Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: A quantitative approach

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na+-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the α, β, and γ ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of α, β, and γ ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (β R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts.

The junction-associated protein zonula occludens-1 (ZO-1) is a member of a family of membrane-associated guanylate kinase homologues thought to be important in signal transduction at sites of cell-cell contact. We present evidence that under certain conditions of cell growth, ZO-1 can be detected in the nucleus. Two different antibodies against distinct portions of the ZO-1 polypeptide reveal nuclear staining in subconfluent, but not confluent, cell cultures. An exogenously expressed, epitope-tagged ZO-1 can also be detected in the nuclei of transfected cells. Nuclear accumulation can be stimulated at sites of wounding in cultured epithelial cells, and immunoperoxidase detection of ZO-1 in tissue sections of intestinal epithelial cells reveals nuclear labeling only along the outer tip of the villus. These results suggest that the nuclear localization of ZO-1 is inversely related to the extent and/or maturity of cell contact. Since cell-cell contacts are specialized sites for signaling pathways implicated in growth and differentiation, we suggest that the nuclear accumulation of ZO-1 may be relevant for its suggested role in membrane-associated guanylate kinase homologue signal transduction.

cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein.

A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function--Mei-41, Mec1p, and Rad3--and as such may be the functional human counterpart of these proteins.

Responsivity and resonant properties of dipole, bowtie, and spiral Seebeck nanoantennas

Seebeck nanoantennas, which are based on the thermoelectric effect, have been proposed for electromagnetic energy harvesting and infrared detection. The responsivity and frequency dependence of three types of Seebeck nanoantennas is obtained by electromagnetic simulation for different materials. Results show that the square spiral antenna has the widest bandwidth and the highest induced current of the three analyzed geometries. However, the geometry that presented the highest temperature gradient was the bowtie antenna, which favors the thermoelectric effect in a Seebeck nanoantenna. The results also show that these types of devices can present a voltage responsivity as high as 36  μV/W36  μV/W for titanium–nickel dipoles resonant at far-infrared wavelengths.

Human Motion Analysis Based on Sequential Modeling of Radar Signal and Stereo Image Features

Falls are one of the greatest threats to elderly health in their daily living routines and activities. Therefore, it is very important to detect falls of an elderly in a timely and accurate manner, so that immediate response and proper care can be provided, by sending fall alarms to caregivers. Radar is an effective non-intrusive sensing modality which is well suited for this purpose, which can detect human motions in all types of environments, penetrate walls and fabrics, preserve privacy, and is insensitive to lighting conditions. Micro-Doppler features are utilized in radar signal corresponding to human body motions and gait to detect falls using a narrowband pulse-Doppler radar. Human motions cause time-varying Doppler signatures, which are analyzed using time-frequency representations and matching pursuit decomposition (MPD) for feature extraction and fall detection. The extracted features include MPD features and the principal components of the time-frequency signal representations. To analyze the sequential characteristics of typical falls, the extracted features are used for training and testing hidden Markov models (HMM) in different falling scenarios. Experimental results demonstrate that the proposed algorithm and method achieve fast and accurate fall detections. The risk of falls increases sharply when the elderly or patients try to exit beds. Thus, if a bed exit can be detected at an early stage of this motion, the related injuries can be prevented with a high probability. To detect bed exit for fall prevention, the trajectory of head movements is used for recognize such human motion. A head detector is trained using the histogram of oriented gradient (HOG) features of the head and shoulder areas from recorded bed exit images. A data association algorithm is applied on the head detection results to eliminate head detection false alarms. Then the three dimensional (3D) head trajectories are constructed by matching scale-invariant feature transform (SIFT) keypoints in the detected head areas from both the left and right stereo images. The extracted 3D head trajectories are used for training and testing an HMM based classifier for recognizing bed exit activities. The results of the classifier are presented and discussed in the thesis, which demonstrates the effectiveness of the proposed stereo vision based bed exit detection approach.

