Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles

HIV-1 Pr55 Gag virus-like particles (VLPs) are strong immunogens with potential as candidate HIV vaccines. VLP immunogenicity can be broadened by making chimaeric Gag molecules: however, VLPs incorporating polypeptides longer than 200 aa fused in frame with Gag have not yet been reported. We constructed a range of gag-derived genes encoding in-frame C-terminal fusions of myristoylation-competent native Pr55Gag and p6-truncated Gag (Pr50Gag) to test the effects of polypeptide length and sequence on VLP formation and morphology, in an insect cell expression system. Fused sequences included a modified reverse transcriptase-Tat-Nef fusion polypeptide (RTTN, 778 aa), and truncated versions of RTTN ranging from 113 aa to 450 aa. Baculovirus-expressed chimaeric proteins were examined by western blot and electron microscopy. All chimaeras formed VLPs which could be purified by sucrose gradient centrifugation. VLP diameter increased with protein MW, from ∼100 nm for Pr55Gag to ∼250 nm for GagRTTN. The presence or absence of the Gag p6 region did not obviously affect VLP formation or appearance. GagRT chimaeric particles were successfully used in mice to boost T-cell responses to Gag and RT that were elicited by a DNA vaccine encoding a GagRTTN polypeptide, indicating the potential of such chimaeras to be used as candidate HIV vaccines. © 2008 Elsevier B.V. All rights reserved.

Inhibition of maize streak virus (MSV) replication by transient and transgenic expression of MSV replication-associated protein mutants

Maize streak disease is a severe agricultural problem in Africa and the development of maize genotypes resistant to the causal agent, Maize streak virus (MSV), is a priority. A transgenic approach to engineering MSV-resistant maize was developed and tested in this study. A pathogen-derived resistance strategy was adopted by using targeted deletions and nucleotide-substitution mutants of the multifunctional MSV replication-associated protein gene (rep). Various rep gene constructs were tested for their efficacy in limiting replication of wild-type MSV by co-bombardment of maize suspension cells together with an infectious genomic clone of MSV and assaying replicative forms of DNA by quantitative PCR. Digitaria sanguinalis, an MSV-sensitive grass species used as a model monocot, was then transformed with constructs that had inhibited virus replication in the transient-expression system. Challenge experiments using leafhopper-transmitted MSV indicated significant MSV resistance - from highly resistant to immune - in regenerated transgenic D. sanguinalis lines. Whereas regenerated lines containing a mutated full-length rep gene displayed developmental and growth defects, those containing a truncated rep gene both were fertile and displayed no growth defects, making the truncated gene a suitable candidate for the development of transgenic MSV-resistant maize. © 2007 SGM.

Viable chimaeric viruses confirm the biological importance of sequence specific maize streak virus movement protein and coat protein interactions

Background. A variety of interactions between up to three different movement proteins (MPs), the coat protein (CP) and genomic DNA mediate the inter- and intra-cellular movement of geminiviruses in the genus Begomovirus. Although movement of viruses in the genus Mastrevirus is less well characterized, direct interactions between a single MP and the CP of these viruses is also clearly involved in both intra- and intercellular trafficking of virus genomic DNA. However, it is currently unknown how specific these MP-CP interactions are, nor how disruption of these interactions might impact on virus viability. Results. Using chimaeric genomes of two strains of Maize streak virus (MSV) we adopted a genetic approach to investigate the gross biological effects of interfering with interactions between virus MP and CP homologues derived from genetically distinct MSV isolates. MP and CP genes were reciprocally exchanged, individually and in pairs, between maize (MSV-Kom)- and Setaria sp. (MSV-Set)-adapted isolates sharing 78% genome-wide sequence identity. All chimaeras were infectious in Zea mays c.v. Jubilee and were characterized in terms of symptomatology and infection efficiency. Compared with their parental viruses, all the chimaeras were attenuated in symptom severity, infection efficiency, and the rate at which symptoms appeared. The exchange of individual MP and CP genes resulted in lower infection efficiency and reduced symptom severity in comparison with exchanges of matched MP-CP pairs. Conclusion. Specific interactions between the mastrevirus MP and CP genes themselves and/or their expression products are important determinants of infection efficiency, rate of symptom development and symptom severity. © 2008 van der Walt et al; licensee BioMed Central Ltd.

