Molecular Analysis of the shaking-b Locus of Drosophila melanogaster

One strategy for the isolation of molecules required for the establishment of specific synapses is to screen for mutations which disrupt identified neuronal connections and subsequently to clone and characterise the genes involved. The giant fibre system of Drosophila melanogaster is an ideal focus for such investigations. This system mediates the fly's jump-escape response, allowing neuronal connectivity mutants to be isolated as a subset of those flies which fail to jump in response to a light-off stimulus. The Passover mutation was isolated in this way, and was subsequently shown to be an allele of the shaking-B (shak-B) locus, thus implicating shaking-B in the establishment of neuronal connectivity, and inspiring the molecular analysis which is reported here. Genetic analysis reveals two distinct functions at the shaking-B locus, one (termed shak-B(neural)) is required for the normal development of the imaginal nervous system, while the other, shak-B(lethal) is an essential function, without which the animals die as embryos or first instar larvae. Most shaking-B alleles disrupt both of these genetic functions, although some (like shak-B passover) are specifically neural, while others (such as shak-B L41) affect only the essential function. The 19E3 polytene region in which shaking-B resides was cloned by chromosome walking from microcloned entry points and the breakpoints of deficiency chromosomes which encroach upon the shak-B region were used to define a 15 kb stretch of walk in which at least some of the gene must lie. Unique DNA fragments from this area were used to probe cDNA libraries and the embryonic cDNA KE2(1.8) was isolated. The KE2(1.8) cDNA was sequenced and found to contain no extensive regions of reading frame, though an internal 122 codon open reading frame (ORF) was implicated by computer algorithms as a likely coding region, and was found be highly homologous to the N terminus of the Drosophila Ogre protein and to part of the C. elegans Unc-7 protein, both of which are implicated in nervous system development. An asymmetric PCR strategy was used to sequence this small ORF from shak-B mutant chromosomes, and a 17 bp deletion which is predicted to abolish translation of the ORF was found to underlie the shak-BL41 allele. Due to the rarity of shak-B cDNAs, a library screening strategy based upon inverse PCR was devised. This technique enabled the isolation of cDNAs representing a further four shak-B transcript forms, while yet another two cDNAs were isolated by conventional means. Sequence analysis of these clones and of the genomic regions from which they were derived has provided a wealth of data regarding the putative products and genomic organisations of these transcripts. The SIPC8 cDNA contains an ORF of 372 residues, implying a protein of 44.4 kDa with extended homology to Ogre and Unc-7. In the neural and lethal shak-B alleles shak-B allelels and shak-B r-9-29 this reading frame was found to be disrupted by a mutation which introduces a stop codon in a downstream exon. This finding, together with the identification of the shak-BEC201 allele suggested that Shak-B(neural) and Shak'B(lethal) proteins have unique N terminal regions but converge upon common C terminal sequences. While the SIPC8 cDNA is disrupted by lesions causing lethal alleles, the P2.4 cDNA isolated by Krishnan and colleagues was found to contain an ORF with a unique N terminus, and a C terminus common to that of the SIPC8 reading frame. The unique N terminus of P2.4 was found (BCrishnan et aL, 1993) to be disrupted by lesions underlying shak-B(neural) mutations including shak-B passover go fulfilling the criteria demanded of a shak-B(neural) transcript. Shak-B proteins contain hydrophobic segments suggestive of transmembrane domains, and assessment of the likely transmembrane dispositions of all putative Shak-B proteins was carried out using optimal computer algorithms. Based on these structural predictions and on the phenotypes and expression patterns of shaking-B and its homologues, the possible functions of Shaking-B proteins are considered

Antibody Reactivity to Merozoite Antigens in Ghanaian Adults Correlates With Growth Inhibitory Activity Against Plasmodium falciparum in Culture.

Background: Plasmodium falciparum uses a repertoire of merozoite-stage proteins for invasion of erythrocytes. Antibodies against some of these proteins halt the replication cycle of the parasite by preventing erythrocyte invasion and are implicated as contributors to protective immunity against malaria. Methods: We assayed antibody reactivity against a panel of 9 recombinant antigens based on erythrocyte-binding antigen (EBA) and reticulocyte-like homolog (Rh) proteins in plasma from children with malaria and healthy adults residing in 3 endemic areas in Ghana using enzyme-linked immunosorbent assay. Purified immunoglobulin (Ig)G from adult plasma samples was also tested for invasion inhibition against 7 different P falciparum culture lines, including clinical isolates. Results: Antibodies against the antigens increased in an age-dependent manner in children. Breadth of reactivity to the different antigens was strongly associated with in vitro parasite growth inhibitory activity of IgG purified from the adults. The strongest predictors of breadth of antibody reactivity were age and transmission intensity, and a combination of reactivities to Rh2, Rh4, and Rh5 correlated strongly with invasion inhibition. Conclusions: Growth inhibitory activity was significantly associated with breadth of antibody reactivity to merozoite antigens, encouraging the prospect of a multicomponent blood-stage vaccine

