In vitro biosynthesis, core glycosylation and membrane integration of opsin

A membrane-integrated , core-glycosylated form of bovine opsin was synthesized in vitro when bovine retina mRNA was translated in a wheat germ cell-free system supplemented with dog pancreas microsomal vesicles; glycosylation and integration of opsin into membranes were coupled to translation. Proteolysis with themolysin was used to probe the orientation of opsin within the dog pancreas microsomal membrane, and to compare it with that of opsin in rod cell disk membranes isolated from bovine retina. Intact microsomal or disk vesicles were required for production of discrete, membrane-associated thermolysin fragments of opsin; no discrete opsin fragments were detected when membranes were incubated with thermolysin in the presence of the nonionic detergent, Triton X-100. The major opsin fragments produced by themosylin treatment of intact microsomal vesicles resembled those from disk vesicles in their size, oligosaccharide content, and order of appearance. In each case, the first cleavage of opsin took place at the COOH-terminus, generating a glycosylated fragment, O’, which was only slightly smaller than intact opsin. Both the microsomal and disk membrane forms of O’ were next cleaved internally; glycosylated fragments of similar sizes in both cases were detected which were derived from the NH(2)-terminal portion of O’. Several smaller NH(2)-terminal fragments of opsin were detected only in thermolysin-treated microsomal membranes, and not in disk membranes. The data suggest that the topology of opsin integrated into dog pancreas microsomal vesicles is similar to that in rod cell disk vesicles, although not identical. In each case, the glycosylated NH(2)-terminal region of opsin is located within the lumen of the vesicle, while discrete COOH-terminal and internal segments of opsin apparently emerge at the outer, cytoplasmic face of the membrane. Thus, opsin in the heterologous microsomal membrane, like its counterpart in the native disk membrane, may cross the bilayer at least three times. The internal domain of the polypeptide that emerges at the outer membrane surface is apparently more highly exposed in the case of opsin in microsomal membranes, evidenced by the additional internal thermolysin cleavage sites detected.     

Effect of fruit maturity,seed weight and storage time on the viability and germination of the seed of candelilla (<i>Euphorbia antisiphylitica</i> Zucc.)

Candelilla (Euphorbia antisiphylitica Zucc.) is a very important plant resource in the arid lands of Northern Mexico. This is because the wax content coating the stem has unique properties which have been useful for multiple applications in the food industry, electronics, cosmetics, etc. However, the intensive exploitation of this resource has caused a great decrease in the populations of this species making necessary to consider strategies for their conservation and sustainable use. One of the primary needs with regeneration purposes is to know their reproductive processes, particularly the biotic and/or abiotic factors that determine the viability and germination of seeds. The present study evaluated the (1) germination and seed viability in relation to the ripeness degree of the fruit at the time of collection, (2) weight of the seed (low, medium and high), and (3) storage time (1, 3, and 5 months). Fruits from four locations, two in the State of Coahuila (Las Coloradas and Candela) and two in the State of Nuevo Leon (Icamole 1 and Icamole 2), were collected. Three germination assays were carried out corresponding to each month of storage. Seed viability was determined by the tetrazolium test. The average weight of the candelilla seeds was 0.0029 ± 0.0010 g, with extreme average values of 0.0018 ± 0.0006 g at Las Coloradas and 0.0036 ± 0.0010 g in Icamole 2. Those seeds with heavier weight obtained from red fruits and with 1 month of storage showed the highest average percentage of viability (66.87 ± 24.19%). At the same time, seeds with around average weight, obtained from red fruits and five months of storage, showed the highest average germination percentage (50.00 ± 9.42%).     

Attentional modulation of central odor processing

Two studies were conducted to investigate the influence of attention on the components of the chemosensory event-related potential (CSERP). In the first study the odors linalool and eugenol were delivered to six male subjects, in the second study three male and two female subjects were presented with their own body odor (axillary hair) and the body odor of a same sex donor. In both studies the odors were presented in an oddball paradigm under ignore and attend conditions via a constant- flow olfactometer. In the ignore condition attention was diverted from the odors with a distractor task, while in the attend condition the subjects were asked to respond to the infrequently occurring odor. In both studies the allocation of attention led to a decrease in the latency of the early components (N1, P2, N2) and to an increase in the amplitude of the late positivities. The modulation of the early components suggests that attentional gating in olfaction might already be effective at an early processing level.      