Maize streak virus: An old and complex 'emerging' pathogen

Maize streak virus (MSV; Genus Mastrevirus, Family Geminiviridae) occurs throughout Africa, where it causes what is probably the most serious viral crop disease on the continent. It is obligately transmitted by as many as six leafhopper species in the Genus Cicadulina, but mainly by C. mbila Naudé and C. storeyi. In addition to maize, it can infect over 80 other species in the Family Poaceae. Whereas 11 strains of MSV are currently known, only the MSV-A strain is known to cause economically significant streak disease in maize. Severe maize streak disease (MSD) manifests as pronounced, continuous parallel chlorotic streaks on leaves, with severe stunting of the affected plant and, usuallly, a failure to produce complete cobs or seed. Natural resistance to MSV in maize, and/or maize infections caused by non-maize-adapted MSV strains, can result in narrow, interrupted streaks and no obvious yield losses. MSV epidemiology is primarily governed by environmental influences on its vector species, resulting in erratic epidemics every 3-10 years. Even in epidemic years, disease incidences can vary from a few infected plants per field, with little associated yield loss, to 100% infection rates and complete yield loss. Taxonomy: The only virus species known to cause MSD is MSV, the type member of the Genus Mastrevirus in the Family Geminiviridae. In addition to the MSV-A strain, which causes the most severe form of streak disease in maize, 10 other MSV strains (MSV-B to MSV-K) are known to infect barley, wheat, oats, rye, sugarcane, millet and many wild, mostly annual, grass species. Seven other mastrevirus species, many with host and geographical ranges partially overlapping those of MSV, appear to infect primarily perennial grasses. Physical properties: MSV and all related grass mastreviruses have single-component, circular, single-stranded DNA genomes of approximately 2700 bases, encapsidated in 22 × 38-nm geminate particles comprising two incomplete T = 1 icosahedra, with 22 pentameric capsomers composed of a single 32-kDa capsid protein. Particles are generally stable in buffers of pH 4-8. Disease symptoms: In infected maize plants, streak disease initially manifests as minute, pale, circular spots on the lowest exposed portion of the youngest leaves. The only leaves that develop symptoms are those formed after infection, with older leaves remaining healthy. As the disease progresses, newer leaves emerge containing streaks up to several millimetres in length along the leaf veins, with primary veins being less affected than secondary or tertiary veins. The streaks are often fused laterally, appearing as narrow, broken, chlorotic stripes, which may extend over the entire length of severely affected leaves. Lesion colour generally varies from white to yellow, with some virus strains causing red pigmentation on maize leaves and abnormal shoot and flower bunching in grasses. Reduced photosynthesis and increased respiration usually lead to a reduction in leaf length and plant height; thus, maize plants infected at an early stage become severely stunted, producing undersized, misshapen cobs or giving no yield at all. Yield loss in susceptible maize is directly related to the time of infection: Infected seedlings produce no yield or are killed, whereas plants infected at later times are proportionately less affected. Disease control: Disease avoidance can be practised by only planting maize during the early season when viral inoculum loads are lowest. Leafhopper vectors can also be controlled with insecticides such as carbofuran. However, the development and use of streak-resistant cultivars is probably the most effective and economically viable means of preventing streak epidemics. Naturally occurring tolerance to MSV (meaning that, although plants become systemically infected, they do not suffer serious yield losses) has been found, which has primarily been attributed to a single gene, msv-1. However, other MSV resistance genes also exist and improved resistance has been achieved by concentrating these within individual maiz genotypes. Whereas true MSV immunity (meaning that plants cannot be symptomatically infected by the virus) has been achieved in lines that include multiple small-effect resistance genes together with msv-1, it has proven difficult to transfer this immunity into commercial maize genotypes. An alternative resistance strategy using genetic engineering is currently being investigated in South Africa. Useful websites: 〈http://www.mcb.uct.ac.za/MSV/mastrevirus.htm〉; 〈http://www. danforthcenter.org/iltab/geminiviridae/geminiaccess/mastrevirus/Mastrevirus. htm〉. © 2009 Blackwell Publishing Ltd.

