Towards a molecular characterization of autism spectrum disorders: an exome sequencing and systems approach.

The hypothetical 'AXAS' gene network model that profiles functional patterns of heterogeneous DNA variants overrepresented in autism spectrum disorder (ASD), X-linked intellectual disability, attention deficit and hyperactivity disorder and schizophrenia was used in this current study to analyze whole exome sequencing data from an Australian ASD cohort. An optimized DNA variant filtering pipeline was used to identify loss-of-function DNA variations. Inherited variants from parents with a broader autism phenotype and de novo variants were found to be significantly associated with ASD. Gene ontology analysis revealed that putative rare causal variants cluster in key neurobiological processes and are overrepresented in functions involving neuronal development, signal transduction and synapse development including the neurexin trans-synaptic complex. We also show how a complex gene network model can be used to fine map combinations of inherited and de novo variations in families with ASD that converge in the L1CAM pathway. Our results provide an important step forward in the molecular characterization of ASD with potential for developing a tool to analyze the pathogenesis of individual affected families.

Detecting new Buffel grass infestations in Australian arid lands: evaluation of methods using high-resolution multispectral imagery and aerial photography.

We assess the feasibility of using airborne imagery for Buffel grass detection in Australian arid lands and evaluate four commonly used image classification techniques (visual estimate, manual digitisation, unsupervised classification and normalised difference vegetation index (NDVI) thresholding) for their suitability to this purpose. Colour digital aerial photography captured at approximately 5 cm of ground sample distance (GSD) and four-band (visible­near-infrared) multispectral imagery (25 cm GSD) were acquired (14 February 2012) across overlapping subsets of our study site. In the field, Buffel grass projected cover estimates were collected for quadrates (10 m diameter), which were subsequently used to evaluate the four image classification techniques. Buffel grass was found to be widespread throughout our study site; it was particularly prevalent in riparian land systems and alluvial plains. On hill slopes, Buffel grass was often present in depressions, valleys and crevices of rock outcrops, but the spread appeared to be dependent on soil type and vegetation communities. Visual cover estimates performed best (r 2 0.39), and pixel-based classifiers (unsupervised classification and NDVI thresholding) performed worst (r 2 0.21). Manual digitising consistently underrepresented Buffel grass cover compared with field- and image-based visual cover estimates; we did not find the labours of digitising rewarding. Our recommendation for regional documentation of new infestation of Buffel grass is to acquire ultra-high-resolution aerial photography and have a trained observer score cover against visual standards and use the scored sites to interpolate density across the region.

Structural characterisation of the exopolysaccharide produced by Streptococcus thermophilus EU20.

Streptococcus thermophilus EU20 when grown on skimmed milk secretes a high-molecular-weight exopolysaccharide that is composed of glucose, galactose and rhamnose in a molar ratio of 2:3:2. Using chemical techniques and 1D and 2D-NMR spectroscopy (1H and 13C) the polysaccharide has been shown to possess a heptasaccharide repeating unit having the following structure: [chemical structure: see text]. Treatment of the polysaccharide with mild acid (0.5 M TFA, 100 degrees C for 1 h) liberates two oligosaccharides; the components correspond to the repeating unit and a hexasaccharide equivalent to the repeating unit minus the terminal alpha-L-Rhap.

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure.

AIMS: To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs). METHODS AND RESULTS: The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random amplification of polymorphic DNA (RAPD). Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure. CONCLUSION: Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups. SIGNIFICANCE AND IMPACT OF THE STUDY: This information should help in an understanding of genetic exchanges in lactic acid bacteria.

Improvements in allelic discrimination of microsatellite markers using denaturing polyacrylamide gel electrophoresis.

Poor resolution, retarded progress of DNA through gels, and variable sizing of DNA fragments between and within gels hinder accurate genotyping of some simple sequence length polymorphism (SSLP) markers with the Perkin Elmer Applied Biosystems 377 Sequenator. These problems are similar to renaturation related problems observed in DNA sequencing gels. PCR products especially susceptible to these problems are shown to have higher melting temperatures (T(m)) than others. Gels containing increased concentrations of denaturants allow greater accuracy in allelic discrimination. This is especially beneficial where quantification is necessary.

