Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes

A postembedding method has been developed for localizing water soluble allergens in rye-grass pollen. This uses dry fixation in glutaraldehyde vapour, followed by 2,2-dimethoxypropane, prior to a 100% ethanol series leading into embedment in LR Gold. This has allowed the attachment of specific monoclonal antibodies to the allergen, which are themselves probed with specific immunogold labels to the antibodies. Wall and cytoplasmic sites have been identified, representing an improvement of fixation and localization of allergens over previous studies employing polyclonal, broad spectrum antibodies.

Rye-grass allergens are labelled in mature pollen grains in the exine (tectum, nexine and central chamber), and in the electron opaque areas of the cytoplasm, especially mitochondria. The allergens are absent from the intine, polysaccharide (P) particles, amyloplasts, Golgi bodies and endoplasmic reticulum. IgE antibodies derived from humans allergic to rye-grass pollen, bind to similar sites in the cytoplasm but only to the outer surface of the pollen grain wall. This method now provides a valuable tool for further developmental studies on the pollen grains, in order to establish the site/s of synthesis of the allergens.

Secure localization with attack detection in wireless sensor networks

Rapid technological advances have enabled the development of low-cost sensor networks for various monitoring tasks, where it is important to estimate the positions of a number of regular sensor nodes whose locations cannot be known apriori. We address the problem of localizing the regular nodes with range-based location references obtained from certain anchor nodes referred to as beacons, particularly in an adverse environment where some of the beacons may be compromised. We propose an innovative modular solution featuring two lightweight modules that are for dedicated functionalities, respectively, but can also be closely integrated. First, we harness simple geometric triangular rules and an efficient voting technique to enable the attack detection module, which identifies and filters out malicious location references. We then develop a secure localization module that computes and clusters certain reference points, and the position of the concerned regular node is estimated with the centroid of the most valuable reference points identified. Extensive simulations show that our attack detection module can detect compromised beacons effectively, and the secure localization module can subsequently provide a dependable localization service in terms of bounded estimation error. The integrated system turns out to be tolerant of malicious attacks even in highly challenging scenarios.

A robust solution to the stereo-vision-based simultaneous localization and mapping problem with steady and moving landmarks

The problem of visual simultaneous localization and mapping (SLAM) is examined in this paper using recently developed ideas and algorithms from modern robust control and estimation theory. A nonlinear model for a stereo-vision-based sensor is derived that leads to nonlinear measurements of the landmark coordinates along with optical flow-based measurements of the relative robot-landmark velocity. Using a novel analytical measurement transformation, the nonlinear SLAM problem is converted into the linear domain and solved using a robust linear filter. Actually, the linear filter is guaranteed stable and the SLAM state estimation error is bounded within an ellipsoidal set. A mathematically rigorous stability proof is given that holds true even when the landmarks move in accordance with an unknown control input. No similar results are available for the commonly employed extended Kalman filter, which is known to exhibit divergence and inconsistency characteristics in practice. A number of illustrative examples are given using both simulated and real vision data that further validate the proposed method.

Optimal sensor placement in linear arrays : part I - AoA based localization

In this paper, we examine the optimal linear separation requirements for AoA sensors, in order to achieve the best performance in estimating the position of a target subjected to noisy measurements. Cramer-Rao inequality and the corresponding Fisher information matrix are used to analyze the sensor-target geometry, in order to characterize localization performance with respect to the linear spacial distribution of sensors.

Apical localization of zinc transporter ZnT4 in human airway epithelial cells and its loss in a murine model of allergic airway inflammation