The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine

Background. One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1) and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1), an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. Results. The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap) alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. Conclusions. We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used. © 2011 Tanzer et al; licensee BioMed Central Ltd.

Generation and characterisation of Cisplatin-resistant non-small cell lung cancer cell lines displaying a stem-like signature

Introduction: Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin. Methods: An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed. Results: Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines. Conclusion: Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer. © 2013 Barr et al.

An influenza virus inspired polymer system for the timed release of siRNA

Small interfering RNA silences specific genes by interfering with mRNA translation, and acts to modulate or inhibit specific biological pathways; a therapy that holds great promise in the cure of many diseases. However, the naked small interfering RNA is susceptible to degradation by plasma and tissue nucleases and due to its negative charge unable to cross the cell membrane. Here we report a new polymer carrier designed to mimic the influenza virus escape mechanism from the endosome, followed by a timed release of the small interfering RNA in the cytosol through a self-catalyzed polymer degradation process. Our polymer changes to a negatively charged and non-toxic polymer after the release of small interfering RNA, presenting potential for multiple repeat doses and long-term treatment of diseases.

Higher harmonic large-amplitude fourier transformed alternating current voltammetry : analytical attributes derived from studies of the oxidation of ferrocenemethanol and uric acid at a glassy carbon electrode

An analytical evaluation of the higher ac harmonic components derived from large amplitude Fourier transformed voltammetry is provided for the reversible oxidation of ferrocenemethanol (FcMeOH) and oxidation of uric acid by an EEC mechanism in a pH 7.4 phosphate buffer at a glassy carbon (GC) electrode. The small background current in the analytically optimal fifth harmonic is predominantly attributed to faradaic current associated with the presence of electroactive functional groups on the GC electrode surface, rather than to capacitive current which dominates the background in the dc, and the initial three ac harmonics. The detection limits for the dc and the first to fifth harmonic ac components are 1.9, 5.89, 2.1, 2.5, 0.8, and 0.5 µM for FcMeOH, respectively, using a sine wave modulation of 100 mV at 21.46 Hz and a dc sweep rate of 111.76 mV s−1. Analytical performance then progressively deteriorates in the sixth and higher harmonics. For the determination of uric acid, the capacitive background current was enhanced and the reproducibility lowered by the presence of surface active uric acid, but the rapid overall 2e− rather than 1e– electron transfer process gives rise to a significantly enhanced fifth harmonic faradaic current which enabled a detection limit of 0.3 µM to be achieved which is similar to that reported using chemically modified electrodes. Resolution of overlapping voltammetric signals for a mixture of uric acid and dopamine is also achieved using higher fourth or fifth harmonic components, under very low background current conditions. The use of higher fourth and fifth harmonics exhibiting highly favorable faradaic to background (noise) current ratios should therefore be considered in analytical applications under circumstances where the electron transfer rate is fast.

Angiogenesis and non-small cell lung cancer

Lung cancer is the commonest cause of cancer death in the western world. Recent evidence suggests that angiogenesis is related to poor prognosis in many solid tumours including non-small cell lung cancer. Angiogenesis is controlled by a complex interaction between growth and apoptotic factors, proteases and adhesion molecules. The angiogenic process may prove a target for novel therapies such as matrix metalloproteinase inhibitors, growth factor antisense RNA, growth factor receptor antagonists and naturally occurring antiangiogenic peptides. These agents may be used alone or in combination with traditional chemotherapy, radiotherapy and surgery. (C) 2000 Elsevier Science Ireland Ltd.