EmotiBlog: a finer-grained and more precise learning of subjectivity expression models

The exponential growth of the subjective information in the framework of the Web 2.0 has led to the need to create Natural Language Processing tools able to analyse and process such data for multiple practical applications. They require training on specifically annotated corpora, whose level of detail must be fine enough to capture the phenomena involved. This paper presents EmotiBlog – a fine-grained annotation scheme for subjectivity. We show the manner in which it is built and demonstrate the benefits it brings to the systems using it for training, through the experiments we carried out on opinion mining and emotion detection. We employ corpora of different textual genres –a set of annotated reported speech extracted from news articles, the set of news titles annotated with polarity and emotion from the SemEval 2007 (Task 14) and ISEAR, a corpus of real-life self-expressed emotion. We also show how the model built from the EmotiBlog annotations can be enhanced with external resources. The results demonstrate that EmotiBlog, through its structure and annotation paradigm, offers high quality training data for systems dealing both with opinion mining, as well as emotion detection.

Sentiment classification for early detection of health alerts in the chemical textile domain

In the chemical textile domain experts have to analyse chemical components and substances that might be harmful for their usage in clothing and textiles. Part of this analysis is performed searching opinions and reports people have expressed concerning these products in the Social Web. However, this type of information on the Internet is not as frequent for this domain as for others, so its detection and classification is difficult and time-consuming. Consequently, problems associated to the use of chemical substances in textiles may not be detected early enough, and could lead to health problems, such as allergies or burns. In this paper, we propose a framework able to detect, retrieve, and classify subjective sentences related to the chemical textile domain, that could be integrated into a wider health surveillance system. We also describe the creation of several datasets with opinions from this domain, the experiments performed using machine learning techniques and different lexical resources such as WordNet, and the evaluation focusing on the sentiment classification, and complaint detection (i.e., negativity). Despite the challenges involved in this domain, our approach obtains promising results with an F-score of 65% for polarity classification and 82% for complaint detection.

The Big BIM battle: BIM adoption in the UK for large and small companies

As BIM adoption continues, the goal of a totally collaborative model with multiple contributors is attainable. Many initiatives such as the 2016 UK government level 2 BIM deadline are putting pressure on the construction industry to speed up the changeover. Clients and collaborators have higher expectations of using digital 3D models to communicate design ideas and solve practical problems. Contractors and clients are benefitting from cost saving scheduling and clash detection offered by BIM. Effective collaboration on the project will also give speed and efficiency gains. Despite this, many businesses of varying sizes are still having problems. The cost of the software and the training provides an obvious barrier for micro-enterprises and could explain a delay in adoption. Many studies have looked at these problems faced by SME and micro-enterprises. Larger companies have different problems. The efforts made by government to encourage them are quite comprehensive, but is anything being done to help smaller sectors and keep the industry cohesive? This limited study examines several companies of varying size and varying project type: architectural design businesses, main contractor, structural engineer and building consultancy. The study examines the barriers to a truly collaborative BIM workflow facing different specialities on a larger project and a contrasting small/medium project. The findings will establish that different barriers for each sector are actually pushing further apart, thus potentially creating a BIM-only construction elite, leaving the small companies remaining on 2D based drawing.

The differential evolution of hot- and cold-dust in galaxies in the last 7.5 GYR

Tese de mestrado em Física, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016

Therapeutic drug monitoring of cefepime with micellar electrokinetic capillary chromatography: Assay improvement, quality assurance, and impact on patient drug levels

