GOGOT: a method for the identification of differentially expressed fragments from cDNA-AFLP data

<p>Abstract</p> <p>Background</p> <p>One-dimensional (1-D) electrophoretic data obtained using the cDNA-AFLP method have attracted great interest for the identification of differentially expressed transcript-derived fragments (TDFs). However, high-throughput analysis of the cDNA-AFLP data is currently limited by the need for labor-intensive visual evaluation of multiple electropherograms. We would like to have high-throughput ways of identifying such TDFs.</p> <p>Results</p> <p>We describe a method, GOGOT, which automatically detects the differentially expressed TDFs in a set of time-course electropherograms. Analysis by GOGOT is conducted as follows: correction of fragment lengths of TDFs, alignment of identical TDFs across different electropherograms, normalization of peak heights, and identification of differentially expressed TDFs using a special statistic. The output of the analysis is a highly reduced list of differentially expressed TDFs. Visual evaluation confirmed that the peak alignment was performed perfectly for the TDFs by virtue of the correction of peak fragment lengths before alignment in step 1. The validity of the automated ranking of TDFs by the special statistic was confirmed by the visual evaluation of a third party.</p> <p>Conclusion</p> <p>GOGOT is useful for the automated detection of differentially expressed TDFs from cDNA-AFLP temporal electrophoretic data. The current algorithm may be applied to other electrophoretic data and temporal microarray data.</p

A normalization strategy applied to HiCEP (an AFLP-based expression profiling) analysis: Toward the strict alignment of valid fragments across electrophoretic patterns

BACKGROUND: Gene expression analysis based on comparison of electrophoretic patterns is strongly dependent on the accuracy of DNA fragment sizing. The current normalization strategy based on molecular weight markers has limited accuracy because marker peaks are often masked by intense peaks nearby. Cumulative errors in fragment lengths cause problems in the alignment of same-length fragments across different electropherograms, especially for small fragments (< 100 bp). For accurate comparison of electrophoretic patterns, further inspection and normalization of electrophoretic data after fragment sizing by conventional strategies is needed. RESULTS: Here we describe a method for the normalization of a set of time-course electrophoretic data to be compared. The method uses Gaussian curves fitted to the complex peak mixtures in each electropherogram. It searches for target ranges for which patterns are dissimilar to the other patterns (called "dissimilar ranges") and for references (a kind of mean or typical pattern) in the set of resultant approximate patterns. It then constructs the optimal normalized pattern whose correlation coefficient against the reference in the range achieves the highest value among various combinations of candidates. We applied the procedure to time-course electrophoretic data produced by HiCEP, an AFLP-based expression profiling method which can detect a slight expression change in DNA fragments. We obtained dissimilar ranges whose electrophoretic patterns were obviously different from the reference and as expected, most of the fragments in the detected ranges were short (< 100 bp). The normalized electrophoretic patterns also agreed well with reference patterns. CONCLUSION: The normalization strategy presented here demonstrates the importance of pre-processing before electrophoretic signal comparison, and we anticipate its usefulness especially for temporal expression analysis by the electrophoretic method

Recombination Activating Gene (RAG)-1 and 2 Encoding Proteins Expressed by the Baculovirus System

We have been attempting to obtain mouse recombination activating gene-1 (RAG-1) and RAG-2 protein for biochemical analyses. First of all, we obtained truncated products of these genes expressed and purified using the E. coli expression system and then established the polyclonal and monoclonal antibodies by means of E. coli expressed peptides as antigens. Subsequently, whole RAG-1 and RAG-2 gene products were expressed the baculovirus expression system. Since it has been difficult to achieve the significant gene expression of full-lenght cDNA, we employed the glutathione S-transferase (GST)-fused gene-expression system which facilitated the massive expression of gene products. This system was also advantageous in that we could detect the expressed protein molecules not only with anti-RAG antibody but also with anti-GST antibody

When good signatures go bad: Applying hydrologic signatures in large sample studies

Hydrologic signatures are quantitative metrics that describe streamflow statistics and dynamics. Signatures have many applications, including assessing habitat suitability and hydrologic alteration, calibrating and evaluating hydrologic models, defining similarity between watersheds and investigating watershed processes. Increasingly, signatures are being used in large sample studies to guide flow management and modelling at continental scales. Using signatures in studies involving 1000s of watersheds brings new challenges as it becomes impractical to examine signature parameters and behaviour in each watershed. For example, we might wish to check that signatures describing flood event characteristics have correctly identified event periods, that signature values have not been biassed by data errors, or that human and natural influences on signature values have been correctly interpreted. In this commentary, we draw from our collective experience to present case studies where naïve application of signatures fails to correctly identify streamflow dynamics. These include unusual precipitation or flow regimes, data quality issues, and signature use in human‐influenced watersheds. We conclude by providing guidance and recommendations on applying signatures in large sample studies

Characteristics of soil and hillslope responses in humid tropical forests in Sumatra, Indonesia

Extensive deforestation in tropical regions may significantly influence the hydrological cycle. However, subsurface runoff processes in thick soil layers in humid tropical forests are poorly understood; thus, the impact of land-use changes in such regions remains unclear. To understand runoff generation mechanisms in the humid tropics, we monitored groundwater and soil moisture dynamics in a forested hillslope in Sumatra, Indonesia. We also conducted field and laboratory experiments to determine soil hydraulic characteristics and used the results to simulate vertical infiltration and groundwater recharge. Although the soil is categorized as silty clay loam, the high infiltrability and high water retention capacity of the soil enabled infiltration during storm events and recharge to groundwater. Within the 4–5 m thick soil layer at the foot of the hillslope, the shallow groundwater table quickly responded to rainfall and did not drop below a depth of 2–3 m, possibly due to continuous flow contributions from the upslope. Overall, this study demonstrates the importance of subsurface flow and vertical infiltration in thick soil layers in humid tropical regions

A successfully treated Brugada syndrome presenting in ventricular fibrillation preceded by fever and concomitant hypercalcemia

Brugada syndrome (BS) is a genetic channelopathy syndrome that causes fatal cardiac dysrhythmias and sudden death. Fever and antiarrhythmics are aggravating factors of BS. There are many reports about BS preceded by fever but fewer reports on BS caused by hypercalcemia (HC). Here, we describe a unique case of BS preceded by concurrent fever and HC. A 46-year-old male visited the emergency department for malaise and fever. During admission, he suddenly developed cardiac arrest and ventricular fibrillation (VF). After resuscitation, electrocardiogram (ECG) showed “coved-type” ST elevation in V1 and V2, which led to the diagnosis of BS. This ST change declined after the fever subsided. He also had HC at the same time. After admission, he developed septic shock. We started treatment assuming that it was caused by the aggravation of ulcerative colitis, and liver abscess was revealed on contrast-enhanced computed tomography. After the infection was controlled, we implanted an implantable cardioverter defibrillator (ICD) and he was discharged. The cause of HC appeared to be an ectopic parathyroid adenoma, and calcium was normalized after tumor resection. In addition, this patient had nonfunctional pituitary adenoma and a nonfunctional adrenal tumor. His condition was indicative of multiple endocrine neoplasia type 1. This patient had BS presenting as VF induced by fever due to liver abscess and early repolarization, increasing the risk of arrhythmic events to carry out ICD implantation. HC can contribute to induce arrhythmia