Targeted and shielded adenovectors for cancer therapy

Conditionally replicative adenovirus (CRAd) vectors are novel vectors with utility as virotherapy agents for alternative cancer therapies. These vectors have already established a broad safety record in humans and overcome some of the limitations of non-replicative adenovirus (Ad) vectors. In addition, one potential problem with these vectors, attainment of tumor or tissue selectivity has widely been addressed. However, two confounding problems limiting efficacy of these drug candidates remains. The paucity of the native Ad receptor on tumor tissues, and host humoral response due to pre-existing titers of neutralizing antibodies against the vector itself in humans have been highlighted in the clinical context. The well-characterized CRAd, AdΔ24-RGD, is infectivity enhanced, thus overcoming the lack of coxsackievirus and adenovirus receptor (CAR), and this agent is already rapidly progressing towards clinical translation. However, the perceived host humoral response potentially will limit gains seen from the infectivity enhancement and therefore a strategy to blunt immunity against the vector is required. On the basis of this caveat a novel strategy, termed shielding, has been developed in which the genetic modification of a virion capsid protein would provide uniformly shielded Ad vectors. The identification of the pIX capsid protein as an ideal locale for genetic incorporation of shielding ligands to conceal the Ad vector from pre-existing neutralizing antibodies is a major progression in the development of shielded CRAds. Preliminary data utilizing an Ad vector with HSV-TK fused to the pIX protein indicates that a shield against neutralizing antibodies can be achieved. The utility of various proteins as shielding molecules is currently being addressed. The creation of AdΔ24S-RGD, an infectivity enhanced and shielded Ad vector will provide the next step in the development of clinically and commercially feasible CRAds that can be dosed multiple times for maximum effectiveness in the fight against cancers in humans.This article is a symposium paper from the Annual Meeting of the "International Society for Cell and Gene Therapy of Cancer", held in Shenzhen, China, on 9–11 December 2005.     

Codon catalog usage and the genome hypothesis.

Frequencies for each of the 61 amino acid codons have been determined in every published mRNA sequence of 50 or more codons. The frequencies are shown for each kind of genome and for each individual gene. A surprising consistency of choices exists among genes of the same or similar genomes. Thus each genome, or kind of genome, appears to possess a "system" for choosing between codons. Frameshift genes, however, have widely different choice strategies from normal genes. Our work indicates that the main factors distinguishing between mRNA sequences relate to choices among degenerate bases. These systematic third base choices can therefore be used to establish a new kind of genetic distance, which reflects differences in coding strategy. The choice patterns we find seem compatible with the idea that the genome and not the individual gene is the unit of selection. Each gene in a genome tends to conform to its species' usage of the codon catalog; this is our genome hypothesis.     

Mutations in Calmodulin Cause Ventricular Tachycardia and Sudden Cardiac Death

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a devastating inherited disorder characterized by episodic syncope and/or sudden cardiac arrest during exercise or acute emotion in individuals without structural cardiac abnormalities. Although rare, CPVT is suspected to cause a substantial part of sudden cardiac deaths in young individuals. Mutations in RYR2, encoding the cardiac sarcoplasmic calcium channel, have been identified as causative in approximately half of all dominantly inherited CPVT cases. Applying a genome-wide linkage analysis in a large Swedish family with a severe dominantly inherited form of CPVT-like arrhythmias, we mapped the disease locus to chromosome 14q31-32. Sequencing CALM1 encoding calmodulin revealed a heterozygous missense mutation (c.161A>T [p.Asn53Ile]) segregating with the disease. A second, de novo, missense mutation (c.293A>G [p.Asn97Ser]) was subsequently identified in an individual of Iraqi origin; this individual was diagnosed with CPVT from a screening of 61 arrhythmia samples with no identified RYR2 mutations. Both CALM1 substitutions demonstrated compromised calcium binding, and p.Asn97Ser displayed an aberrant interaction with the RYR2 calmodulin-binding-domain peptide at low calcium concentrations. We conclude that calmodulin mutations can cause severe cardiac arrhythmia and that the calmodulin genes are candidates for genetic screening of individual cases and families with idiopathic ventricular tachycardia and unexplained sudden cardiac death.     

Controlling the cortical actin motor

Actin is the essential force-generating component of the microfilament system, which powers numerous motile processes in eukaryotic cells and undergoes dynamic remodeling in response to different internal and external signaling. The ability of actin to polymerize into asymmetric filaments is the inherent property behind the site-directed force-generating capacity that operates during various intracellular movements and in surface protrusions. Not surprisingly, a broad variety of signaling pathways and components are involved in controlling and coordinating the activities of the actin microfilament system in a myriad of different interactions. The characterization of these processes has stimulated cell biologists for decades and has, as a consequence, resulted in a huge body of data. The purpose here is to present a cellular perspective on recent advances in our understanding of the microfilament system with respect to actin polymerization, filament structure and specific folding requirements.     