The improvement and performance of a micellar electrokinetic capillary chromatography assay for cefepime in human serum and plasma with a 50 μm id fused-silica capillary elongated from 40 to 60 cm is reported. Sample preparation with dodecylsulfate protein precipitation at pH 4.5, the pH 9.1 separation medium and the applied voltage were as reported previously[16]. The change resulted in a significant lower current, higher resolution and increased detection time intervals. The performance of the assay with multi-level internal calibration was assessed with calibration and control samples. Quality assurance data of a two year period assessed under the new conditions demonstrated the robustness of the assay. In serum samples of patients who received both cefepime and sulfamethoxazole, cefepime could not be detected due to the inseparability of the two compounds. The presence of an interference can be recognized by an increased peak width (width > 0.2 min), the appearance of a shoulder or an unresolved double peak. The patient data gathered during a three year period reveal that introduction of therapeutic drug monitoring led to a 50% reduction of the median drug level. The data suggest that therapeutic drug monitoring can help to minimize the risk of major adverse reactions and to increase drug safety on an individual basis. This article is protected by copyright. All rights reserved.

Afterglows, redshifts, and properties of swift gammaray bursts

We present optical, near-IR, and radio follow-up of 16 Swift bursts, including our discovery of nine afterglows and a redshift determination for three. These observations, supplemented by data from the literature, provide an afterglow recovery rate of 52% in the optical/near-IR, much higher than in previous missions (BeppoSAX, HETE-2, INTEGRAL, and IPN). The optical/near-IR afterglows of Swift events are on average 1.8 mag fainter at t = 12 hr than those of previous missions. The X-ray afterglows are similarly fainter than those of pre-Swift bursts. In the radio the limiting factor is the VLA threshold, and the detection rate for Swift bursts is similar to that for past missions. The redshift distribution of pre-Swift bursts peaked at z similar to 1, whereas the six Swift bursts with measured redshifts are distributed evenly between 0.7 and 3.2. From these results we conclude that ( 1) the pre-Swift distributions were biased in favor of bright events and low-redshift events, ( 2) the higher sensitivity and accurate positions of Swift result in a better representation of the true burst redshift and brightness distributions ( which are higher and dimmer, respectively), and (3) similar to 10% of the bursts are optically dark, as a result of a high redshift and/or dust extinction. We remark that the apparent lack of low-redshift, low-luminosity Swift bursts and the lower event rate than prelaunch estimates ( 90 vs. 150 per year) are the result of a threshold that is similar to that of BATSE. In view of these inferences, afterglow observers may find it advisable to make significant changes in follow-up strategies of Swift events. The faintness of the afterglows means that large telescopes should be employed as soon as the burst is localized. Sensitive observations in RIz and near-IR bands will be needed to discriminate between a typical z similar to 2 burst with modest extinction and a high-redshift event. Radio observations will be profitable for a small fraction (similar to 10%) of events. Finally, we suggest that a search for bright host galaxies in untriggered BAT localizations may increase the chance of finding nearby low-luminosity GRBs.

Development of an oligonucleotide-based SNP detection method on lateral flow strips using hexapet tages

Detection of point mutations or single nucleotide polymorphisms (SNPs) is important in relation to disease susceptibility or detection in pathogens of mutations determining drug resistance or host range. There is an emergent need for rapid detection methods amenable to point-of-care applications. The purpose of this study was to reduce to practice a novel method for SNP detection and to demonstrate that this technology can be used downstream of nucleic acid amplification. The authors used a model system to develop an oligonucleotide-based SNP detection system on nitrocellulose lateral flow strips. To optimize the assay they used cloned sequences of the herpes simplex virus-1 (HSV-1) DNA polymerase gene into which they introduced a point mutation. The assay system uses chimeric polymerase chain reaction (PCR) primers that incorporate hexameric repeat tags ("hexapet tags"). The chimeric sequences allow capture of amplified products to predefined positions on a lateral flow strip. These "hexapet" sequences have minimal cross-reactivity and allow specific hybridization-based capture of the PCR products at room temperature onto lateral flow strips that have been striped with complementary hexapet tags. The allele-specific amplification was carried out with both mutant and wild-type primer sets present in the PCR mix ("competitive" format). The resulting PCR products carried a hexapet tag that corresponded with either a wild-type or mutant sequence. The lateral flow strips are dropped into the PCR reaction tube, and mutant sequence and wild-type sequences diffuse along the strip and are captured at the corresponding position on the strip. A red line indicative of a positive reaction is visible after 1 minute. Unlike other systems that require separate reactions and strips for each target sequence, this system allows multiplex PCR reactions and multiplex detection on a single strip or other suitable substrates. Unambiguous visual discrimination of a point mutation under room temperature hybridization conditions was achieved with this model system in 10 minutes after PCR. The authors have developed a capture-based hybridization method for the detection and discrimination of HSV-1 DNA polymerase genes that contain a single nucleotide change. It has been demonstrated that the hexapet oligonucleotides can be adapted for hybridization on the lateral flow strip platform for discrimination of SNPs. This is the first step in demonstrating SNP detection on lateral flow using the hexapet oligonucleotide capture system. It is anticipated that this novel system can be widely used in point-of-care settings.

