Transcutaneous immunization with novel lipid-based adjuvants induces protection against gastric Helicobacter pylori infection

The development of vaccines to combat pathogens that infect across mucosal surfaces has been a major goal of vaccine research. Successful mucosal vaccination requires the co-administration of adjuvants that can overcome the state of immune tolerance normally associated with mucosal application of proteins. In the case of oral immunization, delivery systems are also required to protect vaccine antigens against destruction by gastric pH and digestive enzymes. Furthermore, adjuvants used for mucosal delivery must be free of neurotoxic effects like those induced by the commonly used experimental mucosal adjuvant cholera toxin. Maintenance of the "cold chain" is also essential for the effectiveness of any vaccine and adjuvants/delivery systems that enhance the stability of a vaccine would offer a significant advantage. Needle-free methods of vaccination that induce protective immunity at multiple mucosal surfaces are also desirable for rapid vaccination of large populations. In the present study we show that transcutaneous immunization (TCI) using Lipid C, a novel lipid-based matrix originally developed for oral immunization, containing soluble Helicobacter sonicate significantly reduces the gastric bacterial burden in mice following gastric challenge with live Helicobacter pylori. Protection is associated with the production of splenic gamma interferon and gastric IgA and was achieved without the co-administration of potent and potentially toxic adjuvants, although protection was further enhanced by inclusion of CpG-ODN and cholera toxin in the lipid delivery system.

Identifying optimal lipid raft characteristics required to promote nanoscale protein-protein interactions on the plasma membrane

The dynamic lateral segregation of signaling proteins into microdomains is proposed to facilitate signal transduction, but the constraints on microdomain size, mobility, and diffusion that might realize this function are undefined. Here we interrogate a stochastic spatial model of the plasma membrane to determine how microdomains affect protein dynamics. Taking lipid rafts as representative microdomains, we show that reduced protein mobility in rafts segregates dynamically partitioning proteins, but the equilibrium concentration is largely independent of raft size and mobility. Rafts weakly impede small-scale protein diffusion but more strongly impede long-range protein mobility. The long-range mobility of raft-partitioning and raft-excluded proteins, however, is reduced to a similar extent. Dynamic partitioning into rafts increases specific interprotein collision rates, but to maximize this critical, biologically relevant function, rafts must be small (diameter, 6 to 14 nm) and mobile. Intermolecular collisions can also be favored by the selective capture and exclusion of proteins by rafts, although this mechanism is generally less efficient than simple dynamic partitioning. Generalizing these results, we conclude that microdomains can readily operate as protein concentrators or isolators but there appear to be significant constraints on size and mobility if microdomains are also required to function as reaction chambers that facilitate nanoscale protein-protein interactions. These results may have significant implications for the many signaling cascades that are scaffolded or assembled in plasma membrane microdomains.

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor α (TNFα) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNFα is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasmamembrane, and we investigated a possible role for lipid rafts in TNFα trafficking and secretion. TNFα surface delivery and secretion were found to be cholesterol- dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasmamembrane, particularly on filopodia. Imaging the early stages of TNFα surface distribution revealed these puncta to be the initial points of TNFα delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.

Agrobacterium-mediated transformation of indica rice cultivars using binary and superbinary vectors

The Agrobacterium-mediated transformation system was extended to two indica cultivars: a widely cultivated breeding line IR-64 and an elite basmati cultivar Karnal Local. Root tips and shoot tips of seedlings, and scutellar-calli derived from mature seeds showed high-efficiency Agrobacterium tumefaciens infection and stable transformation. In addition to the superbinary vector pTOK233 in Agrobacterium strain LBA4404, almost equally high levels of transformation were achieved with a relatively much smaller (13.1 kb) binary vector (pCAMBIA1301) in a supervirulent host strain AGL1. In both cases, as well as in both cultivars, while 60–90% of the infected explants produced calli resistant to the selectable agent hygromycin, 59–75% of such calli tested positive for GUS. A high level (400 μM) of acetosyringone in the preinduction medium for Agrobacterium and a higher level (500 μM) in the cocultivation medium was necessary for an enhancement in transformation frequency of the binary vector to levels comparable to a superbinary. Hygromycin-resistant calli could be produced from all the explants used. Transformants could be regenerated for both cultivars using the superbinary and binary vector, but only for calli of scutellar origin. In addition to the molecular confirmation of hpt and gus gene transfer and transcription, absence of gene sequences outside the transferred DNA (T-DNA) region confirmed absence of any long T-DNA transfer.