Platelet-derived endothelial cell growth factor (thymidine phosphorylase) expression in lung cancer

Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is an enzyme with angiogenic and cell motility properties. Moreover, it is involved in the transformation of fluoropyrimidines into active cytotoxic metabolites, In the present study, the expression of PD-ECGF in normal lung and lung cancer was immunohistochemically evaluated using the P-GF.44C monoclonal antibody. Alveolar and tumoural macrophages mere invariably stained and mere used as an internal control for assessment of the staining. Alveolar epithelium was always negative, whilst bronchiolar epithelium showed occasional positive reactivity. Normal lung and tumour endothelium was occasionally positive, Positive staining in more than 50 per cent of cells was observed in 23/71 squamous carcinomas (32 per cent), 16/38 (42 per cent) adenocarcinomas, and 2/6 (33 per cent) adenosquamous carcinomas. Differentiated areas and areas of squamous metaplasia mere more strongly positive than other tumour areas. All 22 small cell carcinomas and one carcinoid tumour were negative. The present study provides a baseline for future studies in non-small cell lung cancer to correlate PD-ECGF expression with tumour vascularization, prognosis, and response to chemotherapy.

Epigenetic regulation of glucose transporters in non-small cell lung cancer

Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP to provide for their energy needs, known as the Warburg effect. At the same time, overexpression of the insulin receptor in non-small cell lung cancer (NSCLC) is associated with an increased risk of metastasis and decreased survival. The uptake of glucose into cells is carried out via glucose transporters or GLUTs. Of these, GLUT-4 is essential for insulin-stimulated glucose uptake. Following treatment with the epigenetic targeting agents histone deacetylase inhibitors (HDACi), GLUT-3 and GLUT-4 expression were found to be induced in NSCLC cell lines, with minimal responses in transformed normal human bronchial epithelial cells (HBECs). Similar results for GLUT-4 were observed in cells derived from liver, muscle, kidney and pre-adipocytes. Bioinformatic analysis of the promoter for GLUT-4 indicates that it may also be regulated by several chromatin binding factors or complexes including CTCF, SP1 and SMYD3. Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT-4 is dynamically remodeled in response to HDACi. Overall, these results may have value within the clinical setting as (a) it may be possible to use this to enhance fluorodeoxyglucose (18F) positron emission tomography (FDG-PET) imaging sensitivity; (b) it may be possible to target NSCLC through the use of HDACi and insulin mediated uptake of the metabolic targeting drugs such as 2-deoxyglucose (2-DG); or (c) enhance or sensitize NSCLC to chemotherapy. © 2011 by the authors; licensee MDPI, Basel, Switzerland.

An extracellular signal-regulated kinase 2 survival pathway mediates resistance of human mesothelioma cells to asbestos-induced injury

We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR) - linked survival pathways (phosphoinositol-3-kinase/AKT/ mammalian target of rapamycin and extracellular signal - regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death - related gene expression. Our results suggest that EGFR phosphorylation is causally linkedto pERK and pAKT activation by asbestos in normal and SV40 Tag - immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.

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.

Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA

Many examples of extreme virus resistance and posttranscriptional gene silencing of endogenous or reporter genes have been described in transgenic plants containing sense or antisense transgenes. In these cases of either cosuppression or antisense suppression, there appears to be induction of a surveillance system within the plant that specifically degrades both the transgene and target RNAs. We show that transforming plants with virus or reporter gene constructs that produce RNAs capable of duplex formation confer virus immunity or gene silencing on the plants. This was accomplished by using transcripts from one sense gene and one antisense gene colocated in the plant genome, a single transcript that has self-complementarity, or sense and antisense transcripts from genes brought together by crossing. A model is presented that is consistent with our data and those of other workers, describing the processes of induction and execution of posttranscriptional gene silencing.

Sequence and organization of barley yellow dwarf virus genomic RNA

The nucleotide sequence of the genomic RNA of barley yellow dwarf virus, PAV serotype was determined except for the 5′-terminal base, and its genome organization deduced. The 5,677 nucleotide genome contains five large open reading frames (ORFs). The genes for the coat protein (1) and the putative viral RNA-dependent RNA polymerase were identified. The latter shows a striking degree of similarity to that of carnation mottle virus (CarMV). By comparison with corona- and retrovirus RNAs, it is proposed that a translational frameshift is involved in expression of the polymerase. An ORF encoding an Mr 49,797 protein (50K ORF) may be translated by in-frame readthrough of the coat protein stop codon. The coat protein, an overlapping 17K ORF, and a 3′ 6.7K ORF are likely to be expressed via subgenomic mRNAs. © 1988 IRL Press Limited.