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Results for “binary vectorization”

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PubMed1998

Cascaded redundancy reduction.

We describe a method for incrementally constructing a hierarchical generative model of an ensemble of binary data vectors. The model is composed of stochastic, binary, logistic units. Hidden units are added to the model one at a time with the goal of minimizing the information required to describe the data vectors using the model. In addition to the top-down generative weights that define the model, there are bottom-up recognition weights that determine the binary states of the hidden units given a data vector. Even though the stochastic generative model can produce each data vector in many ways, the recognition model is forced to pick just one of these ways. The recognition model therefore underestimates the ability of the generative model to predict the data, but this underestimation greatly simplifies the process of searching for the generative and recognition weights of a new hidden unit.

Algorithms↗

PubMed2004

Anti-liver cancer activity of TNF-related apoptosis-inducing ligand gene and its bystander effects.

AIM: To observe the anti-liver cancer activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene and its bystander effects on hepatocellular carcinoma (HCC) cell line SMMC7721. METHODS: Full-length cDNA of human TRAIL was transferred into SMMC7721 cells with a binary adenoviral vector system. Polymerase-chain reaction following reverse transcription (RT-PCR) was used to determine the expression of TRAIL gene. Effects of the transfected gene on proliferation of SMMC7721 cells were measured by MTT assay. Its influence on apoptosis was demonstrated by fluorescence-activated cell sorting (FACS). The bystander effect was observed by co-culturing the SMMC7721 cells with and without the transfected TRAIL gene at different ratios, and the culture medium supernatant from the transfected cells was also examined for its influence on SMMC7721 cells. RESULTS: The growth-inhibition rate and apoptotic cell fraction in the cells transfected with the TRAIL gene, Bax gene or only LacZ gene were 91.2%, 48.0%, 28.8% and 29.1%, 12.5%, 6.6%, respectively. The growth-inhibition rate of transfection with these three sequences in normal human fibroblasts was 6.1%, 45.5% and 7.6%, respectively, indicating a discriminative inhibition of TRAIL transfection on the cancer cells. In the co-culturing test, addition of the transfected TRAIL to SMMC7721 cells in proportions of 5%, 25%, 50%, 75% and 100%, resulted in a growth-inhibition of 15.9%, 67%, 80.2%, 86.4% and 87.7%, respectively. We failed to observe a significant growth-inhibition effect of the culture medium supernatant on SMMC7721 cells. CONCLUSION: TRAIL gene transferred by a binary adenoviral vector system can inhibit proliferation of SMMC7721 cells and induce their apoptosis. A bystander effect was observed, which seemed not to be mediated by soluble factors.

Adenoviridae↗

PubMed2002

Binary tree-structured vector quantization approach to clustering and visualizing microarray data.

MOTIVATION: With the increasing number of gene expression databases, the need for more powerful analysis and visualization tools is growing. Many techniques have successfully been applied to unravel latent similarities among genes and/or experiments. Most of the current systems for microarray data analysis use statistical methods, hierarchical clustering, self-organizing maps, support vector machines, or k-means clustering to organize genes or experiments into 'meaningful' groups. Without prior explicit bias almost all of these clustering methods applied to gene expression data not only produce different results, but may also produce clusters with little or no biological relevance. Of these methods, agglomerative hierarchical clustering has been the most widely applied, although many limitations have been identified. RESULTS: Starting with a systematic comparison of the underlying theories behind clustering approaches, we have devised a technique that combines tree-structured vector quantization and partitive k-means clustering (BTSVQ). This hybrid technique has revealed clinically relevant clusters in three large publicly available data sets. In contrast to existing systems, our approach is less sensitive to data preprocessing and data normalization. In addition, the clustering results produced by the technique have strong similarities to those of self-organizing maps (SOMs). We discuss the advantages and the mathematical reasoning behind our approach.

Algorithms↗

PubMed2006

Transformation of maize via Agrobacterium tumefaciens using a binary co-integrate vector system.

This chapter describes a stepwise protocol to achieve success in genetic transformation of maize using Agrobacterium tumefaciens as a DNA delivery system. Researchers will be able to effectively transform immature embryos of Hi-II and related genotypes with this protocol. The outcome of the transformation process will be transgenic embryogenic callus tissue, transgenic plants, and transgenic progeny seeds. Recommendations for molecular confirmation and evaluation of transgenic tissue/plants are also provided.