Implications of lipid biology for the pathogenesis of schizophrenia

Objective: Preclinical and clinical data suggest that lipid biology is integral to brain development and neurodegeneration. Both aspects are proposed as being important in the pathogenesis of schizophrenia. The purpose of this paper is to examine the implications of lipid biology, in particular the role of essential fatty acids (EFA), for schizophrenia. Methods: Medline databases were searched from 1966 to 2001 followed by the crosschecking of references. Results: Most studies investigating lipids in schizophrenia described reduced EFA, altered glycerophospholipids and an increased activity of a calcium-independent phospholipase A2 in blood cells and in post-mortem brain tissue. Additionally, in vivo brain phosphorus-31 Magnetic Resonance Spectroscopy (31P-MRS) demonstrated lower phosphomonoesters (implying reduced membrane precursors) in first- and multi-episode patients. In contrast, phosphodiesters were elevated mainly in first-episode patients (implying increased membrane breakdown products), whereas inconclusive results were found in chronic patients. EFA supplementation trials in chronic patient populations with residual symptoms have demonstrated conflicting results. More consistent results were observed in the early and symptomatic stages of illness, especially if EFA with a high proportion of eicosapentaenoic acid was used. Conclusion: Peripheral blood cell, brain necropsy and 31P-MRS analysis reveal a disturbed lipid biology, suggesting generalized membrane alterations in schizophrenia. 31P-MRS data suggest increased membrane turnover at illness onset and persisting membrane abnormalities in established schizophrenia. Cellular processes regulating membrane lipid metabolism are potential new targets for antipsychotic drugs and might explain the mechanism of action of treatments such as eicosapentaenoic acid.

Real, complex, and binary semantic vectors

This paper presents a combined structure for using real, complex, and binary valued vectors for semantic representation. The theory, implementation, and application of this structure are all significant. For the theory underlying quantum interaction, it is important to develop a core set of mathematical operators that describe systems of information, just as core mathematical operators in quantum mechanics are used to describe the behavior of physical systems. The system described in this paper enables us to compare more traditional quantum mechanical models (which use complex state vectors), alongside more generalized quantum models that use real and binary vectors. The implementation of such a system presents fundamental computational challenges. For large and sometimes sparse datasets, the demands on time and space are different for real, complex, and binary vectors. To accommodate these demands, the Semantic Vectors package has been carefully adapted and can now switch between different number types comparatively seamlessly. This paper describes the key abstract operations in our semantic vector models, and describes the implementations for real, complex, and binary vectors. We also discuss some of the key questions that arise in the field of quantum interaction and informatics, explaining how the wide availability of modelling options for different number fields will help to investigate some of these questions.

Drugs for cardiac arrhythmmias, antithrombotic drugs and lipid modulating drugs

13.1 Drugs for cardiac arrhythmias 13.1.1 Introduction to cardiac arrhythmias 13.1.2 Cardiac action potentials 13.1.3 Mechanisms of cardiac arrhythmias 13.1.3 Class I 13.1.4 Class II 13.1.5 Class III 12.1.6 Class IV 13.1.7 Amiodarone 13.1.8 Adenosine 13.2 Antithrombotic drugs 13.2.1 Thrombus formation 13.2.2 Platelet aggregation and anti-platelet drugs 13.2.3 Coagulation 13.2.4 Anticoagulants 13.2.5 Fibrinolysis and fibrinolytics 13.3. Lipid modulating drugs 13.3.1 Cholesterol 13.3.2 Statins 13.3.3 Fibric acid derivatives 13.3.4 Ezetimibe

Gene transfer vectors targeted to human prostate cancer : do we need better preclinical testing systems?

