Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order +35 > +1 > -3 > -8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60-72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in 'glowing silkworms'. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.

Topological mimicry and epitope duplication in the guanylyl cyclase C receptor

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.

Structural basis for the function of anti-idiotypic antibody in immune memory

We had earlier proposed a hypothesis to explain the mechanism of perpetuation of immunological memory based on the operation of idiotypic network in the complete absence of antigen. Experimental evidences were provided for memory maintenance through anti-idiotypic antibody (Ab2) carrying the internal image of the antigen. In the present work, we describe a structural basis for such memory perpetuation by molecular modeling and structural analysis studies. A three-dimensional model of Ab2 was generated and the structure of the antigenic site on the hemagglutinin protein H of Rinderpest virus was modeled using the structural template of hemagglutinin protein of Measles virus. Our results show that a large portion of heavy chain containing the CDR regions of Ab2 resembles the domain of the hemagglutinin housing the epitope regions. The similarity demonstrates that an internal image of the H antigen is formed in Ab2, which provides a structural basis for functional mimicry demonstrated earlier. This work brings out the importance of the structural similarity between a domain of hemagglutinin protein to that of its corresponding Ab2. It provides evidence that Ab2 is indeed capable of functioning as surrogate antigen and provides support to earlier proposed relay hypothesis which has provided a mechanism for the maintenance of immunological memory.

A Pattern Recognition Model of Voice-Based Personal Verification Systems for Forensic Applications

Abstract-The success of automatic speaker recognition in laboratory environments suggests applications in forensic science for establishing the Identity of individuals on the basis of features extracted from speech. A theoretical model for such a verification scheme for continuous normaliy distributed featureIss developed. The three cases of using a) single feature, b)multipliendependent measurements of a single feature, and c)multpleindependent features are explored.The number iofndependent features needed for areliable personal identification is computed based on the theoretcal model and an expklatory study of some speech featues.

Structural basis for the function of anti-idiotypic antibody in immune memory

We had earlier proposed a hypothesis to explain the mechanism of perpetuation of immunological memory based on the operation of idiotypic network in the complete absence of antigen. Experimental evidences were provided for memory maintenance through anti-idiotypic antibody (Ab(2)) carrying the internal image of the antigen. In the present work, we describe a structural basis for such memory perpetuation by molecular modeling and structural analysis studies. A three-dimensional model of Ab(2) was generated and the structure of the antigenic site on the hemagglutinin protein H of Rinderpest virus was modeled using the structural template of hemagglutinin protein of Measles virus. Our results show that a large portion of heavy chain containing the CDR regions of Ab(2) resembles the domain of the hemagglutinin housing the epitope regions. The similarity demonstrates that an internal image of the H antigen is formed in Ab(2), which provides a structural basis for functional mimicry demonstrated earlier. This work brings out the importance of the structural similarity between a domain of hemagglutinin protein to that of its corresponding Ab(2). It provides evidence that Ab(2) is indeed capable of functioning as surrogate antigen and provides support to earlier proposed relay hypothesis which has provided a mechanism for the maintenance of immunological memory.

A Fuzzy Approximation Scheme for Sequential Learning in Pattern Recognition

An adaptive learning scheme, based on a fuzzy approximation to the gradient descent method for training a pattern classifier using unlabeled samples, is described. The objective function defined for the fuzzy ISODATA clustering procedure is used as the loss function for computing the gradient. Learning is based on simultaneous fuzzy decisionmaking and estimation. It uses conditional fuzzy measures on unlabeled samples. An exponential membership function is assumed for each class, and the parameters constituting these membership functions are estimated, using the gradient, in a recursive fashion. The induced possibility of occurrence of each class is useful for estimation and is computed using 1) the membership of the new sample in that class and 2) the previously computed average possibility of occurrence of the same class. An inductive entropy measure is defined in terms of induced possibility distribution to measure the extent of learning. The method is illustrated with relevant examples.

Bayesian and Decision Tree Approaches for Pattern Recognition Including Feature Measurement Costs

The minimum cost classifier when general cost functionsare associated with the tasks of feature measurement and classification is formulated as a decision graph which does not reject class labels at intermediate stages. Noting its complexities, a heuristic procedure to simplify this scheme to a binary decision tree is presented. The optimizationof the binary tree in this context is carried out using ynamicprogramming. This technique is applied to the voiced-unvoiced-silence classification in speech processing.

Fluorescent labelling of strychnine: A novel approach for recognition of strychnine binding sites on neuronal membrane

trychnine was coupled to fluorescein isothiocyanate to mark strychnine binding sites in spinal cord of rat. Specific binding of strychnine could be demonstrated in synaptosomal fraction. Addition of glycine to the strychninised membrane led to a decrease in fluorescence indicating same receptor loci.

Antibody response against endogenous stages of an attenuated strain of Eimeria tenella

The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite lifecycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.

Simplified algorithms based on Haar transforms for signal recognition in protective relays

This letter presents the development of simplified algorithms based on Haar functions for signal extraction in relaying signals. These algorithms, being computationally simple, are better suited for microprocessor-based power system protection relaying. They provide accurate estimates of the signal amplitude and phase.