Agrobacterium tumefaciens↗

PubMed2004

Generation of T-DNA tagging lines with a bidirectional gene trap vector and the establishment of an insertion-site database.

We have developed a binary T-DNA vector, pGA2717, that contains the promoter-less beta-glucuronidase (gus) gene adjacent to the right border and the promoter-less green fluorescence protein (gfp) gene next to the left border of the T-DNA. Therefore, inserting T-DNA into a gene can result in the activation of either gus or gfp. A total of 12 169 T-DNA insertional lines of japonica rice were generated using this binary vector. Out of 3140 lines examined, 0.5% of their mature seeds and 2.0% of the 3-day-old etiolated seedlings were GFP-positive. However, GUS assays of the same materials resulted in the identification of 151 (4.8%) GUS-positive lines. Using DNA gel blot and reverse transcription (RT)-PCR analyses, we confirmed that the GFP-positive lines were a true indication of gene trapping. A fusion transcript was also obtained between gfp and the trapped gene. We isolated 990 genomic sequences flanking T-DNA from our analysis of 2099 transgenic plants. Among the insertions, 625 T-DNAs were integrated into genic regions; 361 were located in intergenic regions. These tagging lines will be valuable in trapping and studying various genes for their expression patterns, as well as providing a useful tool for genetic approaches.

Base Sequence↗

PubMed2025

Development of a plant-based vaccine against brucellosis: stable expression of Brucella abortus OMP25 in transgenic tobacco.

Brucellosis, caused by Brucella species, is a global threat to livestock farming, resulting in economic losses and socio-economic challenges, particularly in rural areas. Despite its impact, no licensed human vaccines are available. Animal vaccination remains the most cost-effective control method, but traditional vaccine production is expensive. Edible vaccines, using plants as bioreactors to produce immunogenic antigens, offer a low-cost alternative by eliminating complex purification processes. This study developed a transgenic plant by expressing the Brucella abortus outer membrane protein OMP25 in tobacco plants. OMP25, a conserved transmembrane protein with high immunogenicity, was cloned into a Gateway pDONR vector via a Boundary Pairing reaction and transferred to a binary destination vector via a Left-Right reaction. The destination vector was introduced into Agrobacterium tumefaciens and subsequently used for Agrobacterium-mediated transformation of tobacco plants. Transgenic plants were selected on media containing kanamycin, and the expression of the transgene was verified through the fluorescence of green fluorescent protein. Microcallus formation and shoot development on selective media confirmed kanamycin resistance and the successful integration of the transgene. After phenotypic selection, genomic DNA was extracted from transgenic plants and analyzed by PCR (Polymerase Chain Reaction) using primers specific to the OMP25 gene. Positive PCR results validated the successful integration of the OMP25 gene into the plant genome. Gene expression was further confirmed at the RNA level through real-time quantitative PCR (qRT-PCR) and at the protein level via Western blot analysis. Future studies will evaluate immune responses in animal models. This approach demonstrates the potential for low-cost, effective vaccines to combat brucellosis, addressing critical economic and public health challenges.

Plants, Genetically Modified↗

PubMed2004

Molecular therapy for peritoneal dissemination of xenotransplanted human MKN-45 gastric cancer cells with adenovirus mediated Bax gene transfer.