Efficacy assessed in follow-ups of clinical trials: methodological conundrum

Increasingly, we see papers describing the long-term follow-up results of randomised clinical trials. Sometimes, like the article by Rantalaiho and colleagues in the previous issue of Arthritis Research & Therapy, the follow-up extends to more than 10 years. It is not uncommon that authors of such articles describe their results as a comparison of the original treatment groups in the original randomised clinical trial. Methodologically, such a comparison is fallible for several reasons. In this editorial, two important sources of bias that may jeopardise the results of such follow-up studies are discussed: confounding by indication and confounding by trial completion.     

Comparison of computational methods for identifying translation initiation sites in EST data

Background

Expressed Sequence Tag (EST) sequences are generally single-strand, single-pass sequences, only 200–600 nucleotides long, contain errors resulting in frame shifts, and represent different parts of their parent cDNA. If the cDNAs contain translation initiation sites, they may be suitable for functional genomics studies. We have compared five methods to predict translation initiation sites in EST data: first-ATG, ESTScan, Diogenes, Netstart, and ATGpr.

Results

A dataset of 100 EST sequences, 50 with and 50 without, translation initiation sites, was created. Based on analysis of this dataset, ATGpr is found to be the most accurate for predicting the presence versus absence of translation initiation sites. With a maximum accuracy of 76%, ATGpr more accurately predicts the position or absence of translation initiation sites than NetStart (57%) or Diogenes (50%). ATGpr similarly excels when start sites are known to be present (90%), whereas NetStart achieves only 60% overall accuracy. As a baseline for comparison, choosing the first ATG correctly identifies the translation initiation site in 74% of the sequences. ESTScan and Diogenes, consistent with their intended use, are able to identify open reading frames, but are unable to determine the precise position of translation initiation sites.

Conclusions

ATGpr demonstrates high sensitivity, specificity, and overall accuracy in identifying start sites while also rejecting incomplete sequences. A database of EST sequences suitable for validating programs for translation initiation site prediction is now available. These tools and materials may open an avenue for future improvements in start site prediction and EST analysis.

     

Identifying and quantifying metabolites by scoring peaks of GC-MS data

Background

Metabolomics is one of most recent omics technologies. It has been applied on fields such as food science, nutrition, drug discovery and systems biology. For this, gas chromatography-mass spectrometry (GC-MS) has been largely applied and many computational tools have been developed to support the analysis of metabolomics data. Among them, AMDIS is perhaps the most used tool for identifying and quantifying metabolites. However, AMDIS generates a high number of false-positives and does not have an interface amenable for high-throughput data analysis. Although additional computational tools have been developed for processing AMDIS results and to perform normalisations and statistical analysis of metabolomics data, there is not yet a single free software or package able to reliably identify and quantify metabolites analysed by GC-MS.

Results

Here we introduce a new algorithm, PScore, able to score peaks according to their likelihood of representing metabolites defined in a mass spectral library. We implemented PScore in a R package called MetaBox and evaluated the applicability and potential of MetaBox by comparing its performance against AMDIS results when analysing volatile organic compounds (VOC) from standard mixtures of metabolites and from female and male mice faecal samples. MetaBox reported lower percentages of false positives and false negatives, and was able to report a higher number of potential biomarkers associated to the metabolism of female and male mice.

Conclusions

Identification and quantification of metabolites is among the most critical and time-consuming steps in GC-MS metabolome analysis. Here we present an algorithm implemented in a R package, which allows users to construct flexible pipelines and analyse metabolomics data in a high-throughput manner.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0374-2) contains supplementary material, which is available to authorized users.     

Experience in the application of Java Technologies in telemedicine

Java language has been demonstrated to be an effective tool in supporting medical image viewing in Russia. This evaluation was completed by obtaining a maximum of 20 images, depending on the client's computer workstation from one patient using a commercially available computer tomography (CT) scanner. The images were compared against standard CT images that were viewed at the site of capture. There was no appreciable difference. The client side is a lightweight component that provides an intuitive interface for end users. Each image is loaded in its own thread and the user can begin work after the first image has been loaded. This feature is especially useful on slow connection speed, 9.6 Kbps for example. The server side, which is implemented by the Java Servlet Engine works more effective than common gateway interface (CGI) programs do. Advantages of the Java Technology place this program on the next level of application development. This paper presents a unique application of Java in telemedicine.     

Application of stains in clinical microbiology

Stains have been used for diagnosing infectious diseases since the late 1800s. The Gram stain remains the most commonly used stain because it detects and differentiates a wide range of pathogens. The next most commonly used diagnostic technique is acid-fast staining that is used primarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agents grow slowly on culture media or may not grow at all; stains may be the only method to detect these organisms in clinical specimens. In the hands of experienced clinical microscopists, stains provide rapid and cost-effective information for preliminary diagnosis of infectious diseases. A review of the most common staining methods used in the clinical microbiology laboratory is presented here.