Destruction of cancer cells by genetically modified viral and nonviral vectors has been the aim of many research programs. The ability to target cytotoxic gene therapies to the cells of interest is an essential prerequisite, and the treatment has always had the potential to provide better and more long-lasting therapy than existing chemotherapies. However, the potency of these infectious agents requires effective testing systems, in which hypotheses can be explored both in vitro and in vivo before the establishment of clinical trials in humans. The real prospect of off-target effects should be eliminated in the preclinical stage, if current prejudices against such therapies are to be overcome. In this review we have set out, using adenoviral vectors as a commonly used example, to discuss some of the key parameters required to develop more effective testing, and to critically assess the current cellular models for the development and testing of prostate cancer biotherapy. Only by developing models that more closely mirror human tissues will we be able to translate literature publications into clinical trials and hence into acceptable alternative treatments for the most commonly diagnosed cancer in humans.

Empirical analysis of the effect of dimension reduction and word order on semantic vectors

The aim of this paper is to provide a comparison of various algorithms and parameters to build reduced semantic spaces. The effect of dimension reduction, the stability of the representation and the effect of word order are examined in the context of the five algorithms bearing on semantic vectors: Random projection (RP), singular value decom- position (SVD), non-negative matrix factorization (NMF), permutations and holographic reduced representations (HRR). The quality of semantic representation was tested by means of synonym finding task using the TOEFL test on the TASA corpus. Dimension reduction was found to improve the quality of semantic representation but it is hard to find the optimal parameter settings. Even though dimension reduction by RP was found to be more generally applicable than SVD, the semantic vectors produced by RP are somewhat unstable. The effect of encoding word order into the semantic vector representation via HRR did not lead to any increase in scores over vectors constructed from word co-occurrence in context information. In this regard, very small context windows resulted in better semantic vectors for the TOEFL test.

Advanced Engineering of Lipid Metabolism in Nicotiana benthamiana Using a Draft Genome and the V2 Viral Silencing-Suppressor Protein

The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18:1 to elongation reactions beginning with 18:1 as substrate. These V2-based leaf assays produced ~50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays. © 2012 Naim et al.

Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants

A series of improved vectors have been constructed that are suitable for use in Agrobacterium tumefaciens-mediated monocot transformation. These binary vectors have several useful features, including the selectable marker genes bar (phosphinothricin resistance) or hph (hygromycin resistance) driven by either the cauliflower mosaic virus (CaMV) 35S promoter or the maize ubiquitin promoter, a high-copy-number replication origin that allows reliable mini-prep DNA isolation from Escherichia coli, and a polylinker sequence into which target genes can be easily inserted. A significant improvement has been made to the hph gene by the introduction of an intron into its coding region. The presence of the intron abolishes hph expression in A. tumefaciens, rendering the bacterium susceptible to the selective agent hygromycin B. The use of such an intron-hph vector thus enables the antibiotic in plant culture media to function as both a selective agent for transformed tissue and as a contraselective agent for A. tumefaciens growth, thus minimising the overgrowth of A. tumefaciens on plant tissues during transformation. Furthermore, the intron appears to be correctly spliced in plant cells and significantly enhances hph expression in transformed rice tissue. In our experiments, the use of the intron-hph vector increased the frequency of rice transformation and has enabled the production of transgenic barley.

Vectors and methods for hairpin RNA and artificial microRNA-mediated gene silencing in plants

In plant cells, DICER-LIKE4 processes perfectly double-stranded RNA (dsRNA) into short interfering (si) RNAs, and DICER-LIKE1 generates micro (mi) RNAs from primary miRNA transcripts (pri-miRNA) that form fold-back structures of imperfectly dsRNA. Both si and miRNAs direct the endogenous endonuclease, ARGONAUTE1 to cleave complementary target single-stranded RNAs and either small RNA (sRNA)-directed pathway can be harnessed to silence genes in plants. A routine way of inducing and directing RNA silencing by siRNAs is to express self-complementary single-stranded hairpin RNA (hpRNA), in which the duplexed region has the same sequence as part of the target gene's mRNA. Artificial miRNA (amiRNA)-mediated silencing uses an endogenous pri-miRNA, in which the original miRNA/miRNA* sequence has been replaced with a sequence complementary to the new target gene. In this chapter, we describe the plasmid vector systems routinely used by our research group for the generation of either hpRNA-derived siRNAs or amiRNAs.

High-throughput vectors for efficient gene silencing in plants

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs, pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.