BACKGROUND: Gene therapy is an innovative therapeutic approach for cancer. An adenoviral vector expressing the tumour suppressor p53 gene (Ad/p53) is currently under clinical evaluation for various cancers. We recently developed a binary adenoviral vector system that can express the strong proapoptotic gene Bax (Ad/PGK-GV16+Ad/GT-Bax: Ad/Bax). AIMS: To evaluate the potential of Bax gene therapy for gastric cancer, we assessed its antitumour effect in comparison with that of p53. METHODS: The human gastric cancer cell lines MKN-1, MKN-7, MKN-28, and MKN-45 were treated with Ad/Bax or Ad/p53, and cell viability, transgene expression, and caspase activation were assessed in vitro. To compare the antitumour effects of Ad/Bax and Ad/p53 treatment in vivo, subcutaneous tumours and peritoneal dissemination of MKN-45 cells were generated in nude mice. Each mouse underwent intratumoral or intraperitoneal administration of viruses and the growth of implanted tumours was observed after treatment. RESULTS: Treatment with Ad/Bax and Ad/p53 resulted in marked Bax and p53 protein expression and effective apoptosis induction in MKN-1, MKN-7, and MKN-28 cells in vitro. In contrast, MKN-45 cells showed resistance to Ad/p53 and only treatment with Ad/Bax resulted in activation of caspase 3 expression and massive apoptosis. Ad/Bax treatment was more effective in suppressing both subcutaneous and peritoneally disseminated MKN-45 tumours compared with Ad/p53 treatment. CONCLUSION: Ad/Bax treatment significantly inhibited the growth of even p53 resistant gastric cancer in vitro and in vivo. Therefore, adenovirus mediated Bax gene transfer may be useful in gene therapy for gastric cancers.

Adenoviridae↗

PubMed2004

Agrobacterium-mediated transformation of polyploid cereals. The efficiency of selection and transgene expression in wheat.

Three combinations of Agrobacterium tumefaciens strains and vectors were used in the transformation of selected Polish wheat cultivars. The combinations were: two hypervirulent strains, AGL1, containing the pDM805 binary plasmid, and EHA101, containing pGAH; and the common Agro strain LBA4404, harboring the super-binary pTOK233 vector. pDM805 contained bar under the control of Ubi1 promoter, pGAH had nptII under nos, and pTOK233 had hpt under 35S. Additionally, pDM805 and pTOK233 carried the gus reporter gene under the Act1 promoter or 35S promoter, respectively. The highest selection rate was 12.6% and was obtained with EHA101(pGAH) on a kanamycin-containing medium. Sixty-five of the plants grown on that medium were PCR positive. The second best combination was LBA4404(pTOK233) and kanamycin selection, which gave an average transformation rate of 2.3%. Phosphinothricin selection gave 1.0% transformation efficiency, while hygromycin, depending on the strain/vector used, gave from 0.2 to 0.4%. PCR tests in T1 revealed that 67% of the lines showed a 3:1 segregation ratio, and 11% a 15:1 ratio, while in 22%, segregation was non-Mendelian. The high number of T0 transgenic plants containing one copy of the transgene was confirmed via Southern blot analysis. Kanamycin resistance in the T1 generation was very low; in some lines, all the progeny were kanamycin sensitive. GUS expression, only tested in young T1 plants, was in agreement with Mendelian segregation in three out of the twelve tested. The factors influencing the efficiency of selection and transgene expression are discussed in this paper.

Agrobacterium tumefaciens↗

PubMed2006

Megaprimer-mediated domain swapping for construction of chimeric viruses.

Clones that encode viral genomes constructed from two viruses with contrasting biological properties have been widely used in studies of viral-host interactions, particularly when the objective is to determine the identity of the viral component recognized by the host in a resistant response, known as the avirulence factor. This paper presents an efficient method based on megaprimer-mediated domain swapping for the construction of clones encoding chimeric viral genomes as a versatile and widely applicable alternative to conventional restriction enzyme digestion and ligation methods. Potato virus X (PVX)-derived vectors expressing genes encoding fluorescent proteins were used to demonstrate this concept. The cyan fluorescent protein (CFP) gene was cloned into a binary PVX vector and subsequently replaced with the yellow fluorescent protein (YFP) gene using the megaprimer amplification reaction. DNA fragments up to 1480 bp could be replaced efficiently and quickly. Most viral clones showed the expected change in phenotype without altered infectivity. Sequence analysis revealed mutations were not introduced into the four domain-swapped plasmids. This approach will provide a valuable tool for determining which domains of a viral genome are essential for infectivity, avirulence, or otherwise determine biologically significant properties of plant viruses.

Bacterial Proteins↗

PubMed2004

[Construction of expression vector with antisense Rubisco activase gene and its genetic transformation in rice].

