic analysis of faecal material represents a relatively non-invasive way to study animal diet and has been widely adopted in ecological research. Due to the heterogeneous nature of faecal material the primary obstacle, common to all genetic approaches, is a means to dissect the constituent DNA sequences. Traditionally, bacterial cloning of PCR amplified products was employed; less common has been the use of species-specific quantitative PCR (qPCR) assays. Currently, with the advent of High-Throughput Sequencing (HTS) technologies and indexed primers it has become possible to conduct genetic audits of faecal material to a much greater depth than previously possible. To date, no studies have systematically compared the estimates obtained by HTS with that of qPCR. What are the relative strengths and weaknesses of each technique and how quantitative are deep-sequencing approaches that employ universal primers? Using the locally threatened Little Penguin (Eudyptula minor) as a model organism, it is shown here that both qPCR and HTS techniques are highly correlated and produce strikingly similar quantitative estimates of fish DNA in faecal material, with no statistical difference. By designing four species-specific fish qPCR assays and comparing the data to the same four fish in the HTS data it was possible to directly compare the strengths and weaknesses of both techniques. To obtain reproducible quantitative data one of the key, and often overlooked, steps common to both approaches is ensuring that efficient DNA isolation methods are employed and that extracts are free of inhibitors. Taken together, the methodology chosen for long-term faecal monitoring programs is largely dependent on the complexity of the prey species present and the level of accuracy that is desired. Importantly, these methods should not be thought of as mutually exclusive, as the use of both HTS and qPCR in tandem will generate datasets with the highest fidelity.     

Label-free electrochemical DNA sensor based on functionalised conducting copolymer

A simple and label-free electrochemical sensor for recognition of the DNA hybridization event was prepared based on a new functionalised conducting copolymer, poly[pyrrole-co-4-(3-pyrrolyl) butanoic acid]. This precursor copolymer can be easily electrodeposited on the electrode surface and shows high electroactivity in an aqueous medium. An amino-substituted oligonucleotide (ODN) probe was covalently grafted onto the surface of the copolymer in a one step procedure and tested on hybridization with complementary ODN segments. The cyclic voltammogram of ODN probe-modified copolymer showed very little change when incubated in presence of non-complementary ODN, while a significant, and reproducible, modification of the voltammogram was observed after addition of complementary ODN. The AC impedance spectrum showed an increased charge transfer resistance (Rct) and double layer capacitance of the sensor film after hybridisation. Sensors with thinner films showed higher sensitivity than thicker films, suggesting that hybridisation at or near the surface of the film produces a larger change in electrical properties than that within the body of the film.     

Increasing sensitivity of Ca2+ spark detection in noisy images by application of a matched-filter object detection algorithm

Microscopic calcium (Ca²⁺) events (such as Ca²⁺ sparks) are an important area for study, as they help clarify the mechanism(s) underlying intracellular signaling. In the heart, Ca²⁺ sparks occur as a result of Ca²⁺ release from the sarcoendoplasmic reticulum, via ryanodine receptor channels. Measurement of Ca²⁺ spark properties can provide valuable information about the control of ryanodine receptor channel gating in situ, but requires high spatiotemporal resolution imaging, which produces noisy datasets that are problematic for spark detection. Automated detection algorithms may overcome visual detection bias, but missed and false-positive events can distort the distribution of measured Ca²⁺ spark properties. We present a sensitive and reliable method for the automated detection of Ca²⁺ sparks in datasets obtained using confocal line-scanning or total internal reflection fluorescence microscopy. This matched-filter detection algorithm (MFDA) employs a user-defined object, chosen to mimic Ca²⁺ spark properties, and the experimental dataset is searched for instances of the object. Detection certainty is provided by nonparametric statistical testing. The supplied codes can also refine the search object on the basis of those detected to further increase detection sensitivity. In comparison to a commonly used, intensity-thresholding algorithm, the MFDA is more sensitive and reliable, particularly at low signal/noise ratios. The MFDA can also be easily adapted to other signal-detection problems in noisy datasets.     

Fertile transgenic plants obtained from tritordeum inflorescences by tissue electroporation

 Tissue electroporation was applied to a member of the Triticeae family, namely tritordeum (Hordeum chilense Roem.×Triticum turgidum L. Conv. durum), for the generation of fertile transgenic plants. Two transgenic plants were recovered following the treatment of 361 explants of immature inflorescences (although they were subsequently found to result from the same transformation event). The expression of both inserted marker genes (uidA and bar) was confirmed using standard assays, while transgene integration was confirmed using PCR and Southern hybridization analyses. Integration pattern, segregation ratio and the inheritance of transgene expression in T₁ progeny were consistent for the presence of a single transgene locus containing five to ten plasmid insertions. Although this procedure has been applied to other cereal species, stable transformation of the Triticeae using tissue electroporation has not previously been reported. Received: 28 October 1999 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

Transformation of barley scutellum protoplasts: regeneration of fertile transgenic plants

 A system for barley transformation via polyethyleneglycol-mediated DNA uptake into protoplasts isolated directly from scutella and the regeneration of transgenic plants is reported. Scutellum protoplasts (cv. Clipper, an Australian malting cultivar) were co-transformed with plasmids Act 1-DGUS, containing the marker uidA gene, and pCaIneo, which contains the selectable marker neomycin phosphotransferase gene. Protoplast-derived calluses were selected on medium containing the antibiotic G418 (25 and 15 mg.l^–1) and macroscopic antibiotic resistant colonies were recovered. Fertile plants were regenerated from a callus line and molecular analysis confirmed transgene integration. Received: 11 October 1999 / Revision received: 11 February 2000 / Accepted: 11 February 2000     

Floristic variation and willow carr development within a southwest England wetland

Abstract. Woodland colonization on wetlands is considered to have a detrimental effect on their ecological value, even though detailed analysis of this process is lacking. This paper provides an evaluation of the ecological changes resulting from succession of poor fen (base‐poor mire) to willow wet woodland on Goss Moor NNR in Cornwall, UK. Different ages of willow carr were associated with eight understorey communities. During willow colonization, in the ground flora, there was a progressive decrease in poor fen species and an associated increase in woodland species, which appeared to be related to an increase in canopy cover and therefore shade. The most diverse community was found to be the most recent willow and was dominated by poor fen species. The oldest willow was the second most diverse and was associated with a reduction in poor fen species and an increase in woodland species. Architectural features were used successfully to assess the general condition and structure of willow. Tree height and DBH were identified as useful parameters to accurately assess willow age in the field. The implications of active intervention to remove willow in order to conserve the full range of communities within the hydrosere are discussed.     

Cloning of a pea cDNA encoding a polypeptide of the light-harvesting complex associated with photosystem I using a monoclonal antibody

A monoclonal antibody (MAb UB42) is described that binds to thylakoids in pea chloroplasts, as shown by EM-immunogold labelling. The antibody recognised proteins of ca. 23–29 kDa in western blots of a pea leaf homogenate. A cDNA library was prepared from pea epidermal cells in the vector ZAP II, and immunoscreening of the library with UB42 led to the isolation of a clone, pUB42. This was sequenced and had an open reading frame of 269 codons encoding a predicted polypeptide of 28.9 kDa. The sequence showed extensive homology with three closely related polypeptides belonging to a family of chlorophyll a/b-binding proteins from the light harvesting complex of photosytem I (LHCI). Collectively, the results suggest that MAb UB42 recognises an epitope on the type II chlorophyll a/b-binding protein from LHCI and that clone pUB42 encodes this protein.