The apical cytoplasm of airway epithelium (AE) contains abundant labile zinc (Zn) ions that are involved in the protection of AE from oxidants and inhaled noxious substances. A major question is how dietary Zn traffics to this compartment. In rat airways, in vivo selenite autometallographic (Se-AMG)-electron microscopy revealed labile Zn-selenium nanocrystals in structures resembling secretory vesicles in the apical cytoplasm. This observation was consistent with the starry-sky Zinquin fluorescence staining of labile Zn ions confined to the same region. The vesicular Zn transporter ZnT4 was likewise prominent in both the apical and basal parts of the epithelium both in rodent and human AE, although the apical pools were more obvious. Expression of ZnT4 mRNA was unaffected by changes in the extracellular Zn concentration. However, levels increased 3-fold during growth of cells in air liquid interface cultures and decreased sharply in the presence of retinoic acid. When comparing nasal versus bronchial human AE cells, there were significant positive correlations between levels of ZnT4 from the same subject, suggesting that nasal brushings may allow monitoring of airway Zn transporter expression. Finally, there were marked losses of both basally-located ZnT4 protein and labile Zn in the bronchial epithelium of mice with allergic airway inflammation. This study is the first to describe co-localization of zinc vesicles with the specific zinc transporter ZnT4 in airway epithelium and loss of ZnT4 protein in inflamed airways. Direct evidence that ZnT4 regulates Zn levels in the epithelium still needs to be provided. We speculate that ZnT4 is an important regulator of zinc ion accumulation in secretory apical vesicles and that the loss of labile Zn and ZnT4 in airway inflammation contributes to AE vulnerability in diseases such as asthma.

Efficient algorithms for subwindow search in object detection and localization

Recently, a simple yet powerful branch-and-bound method called Efficient Subwindow Search (ESS) was developed to speed up sliding window search in object detection. A major drawback of ESS is that its computational complexity varies widely from O(n²) to O(n⁴) for n × n matrices. Our experimental experience shows that the ESS's performance is highly related to the optimal confidence levels which indicate the probability of the object's presence. In particular, when the object is not in the image, the optimal subwindow scores low and ESS may take a large amount of iterations to converge to the optimal solution and so perform very slow. Addressing this problem, we present two significantly faster methods based on the linear-time Kadane's Algorithm for 1D maximum subarray search. The first algorithm is a novel, computationally superior branchand- bound method where the worst case complexity is reduced to O(n³). Experiments on the PASCAL VOC 2006 data set demonstrate that this method is significantly and consistently faster (approximately 30 times faster on average) than the original ESS. Our second algorithm is an approximate algorithm based on alternating search, whose computational complexity is typically O(n²). Experiments shows that (on average) it is 30 times faster again than our first algorithm, or 900 times faster than ESS. It is thus wellsuited for real time object detection.

Biosynthesis, localization, and macromolecular arrangement of the plasmodium falciparum translocon of exported proteins (PTEX)

To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA+ ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ∼600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.

Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy

ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking. In the patients' fibroblasts, total internal reflection fluorescence microscopy indicated a shift in steady-state equilibrium of ATP7AT994I and ATP7AP1386S, with exaggerated PM localization. Transfection of Hek293T cells and NSC-34 motor neurons with the mutant alleles tagged with the Venus fluorescent protein also revealed excess PM localization. Endocytic retrieval of the mutant alleles from the PM to the TGN was impaired. Immunoprecipitation assays revealed an abnormal interaction between ATP7AT994I and p97/VCP, an ubiquitin-selective chaperone which is mutated in two autosomal dominant forms of motor neuron disease: amyotrophic lateral sclerosis and inclusion body myopathy with early-onset Paget disease and fronto-temporal dementia. Small-interfering RNA (SiRNA) knockdown of p97/VCP corrected ATP7AT994I mislocalization. Flow cytometry documented that non-permeabilized ATP7AP1386S fibroblasts bound a carboxyl-terminal ATP7A antibody, consistent with relocation of the ATP7A di-leucine endocytic retrieval signal to the extracellular surface and partially destabilized insertion of the eighth transmembrane helix. Our findings illuminate the mechanisms underlying ATP7A-related DMN and establish a link between p97/VCP and genetically distinct forms of motor neuron degeneration.

An in vitro comparative assessment with a series of new triphenyltin(IV) 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates endowed with anticancer activities: Structural modifications, analysis of efficacy and cytotoxicity involving human tumor cell lines