In this research, Rubisco activase gene (rca) was amplified using specific primers and inserted into pGEM T-easy vector, and then cut with EcoRI after confirming by sequencing. The fragment was subcloned into pBluescript KS+, digested with the enzyme BamHI and inserted into the binary expression vector pCAMBIA1301, and the resulting construction with antisense rca was named pCAMR02. The pCAMR02 vector was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation and transformed to embryos of rice (Oryza. Sativa L.ssp.japonica) cultivar Zhonghua11 via Agrobacterium tumefaciens system. Plantlets were regenerated in vitro by resistance selection on medium containing various concentrations of hygromycin. Both GUS histochemical assays and PCR amplification demonstrated that antisense rca was integrated into T0 genomes and inherited to T1. The measurement of phenotypes of transgenic rice plants with antisense rca showed that most of them could hardly survive at ambient CO2 concentration, even could not grow. The antisense plants that survived under natural condition were dwarf and grew slower than the wild-type controls, and their contents of RCA and Rubisco changed significantly. These plants generated in this experiment will be used to study the relationship between RCA and Rubisco and their regulation.

Agrobacterium tumefaciens↗

PubMed2002

A pattern-mixture model for longitudinal binary responses with nonignorable nonresponse.

Longitudinal studies frequently incur outcome-related nonresponse. In this article, we discuss a likelihood-based method for analyzing repeated binary responses when the mechanism leading to missing response data depends on unobserved responses. We describe a pattern-mixture model for the joint distribution of the vector of binary responses and the indicators of nonresponse patterns. Specifically, we propose an extension of the multivariate logistic model to handle nonignorable nonresponse. This method yields estimates of the mean parameters under a variety of assumptions regarding the distribution of the unobserved responses. Because these models make unverifiable identifying assumptions, we recommended conducting sensitivity analyses that provide a range of inferences, each of which is valid under different assumptions for nonresponse. The methodology is illustrated using data from a longitudinal study of obesity in children.

Adolescent↗

PubMed2005

Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters.

BACKGROUND: The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX. METHODS: We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX. RESULTS: We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX. CONCLUSIONS: Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.

Animals↗

PubMed1989

Phleomycin resistance as a dominant selectable marker for plant cell transformation.

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance ('Ble') genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however, the highest frequency of rooted plants was obtained when transformation was carried out with the 'Sh Ble' gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.

Bleomycin↗

PubMed2003

CNN universal machine as classificaton platform: an art-like clustering algorithm.

Fast and robust classification of feature vectors is a crucial task in a number of real-time systems. A cellular neural/nonlinear network universal machine (CNN-UM) can be very efficient as a feature detector. The next step is to post-process the results for object recognition. This paper shows how a robust classification scheme based on adaptive resonance theory (ART) can be mapped to the CNN-UM. Moreover, this mapping is general enough to include different types of feed-forward neural networks. The designed analogic CNN algorithm is capable of classifying the extracted feature vectors keeping the advantages of the ART networks, such as robust, plastic and fault-tolerant behaviors. An analogic algorithm is presented for unsupervised classification with tunable sensitivity and automatic new class creation. The algorithm is extended for supervised classification. The presented binary feature vector classification is implemented on the existing standard CNN-UM chips for fast classification. The experimental evaluation shows promising performance after 100% accuracy on the training set.

Algorithms↗

PubMed1994

Performance of generalized estimating equations in practical situations.

Moment methods for analyzing repeated binary responses have been proposed by Liang and Zeger (1986, Biometrika 73, 13-22), and extended by Prentice (1988, Biometrics 44, 1033-1048). In their generalized estimating equations (GEE), both Liang and Zeger (1986) and Prentice (1988) estimate the parameters associated with the expected value of an individual's vector of binary responses as well as the correlations between pairs of binary responses. In this paper, we discuss one-step estimators, i.e., estimators obtained from one step of the generalized estimating equations, and compare their performance to that of the fully iterated estimators in small samples. In simulations, we find the performance of the one-step estimator to be qualitatively similar to that of the fully iterated estimator. When the sample size is small and the association between binary responses is high, we recommend using the one-step estimator to circumvent convergence problems associated with the fully iterated GEE algorithm. Furthermore, we find the GEE methods to be more efficient than ordinary logistic regression with variance correction for estimating the effect of a time-varying covariate.

Air Pollution↗

PubMed2004

Cascades of response vectors of olfactory receptor neurons in Xenopus laevis tadpoles.