Host recognition by a polyphagous lepidopteran (Helicoverpa armigera): Primary host plants, host produced volatiles and neurosensory stimulation

An important question in the host-finding behaviour of a polyphagous insect is whether the insect recognizes a suite or template of chemicals that are common to many plants? To answer this question, headspace volatiles of a subset of commonly used host plants (pigeon pea, tobacco, cotton and bean) and nonhost plants (lantana and oleander) of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) are screened by gas chromatography (GC) linked to a mated female H. armigera electroantennograph (EAG). In the present study, pigeon pea is postulated to be a primary host plant of the insect, for comparison of the EAG responses across the test plants. EAG responses for pigeon pea volatiles are also compared between females of different physiological status (virgin and mated females) and the sexes. Eight electrophysiologically active compounds in pigeon pea headspace are identified in relatively high concentrations using GC linked to mass spectrometry (GC-MS). These comprised three green leaf volatiles [(2E)-hexenal, (3Z)-hexenylacetate and (3Z)-hexenyl-2-methylbutyrate] and five monoterpenes (α-pinene, β-myrcene, limonene, E-β-ocimene and linalool). Other tested host plants have a smaller subset of these electrophysiologically active compounds and even the nonhost plants contain some of these compounds, all at relatively lower concentrations than pigeon pea. The physiological status or sex of the moths has no effect on the responses for these identified compounds. The present study demonstrates how some host plants can be primary targets for moths that are searching for hosts whereas the other host plants are incidental or secondary targets.

Repeatable Condition-Invariant Visual Odometry for Sequence-Based Place Recognition

This paper describes a vision-only system for place recognition in environments that are tra- versed at different times of day, when chang- ing conditions drastically affect visual appear- ance, and at different speeds, where places aren’t visited at a consistent linear rate. The ma- jor contribution is the removal of wheel-based odometry from the previously presented algo- rithm (SMART), allowing the technique to op- erate on any camera-based device; in our case a mobile phone. While we show that the di- rect application of visual odometry to our night- time datasets does not achieve a level of perfor- mance typically needed, the VO requirements of SMART are orthogonal to typical usage: firstly only the magnitude of the velocity is required, and secondly the calculated velocity signal only needs to be repeatable in any one part of the environment over day and night cycles, but not necessarily globally consistent. Our results show that the smoothing effect of motion constraints is highly beneficial for achieving a locally consis- tent, lighting-independent velocity estimate. We also show that the advantage of our patch-based technique used previously for frame recogni- tion, surprisingly, does not transfer to VO, where SIFT demonstrates equally good performance. Nevertheless, we present the SMART system us- ing only vision, which performs sequence-base place recognition in extreme low-light condi- tions where standard 6-DOF VO fails and that improves place recognition performance over odometry-less benchmarks, approaching that of wheel odometry.

An Adaptive Scheme for Learning the Probability Threshold in Pattern Recognition

The statistical minimum risk pattern recognition problem, when the classification costs are random variables of unknown statistics, is considered. Using medical diagnosis as a possible application, the problem of learning the optimal decision scheme is studied for a two-class twoaction case, as a first step. This reduces to the problem of learning the optimum threshold (for taking appropriate action) on the a posteriori probability of one class. A recursive procedure for updating an estimate of the threshold is proposed. The estimation procedure does not require the knowledge of actual class labels of the sample patterns in the design set. The adaptive scheme of using the present threshold estimate for taking action on the next sample is shown to converge, in probability, to the optimum. The results of a computer simulation study of three learning schemes demonstrate the theoretically predictable salient features of the adaptive scheme.

Multi Pattern Dynamic Time Warping For Automatic Speech Recognition

We are addressing the problem of jointly using multiple noisy speech patterns for automatic speech recognition (ASR), given that they come from the same class. If the user utters a word K times, the ASR system should try to use the information content in all the K patterns of the word simultaneously and improve its speech recognition accuracy compared to that of the single pattern based speech recognition. T address this problem, recently we proposed a Multi Pattern Dynamic Time Warping (MPDTW) algorithm to align the K patterns by finding the least distortion path between them. A Constrained Multi Pattern Viterbi algorithm was used on this aligned path for isolated word recognition (IWR). In this paper, we explore the possibility of using only the MPDTW algorithm for IWR. We also study the properties of the MPDTW algorithm. We show that using only 2 noisy test patterns (10 percent burst noise at -5 dB SNR) reduces the noisy speech recognition error rate by 37.66 percent when compared to the single pattern recognition using the Dynamic Time Warping algorithm.

Odour emissions from anaerobic piggery ponds. 2: Improving estimates of emission rate through recognition of spatial variability

Odour emission rates were measured for seven different anaerobic ponds treating piggery wastes at six to nine discrete locations across the surface of each pond on each sampling occasion over a thirteen month period. Significant variability in emission rates were observed for each pond. Measurement of a number of water quality variables in pond liquor samples collected at the same time and from the same locations as the odour samples indicated that the composition of the pond liquor was also variable. The results indicated that spatial variability was a real phenomenon and could have a significant impact on odour assessment practices. Considerably more odour samples would be required to characterise pond emissions than currently recommended by most practitioners, or regulatory agencies.