Differences in surfactant lipids collected from pleural and pulmonary lining fluids

Purpose. The type and relative importance of saturated and unsaturated phospholipid components of surfactant within the epithelial lining fluid (ELF) of the inner and outer surfaces of the lung is not known. Methods. Seven healthy dogs were anesthetized and a bronchoalveolar lavage (BAL) was performed, immediately followed by a pleural lavage (PL). Lipid was extracted from lavage fluid and then analyzed for saturated, primarily dipalmitoylphosphatidylcholine (DPPC), and unsaturated phosphatidylcholine (PC) species using high-performance liquid chromatography (HPLC) with combined fluorescence and ultraviolet detection. Dilution of ELF in lavage fluids was corrected for using the urea method. Results. DPPC (494.7 +/- 213.9 mu g/mL) was the predominant PC present in ELF collected from the alveolar surface. In contrast, significantly higher (p = 0.028) proportions of unsaturated PC species were measured in PL fluid (similar to 105 mu g/mL), particularly stearoyl-linoleoyl-phosphatidylcholine (SLPC), which could not be measured in fluid collected from the alveoli, compared to DPPC (2.6 +/- 2.0 mu g/mL). Conclusions. This study indicates that unsaturated PC species seem to be more important than saturated species, particularly DPPC, in the pleural cavity, which has implications for surfactant replenishment following pleural disease or thoracic surgery.

Epitope-blocking enzyme-linked immunosorbent assay for detection of antibodies to Ross River virus in vertebrate sera

We describe the development of an epitope-blocking enzyme-linked immunosorbent assay (ELISA) for the sensitive and rapid detection of antibodies to Ross River virus (RRV) in human sera and known vertebrate host species. This ELISA provides an alternative method for the serodiagnosis of RRV infections.

Seizure detection algorithm for neonates based on wave-sequence analysis

Objective: The description and evaluation of the performance of a new real-time seizure detection algorithm in the newborn infant. Methods: The algorithm includes parallel fragmentation of EEG signal into waves; wave-feature extraction and averaging; elementary, preliminary and final detection. The algorithm detects EEG waves with heightened regularity, using wave intervals, amplitudes and shapes. The performance of the algorithm was assessed with the use of event-based and liberal and conservative time-based approaches and compared with the performance of Gotman's and Liu's algorithms. Results: The algorithm was assessed on multi-channel EEG records of 55 neonates including 17 with seizures. The algorithm showed sensitivities ranging 83-95% with positive predictive values (PPV) 48-77%. There were 2.0 false positive detections per hour. In comparison, Gotman's algorithm (with 30 s gap-closing procedure) displayed sensitivities of 45-88% and PPV 29-56%; with 7.4 false positives per hour and Liu's algorithm displayed sensitivities of 96-99%, and PPV 10-25%; with 15.7 false positives per hour. Conclusions: The wave-sequence analysis based algorithm displayed higher sensitivity, higher PPV and a substantially lower level of false positives than two previously published algorithms. Significance: The proposed algorithm provides a basis for major improvements in neonatal seizure detection and monitoring. Published by Elsevier Ireland Ltd. on behalf of International Federation of Clinical Neurophysiology.