Olfactory receptor neurons (ORNs) of Xenopus laevis tadpoles respond to water-born stimuli such as amino acids. Their sensitivity spectra with respect to amino acids have recently been shown to become more selective over ontogenetic stages [Manzini & Schild (2004) J. Gen. Physiol., 123, 99-107]. In this paper, we undertake a theoretical analysis of this data set and determine the correlational relationships among odorant responses represented as binary response vectors. We first show that, on the one hand, the number of 204 ORN classes (out of 283 recorded ORNs) cannot be explained by a random expression pattern of olfactory receptors (ORs). On the other hand, this number does not appear to be reconcilable with the idea that individual ORNs express one type of OR each. The covariance matrix of stimulus responses shows that the responses to some stimuli are correlated to those of others. Furthermore, the response vectors show positive as well as negative correlations among each other. While the positive correlations can partly be explained by the differing response frequencies to the odorants used, the negative ones cannot. Finally, we analyse the similarity among responses using the Hamming distance as a distance measure, the result being that most response vectors differ from others by small Hamming distances. Such vectors are shown to form pattern cascades, possibly reflecting a decreasing number of ORs being expressed over ontogenetic stages.

Amino Acid Sequence↗

PubMed2006

[Construction of a plant effective expression vector containing the gene of hepatitis B virus surface antigen].

OBJECTIVE: To construct a plant effective expression vector driven by a fruit specific promoter for the expression of hepatitis B virus surface antigen (HBsAg), to further improve the expression of exogenous gene in plant, and to prepare for the development of an effective anti-hepatitis vaccine. METHODS: Tomato fruit-specific promoters' gene 2A12 and E8 were respectively introduced to pBPFOmega7 to form pB2A12 and pBE8. The DNA fragment containing HBsAg-s gene from plasmid YEP-HBs was inserted respectively into pB2A12 and pBE8 to form pB2A12-HBs and pBE8-HBs. The fragment containing "p35S+2A12+Omega+HBsAg-s+Tnos" of the pB2A12-HBs was sub-cloned into plasmid pCAMBIA1301 to yield the reconstructed plant binary expression plasmid pCAM2A12-HBs, and the fragment containing "p35S+E8+Omega+HBsAg-s+Tnos" of the pBE8-HBs was sub-cloned into plasmid pCAMBIA1301 to yield the plasmid pCAME8-HBs. The inserted gene HBsAg and fruit-specific promoters in the reconstructed plant binary expression vectors were confirmed by sequencing. Then, pCAM2A12-HBs and pCAME8-HBs were directly introduced into Agrobacterium tumefaciens strain EHA105. RESULTS: Digestion with restriction enzymes proved that all recombinant vectors had the inserts with expected length of the target fragments, and the sequencing results were confirmed correct. CONCLUSION: In this study, plant expression vector containing HBsAg gene driven by fruit specific promoter and CaMV35s promoter was successfully constructed.

Cloning, Molecular↗

PubMed2024

Proteome-scale prediction of molecular mechanisms underlying dominant genetic diseases.

Many dominant genetic disorders result from protein-altering mutations, acting primarily through dominant-negative (DN), gain-of-function (GOF), and loss-of-function (LOF) mechanisms. Deciphering the mechanisms by which dominant diseases exert their effects is often experimentally challenging and resource intensive, but is essential for developing appropriate therapeutic approaches. Diseases that arise via a LOF mechanism are more amenable to be treated by conventional gene therapy, whereas DN and GOF mechanisms may require gene editing or targeting by small molecules. Moreover, pathogenic missense mutations that act via DN and GOF mechanisms are more difficult to identify than those that act via LOF using nearly all currently available variant effect predictors. Here, we introduce a tripartite statistical model made up of support vector machine binary classifiers trained to predict whether human protein coding genes are likely to be associated with DN, GOF, or LOF molecular disease mechanisms. We test the utility of the predictions by examining biologically and clinically meaningful properties known to be associated with the mechanisms. Our results strongly support that the models are able to generalise on unseen data and offer insight into the functional attributes of proteins associated with different mechanisms. We hope that our predictions will serve as a springboard for researchers studying novel variants and those of uncertain clinical significance, guiding variant interpretation strategies and experimental characterisation. Predictions for the human UniProt reference proteome are available at https://osf.io/z4dcp/.

Humans↗