Comparison of transcript profiles in different life stages of the nematode Globodera pallida under different host potato genotypes

The potato cyst nematodes (PCNs) Globodera pallida and Globodera rostochiensis are important parasites of potato. PCNs undergo complex biotrophic interactions with their hosts that involve gene expression changes in both the nematode and the host plant. The aim of this study was to determine key genes that are differentially expressed in Globodera pallida life cycle stages and during the initiation of the feeding site in susceptible and partially resistant potato genotypes. For this purpose, two microarray experiments were designed: (i) a comparison of eggs, infective second‐stage juveniles (J2s) and sedentary parasitic‐stage J2s (SJ2); (ii) a comparison of SJ2s at 8 days after inoculation (DAI) in the susceptible cultivar (Desirée) and two partially resistant lines. The results showed differential expression of G. pallida genes during the stages studied, including previously characterized effectors. In addition, a large number of genes changed their expression between SJ2s in the susceptible cultivar and those infecting partially resistant lines; the number of genes with modified expression was lower when the two partially resistant lines were compared. Moreover, a histopathological study was performed at several time points (7, 14 and 30 DAI) and showed the similarities between both partially resistant lines with a delay and degeneration in the formation of the syncytia in comparison with the susceptible cultivar. Females at 30 DAI in partially resistant lines showed a delay in their development in comparison with those in the susceptible cultivar.     

Fc Gamma Receptor IIIB (FcγRIIIB) Polymorphisms Are Associated with Clinical Malaria in Ghanaian Children

Plasmodium falciparum malaria kills nearly a million people annually. Over 90% of these deaths occur in children under five years of age in sub-Saharan Africa. A neutrophil mediated mechanism, the antibody dependent respiratory burst (ADRB), was recently shown to correlate with protection from clinical malaria. Human neutrophils constitutively express Fc gamma receptor-FcγRIIA and FcγRIIIB by which they interact with immunoglobulin (Ig) G (IgG)-subclass antibodies. Polymorphisms in exon 4 of FCGR2A and exon 3 of FCGR3B genes encoding FcγRIIA and FcγRIIIB respectively have been described to alter the affinities of both receptors for IgG. Here, associations between specific polymorphisms, encoding FcγRIIA p.H166R and FcγRIIIB-NA1/NA2/SH variants with clinical malaria were investigated in a longitudinal malaria cohort study. FcγRIIA-p.166H/R was genotyped by gene specific polymerase chain reaction followed by allele specific restriction enzyme digestion. FCGR3B-exon 3 was sequenced in 585 children, aged 1 to 12 years living in a malaria endemic region of Ghana. Multivariate logistic regression analysis found no association between FcγRIIA-166H/R polymorphism and clinical malaria. The A-allele of FCGR3B-c.233C>A (rs5030738) was significantly associated with protection from clinical malaria under two out of three genetic models (additive: p = 0.0061; recessive: p = 0.097; dominant: p = 0.0076) of inheritance. The FcγRIIIB-SH allotype (CTGAAA) containing the 233A-allele (in bold) was associated with protection from malaria (p = 0.049). The FcγRIIIB-NA2*03 allotype (CTGCGA), a variant of the classical FcγRIIIB-NA2 (CTGCAA) was associated with susceptibility to clinical malaria (p = 0.0092). The present study is the first to report an association between a variant of FcγRIIIB-NA2 and susceptibility to clinical malaria and provides justification for further functional characterization of variants of the classical FcγRIIIB allotypes. This would be crucial to the improvement of neutrophil mediated functional assays such as the ADRB assay aimed at assessing the functionality of antibodies induced by candidate malaria vaccines.     

The surf zone: a semi-permeable barrier to onshore recruitment of invertebrate larvae?

The supply of larvae to the shore is important for population replenishment and intertidal community dynamics but its variability at most scales is not well understood. We tested the relationship between nearshore mussel larval abundance and intertidal settlement rates over several years at multiple spatiotemporal scales in Oregon and New Zealand. Abundance of competent larvae nearshore and intertidal recruitment rates were simultaneously quantified using collectors mounted at different depths on moorings 50-1100 m from shore, and at adjacent rocky intertidal sites. Total mussel larval abundance and oceanographic conditions were also measured in some locations. At all scales, abundance of nearshore mussel larvae was unrelated to intertidal recruitment rates. In the intertidal, patterns of mussel recruitment were persistent in space, with sites of consistently high or low recruitment. In contrast, nearshore competent larval abundance showed generally similar abundances among sites except for a high, and spatially-inconsistent, variability in Oregon during 1998 only. On moorings, recruitment tended to be greater on midwater collectors than shallower or deeper. Finally, on moorings larval abundance in traps and recruitment on collectors was unrelated. These results suggest that (1) among sites, the size of the nearshore larval pool is relatively uniform while onshore recruitment varies and is unrelated to larval abundance, (2) temporal variability in nearshore larval availability is not strongly expressed onshore, (3) vertical stratification of competent larvae nearshore is strong and may influence transport and recruitment, and (4) within-coast variability in onshore recruitment is strongly driven by processes occurring locally within the surf zone that need to be studied to understand coastal recruitment dynamics.     