Public values for energy system change

In this paper we discuss the importance of framing the question of public acceptance of sustainable energy transitions in terms of values and a ‘whole-system’ lens. This assertion is based on findings arising from a major research project examining public values, attitudes and acceptability with regards to whole energy system change using a mixed-method (six deliberative workshops, n=68, and a nationally representative survey, n = 2441), interdisciplinary approach. Through the research we identify a set of social values associated with desirable energy futures in the UK, where the values represent identifiable cultural resources people draw on to guide their preference formation about particular aspects of energy system change. As such, we characterise public perspectives as being underpinned by six value clusters relating to efficiency and wastefulness, environment and nature, security and stability, social justice and fairness, autonomy and power, and processes and change. We argue that this ‘value system’ provides a basis for understanding core reasons for public acceptance or rejection of different energy system aspects and processes. We conclude that a focus on values that underpin more specific preferences for energy system change brings insights that could provide a basis for improved dialogue, more robust decision- making, and for anticipating likely points of conflict in energy transitions

A defined mechanistic correlate of protection against Plasmodium falciparum malaria in non-human primates.

Malaria vaccine design and prioritization has been hindered by the lack of a mechanistic correlate of protection. We previously demonstrated a strong association between protection and merozoite-neutralizing antibody responses following vaccination of non-human primates against Plasmodium falciparum reticulocyte binding protein homolog 5 (PfRH5). Here, we test the mechanism of protection. Using mutant human IgG1 Fc regions engineered not to engage complement or FcR-dependent effector mechanisms, we produce merozoite-neutralizing and non-neutralizing anti-PfRH5 chimeric monoclonal antibodies (mAbs) and perform a passive transfer-P. falciparum challenge study in Aotus nancymaae monkeys. At the highest dose tested, 6/6 animals given the neutralizing PfRH5-binding mAb c2AC7 survive the challenge without treatment, compared to 0/6 animals given non-neutralizing PfRH5-binding mAb c4BA7 and 0/6 animals given an isotype control mAb. Our results address the controversy regarding whether merozoite-neutralizing antibody can cause protection against P. falciparum blood-stage infections, and highlight the quantitative challenge of achieving such protection

Molecular Mechanisms of Microcystin Toxicity in Animal Cells

Microcystins (MC) are potent hepatotoxins produced by the cyanobacteria of the genera Planktothrix, Microcystis, Aphanizomenon, Nostoc and Anabaena. These cyclic heptapeptides have strong affinity to serine/threonine protein phosphatases (PPs) thereby acting as an inhibitor of this group of enzymes. Through this interaction a cascade of events responsible for the MC cytotoxic and genotoxic effects in animal cells may take place. Moreover MC induces oxidative stress in animal cells and together with the inhibition of PPs, this pathway is considered to be one of the main mechanisms of MC toxicity. In recent years new insights on the key enzymes involved in the signal-transduction and toxicity have been reported demonstrating the complexity of the interaction of these toxins with animal cells. Key proteins involved in MC up-take, biotransformation and excretion have been identified, demonstrating the ability of aquatic animals to metabolize and excrete the toxin. MC have shown to interact with the mitochondria. The consequences are the dysfunction of the organelle, induction of reactive oxygen species (ROS) and cell apoptosis. MC activity leads to the differential expression/activity of transcriptional factors and protein kinases involved in the pathways of cellular differentiation, proliferation and tumor promotion activity. This activity may result from the direct inhibition of the protein phosphatases PP1 and PP2A. This review aims to summarize the increasing data regarding the molecular mechanisms of MC toxicity in animal systems, reporting for direct MC interacting proteins and key enzymes in the process of toxicity biotransformation/excretion of these cyclic peptides

Mutational heterogeneity in cancer and the search for new cancer genes

Major international projects are now underway aimed at creating a comprehensive catalog of all genes responsible for the initiation and progression of cancer. These studies involve sequencing of matched tumor–normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here, we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false positive findings that overshadow true driver events. Here, we show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumor-normal pairs and discover extraordinary variation in (i) mutation frequency and spectrum within cancer types, which shed light on mutational processes and disease etiology, and (ii) mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and allow true cancer genes to rise to attention