Four new triphenyltin(IV) complexes of composition Ph₃SnLH (where LH = 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoate) (1–4) were synthesized and characterized by spectroscopic (¹H, ¹³C and ¹¹⁹Sn NMR, IR, ¹¹⁹Sn Mössbauer) techniques in combination with elemental analysis. The ¹¹⁹Sn NMR spectroscopic data indicate a tetrahedral coordination geometry in non-coordinating solvents. The crystal structures of three complexes, Ph₃SnL¹H (1), Ph₃SnL³H (3), Ph₃SnL⁴H (4), were determined. All display an essentially tetrahedral geometry with angles ranging from 93.50(8) to 124.5(2)°; ¹¹⁹Sn Mössbauer spectral data support this assignment. The cytotoxicity studies were performed with complexes 1–4, along with a previously reported complex (5) in vitro across a panel of human tumor cell lines viz., A498, EVSA-T, H226, IGROV, M19 MEL, MCF-7 and WIDR. The screening results were compared with the results from other related triphenyltin(IV) complexes (6–7) and tributyltin(IV) complexes (8–11) having 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates framework. In general, the complexes exhibit stronger cytotoxic activity. The results obtained for 1–3 are also comparable to those of its o-analogs i.e. 4–7, except 5, but the advantage is the former set of complexes demonstrated two folds more cytotoxic activity for the cell line MCF-7 with ID₅₀ values in the range 41–53 ng/ml. Undoubtedly, the cytotoxic results of complexes 1–3 are far superior to CDDP, 5-FU and ETO, and related tributyltin(IV) complexes 8–11. The quantitative structure-activity relationship (QSAR) studies for the cytotoxicity of triphenyltin(IV) complexes 1–7 and tributyltin(IV) complexes 8–11 is also discussed against a panel of human tumor cell lines.

Contribution of fibroblast and mast cell (afferent) and tumor (efferent) IL-6 effects within the tumor microenvironment

Hyperactive inflammatory responses following cancer initiation have led to cancer being described as a ‘wound that never heals’. These inflammatory responses elicit signals via NFκB leading to IL-6 production, and IL-6 in turn has been shown to induce epithelial to mesenchymal transition in breast cancer cells in vitro, implicating a role for this cytokine in cancer cell invasion. We previously have shown that conditioned medium derived from cancer-associated fibroblasts induced an Epithelial to Mesenchymal transition (EMT) in PMC42-LA breast cancer cells and we have now identify IL-6 as present in this medium. We further show that IL-6 is expressed approximately 100 fold higher in a cancer-associated fibroblast line compared to normal fibroblasts. Comparison of mouse-specific (stroma) and human-specific (tumor) IL-6 mRNA expression from MCF-7, MDA MB 468 and MDA MB 231 xenografts also indicated the stroma rather than tumor as a significantly higher source of IL-6 expression. Mast cells (MCs) feature in inflammatory cancer-associated stroma, and activated MCs secrete IL-6. We observed a higher MC index (average number of mast cells per xenograft section/average tumor size) in MDA MB 468 compared to MDA MB 231 xenografts, where all MC were observed to be active (degranulating). This higher MC index correlated with greater mouse-specific IL-6 expression in the MDA MB 468 xenografts, implicating MC as an important source of stromal IL-6. Furthermore, immunohistochemistry on these xenografts for pSTAT3, which lies downstream of the IL-6 receptor indicated frequent correlations between pSTAT3 and mast cell positive cells. Analysis of publically available databases for IL-6 expression in patient tissue revealed higher IL-6 in laser capture microdissected stroma compared to adjacent tissue epithelium from patients with inflammatory breast cancer (IBC) and invasive non-inflammatory breast cancer (non-IBC) and we show that IL-6 expression was significantly higher in Basal versus Luminal molecular/phenotypic groupings of breast cancer cell lines. Finally, we discuss how afferent and efferent IL-6 pathways may participate in a positive feedback cycle to dictate tumor progression.

Localization with ghost elimination of emitters via time-delay-of-arrival measurements

This paper investigates the theoretical requirements for unique localization of multiple emitters using time delay of arrival(TDoA) subjected to the data-association problem. Specifically, an examination is carried out to find the necessary fundamental requirements to solve the so-called ghost node problem pertaining to sensor arrays.

Multiple emitter localization using range only measurements considering geometrical constraints

This paper proposes a constrained optimization approach to improve the accuracy of a Time-of-Arrival (ToA) based multiple target localization system. Instead of using an overdetermined measurement system, this paper uses local distance measurements between the targets/emitters as the geometric constraint.Computer simulations are used to evaluate the performance of the geometrically constrained optimization method.

Optimal sensor placement in range based localization for linear arrays

This paper investigates the linear separation requirements for range sensors in order to achieve the optimal performance in estimating the position of a target from multiple and typically noisy sensor measurements. We analyze the sensor-target geometry in terms of the Cramer-Rao inequality and the corresponding Fisher information matrix, in order to characterize localization performance with respect to the linear special distribution.