Is conservation triage just smart decision making?

Conservation efforts and emergency medicine face comparable problems: how to use scarce resources wisely to conserve valuable assets. In both fields, the process of prioritising actions is known as triage. Although often used implicitly by conservation managers, scientists and policymakers, triage has been misinterpreted as the process of simply deciding which assets (e.g. species, habitats) will not receive investment. As a consequence, triage is sometimes associated with a defeatist conservation ethic. However, triage is no more than the efficient allocation of conservation resources and we risk wasting scarce resources if we do not follow its basic principles.     

A comparison of sequence-based polymorphism and haplotype content in transcribed and anonymous regions of the barley genome.

Direct estimates of sequence diversity provides an abundant source of DNA polymorphisms based on single nucleotide polymorphisms (SNPs). The frequency and distribution of nucleotide diversity within 23 genes associated with grain germination in barley were determined in a sample of accessions representing European cultivars, landraces, and wild barley accessions from throughout the fertile crescent. The overall nucleotide diversity ranged from 0.0021 to 0.0189 with a single nucleotide change being detected every 78 bp and insertion-deletion events being observed every 680 bp. Within the cultivated (H. vulgare) genepool, a small number of haplotypes were detected, the total number of haplotypes observed in H. spontaneum was almost double that detected in H. vulgare (46 and 26, respectively). Distinct haplotypes were observed in the H. spontaneum and landrace genepools, which are highly divergent from H. vulgare. A comparison of SNP-based haplotype data with EST-derived SSRs and genomic SSRs revealed a similar trend of decreasing variability in the cultivated genepool. However, the number of unique alleles identified in the cultivated sample was much greater with genomic SSRs (18%) compared with only 2.1% for SNPs and 3.8% for EST-derived SSRs. The potential utility of SNPs and EST-derived SSRs for association mapping in barley is discussed.     

Estimating seasonal and annual carbon inputs,and root decomposition rates in a temperate pasture following field 14C pulse-labelling

Using a ¹⁴C pulse-labelling technique, we studied the seasonal changes in assimilation and partitioning of photoassimilated C in the plant–root–soil components of a temperate pasture. Pasture and soil samples were taken after 4-h, and 35-day chase periods, to examine these seasonal ¹⁴C fluxes. Total C and ¹⁴C were determined in the shoot, root and soil system. The amounts of C translocated annually to roots and soil were also estimated from the seasonal ¹⁴C distribution and pasture growth. The in situ field decomposition of newly formed roots during different seasons, also using ¹⁴C-labelling, was studied for one year in undisturbed rhizosphere soil. The ¹⁴C-labelled roots were sampled five times and decomposition rates were calculated assuming first-order decomposition.Annual pasture production at the site was 16 020 kg DM ha^–1, and pasture growth varied with season being highest (75–79 kg ha^–1 d^–1) in spring and lowest (18–20 kg ha^–1 d^–1) in winter. The above- and below-ground partitioning of ¹⁴C also varied with the season. The respiratory ¹⁴C–CO₂ losses, calculated as the difference between the total amounts of ¹⁴C recovered in the soil-plant system at 4 h and 35 days, were high (66–70%) during the summer, autumn and winter season, and low (37–39%) during the spring and late-spring season. Pasture plants partitioned more C below-ground during spring compared with summer, autumn and winter seasons. Overall, at this high fertility dairy pasture site, 18 220 kg C/ha was respired, 6490 kg remained above-ground in the shoot, and 6820 kg was translocated to roots and 1320 kg to soil. Root decomposition rate constant (k) differed widely with the season and were the highest for the autumn roots. The half-life was highest (111 days) for autumn roots and lowest (64 days) for spring roots. About one-third of the root label measured in the spring season disappeared in the first 5 weeks after the initial 35 Day of allocation period. The late spring, summer, late summer and winter roots had intermediate half-lives (88–94 days). These results indicate that seasonal changes in root growth and decomposition should be accounted for to give a better quantification of root turnover.