HIV Antigen Incorporation within Adenovirus Hexon Hypervariable 2 for a Novel HIV Vaccine Approach

Adenoviral (Ad) vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. However, in some cases these conventional Ad-based vaccines have had sub-optimal clinical results. These sub-optimal results are attributed in part to pre-existing Ad serotype 5 (Ad5) immunity. In order to circumvent the need for antigen expression via transgene incorporation, the “antigen capsid-incorporation” strategy has been developed and used for Ad-based vaccine development in the context of a few diseases. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The major capsid protein hexon has been utilized for these capsid incorporation strategies due to hexon's natural role in the generation of anti-Ad immune response and its numerical representation within the Ad virion. Using this strategy, we have developed the means to incorporate heterologous peptide epitopes specifically within the major surface-exposed domains of the Ad capsid protein hexon. Our study herein focuses on generation of multivalent vaccine vectors presenting HIV antigens within the Ad capsid protein hexon, as well as expressing an HIV antigen as a transgene. These novel vectors utilize HVR2 as an incorporation site for a twenty-four amino acid region of the HIV membrane proximal ectodomain region (MPER), derived from HIV glycoprotein gp41 (gp41). Our study herein illustrates that our multivalent anti-HIV vectors elicit a cellular anti-HIV response. Furthermore, vaccinations with these vectors, which present HIV antigens at HVR2, elicit a HIV epitope-specific humoral immune response

Functional Memory B Cells and Long-Lived Plasma Cells Are Generated after a Single Plasmodium chabaudi Infection in Mice

Antibodies have long been shown to play a critical role in naturally acquired immunity to malaria, but it has been suggested that Plasmodium-specific antibodies in humans may not be long lived. The cellular mechanisms underlying B cell and antibody responses are difficult to study in human infections; therefore, we have investigated the kinetics, duration and characteristics of the Plasmodium-specific memory B cell response in an infection of P. chabaudi in mice. Memory B cells and plasma cells specific for the C-terminal region of Merozoite Surface Protein 1 were detectable for more than eight months following primary infection. Furthermore, a classical memory response comprised predominantly of the T-cell dependent isotypes IgG2c, IgG2b and IgG1 was elicited upon rechallenge with the homologous parasite, confirming the generation of functional memory B cells. Using cyclophosphamide treatment to discriminate between long-lived and short-lived plasma cells, we demonstrated long-lived cells secreting Plasmodium-specific IgG in both bone marrow and in spleens of infected mice. The presence of these long-lived cells was independent of the presence of chronic infection, as removal of parasites with anti-malarial drugs had no impact on their numbers. Thus, in this model of malaria, both functional Plasmodium-specific memory B cells and long-lived plasma cells can be generated, suggesting that defects in generating these cell populations may not be the reason for generating short-lived antibody responses

Systems Biology Approach Predicts Antibody Signature Associated with Brucella melitensis Infection in Humans

A complete understanding of the factors that determine selection of antigens recognized by the humoral immune response following infectious agent challenge is lacking. Here we illustrate a systems biology approach to identify the antibody signature associated with Brucella melitensis (Bm) infection in humans and predict proteomic features of serodiagnostic antigens. By taking advantage of a full proteome microarray expressing previously cloned 1406 and newly cloned 1640 Bm genes, we were able to identify 122 immunodominant antigens and 33 serodiagnostic antigens. The reactive antigens were then classified according to annotated functional features (COGs), computationally predicted features (e.g., subcellular localization, physical properties), and protein expression estimated by mass spectrometry (MS). Enrichment analyses indicated that membrane association and secretion were significant enriching features of the reactive antigens, as were proteins predicted to have a signal peptide, a single transmembrane domain, and outer membrane or periplasmic location. These features accounted for 67% of the serodiagnostic antigens. An overlay of the seroreactive antigen set with proteomic data sets generated by MS identified an additional 24%, suggesting that protein expression in bacteria is an additional determinant in the induction of Brucella-specific antibodies. This analysis indicates that one-third of the proteome contains enriching features that account for 91% of the antigens recognized, and after B. melitensis infection the immune system develops significant antibody titers against 10% of the proteins with these enriching features. This systems biology approach provides an empirical basis for understanding the breadth and specificity of the immune response to B. melitensis and a new framework for comparing the humoral responses against other microorganisms