Heterofermentative metabolism of glucose and ribose and utilisation of citrate by the smooth biotype of Lactobacillus amylovorus NCFB 2745.

Lactobacillus amylovorus NCFB 2745 exhibits a rough colony morphology, ferments glucose homofermentatively and cannot utilise ribose. After five transfers in de Man Rogosa and Sharpe media (containing glucose and citrate) Lb. amylovorus 2745 appears smooth on agar plates; smooth cultures reverted to rough by culturing in aerobic conditions. The smooth type shows patterns of fermentation that are typical of a heterofermentative lactobacillus. Thus, the smooth morphotype produces CO2 and ethanol in addition to lactate and is able to ferment ribose. The switch in metabolism to the smooth form is accompanied by an increase in phosphoketolase and a reduction in aldolase enzyme activities. Citrate also has effects on growth rates and end-metabolites.

Structural and antigenic properties of merozoite surface protein 4 of Plasmodium falciparum.

Merozoite surface protein 4 (MSP4) of Plasmodium falciparum is a glycosylphosphatidylinositol-anchored integral membrane protein of 272 residues that possesses a single epidermal growth factor (EGF)-like domain near the carboxyl terminus. We have expressed both full-length MSP4 and a number of fragments in Escherichia coli and have used these recombinant proteins to raise experimental antisera. All recombinant proteins elicited specific antibodies that reacted with parasite-derived MSP4 by immunoblotting. Antibody reactivity was highly dependent on the protein conformation. For example, reduction and alkylation of MSP4 almost completely abolished the reactivity of several antibody preparations, including specificities directed to regions of the protein that do not contain cysteine residues and are far removed from the cysteine-containing EGF-like domain. This indicated the presence of conformation-dependent epitopes in MSP4 and demonstrated that proper folding of the EGF-like domain influenced the antigenicity of the entire molecule. The recombinant proteins were used to map epitopes recognized by individuals living in areas where malaria is endemic, and at least four distinct regions are naturally antigenic during infection. Binding of human antibodies to the EGF-like domain was essentially abrogated after reduction of the recombinant protein, indicating the recognition of conformational epitopes by the human immune responses. This observation led us to examine the importance of conformation dependence in responses to other integral membrane proteins of asexual stages. We analyzed the natural immune responses to a subset of these antigens and demonstrated that there is diminished reactivity to several antigens after reduction. These studies demonstrate the importance of reduction-sensitive structures in the maintenance of the antigenicity of several asexual-stage antigens and in particular the importance of the EGF-like domain in the antigenicity of MSP4.

Temporal expression of an H2-linked locus in host response to mouse malaria.

The action of host genes in response to malarial infection is complex. Two mouse loci, Char1, and Char2, have previously been shown to control peak parasitemia and host survival. Recent analysis of host response to mouse malaria has demonstrated that the action of several loci is time dependent. Char1 and Char2 act prior to peak parasitemia. Analysis of additional crosses revealed significant linkage to Chromosome 17 on the day following peak parasitemia. This H2-linked locus acts late in infection and is therefore crucial in clearing parasites from the circulation. The cloning of this gene will lead to a greater understanding of the host-parasite interaction, and the kinetics of host gene expression during an immune response.

Close linkage of three merozoite surface protein genes on chromosome 2 of Plasmodium falciparum.

We have analysed a 10.5 kb region of chromosome 2 in Plasmodium falciparum that encompasses the coding region of four genes. Three genes are arranged in a head-to-tail orientation and encode the merozoite surface proteins MSP2 and MSP4 as well as a previously unreported sequence that encodes a polypeptide with the characteristics of a merozoite surface protein, now designated MSP5. The fourth gene, asl, is arranged in a tail-to-tail orientation with msp2 and has homology with prokaryotic and eukaryotic genes encoding adenylosuccinate lyase (ASL), an enzyme involved in purine biosynthesis and salvage. The genes, arranged in the order msp4, msp5, msp2 and asl, are separated by intergenic distances of 1021, 1017 and 722 bp, respectively. msp4 and msp5 are clearly related genes, each being composed of 2 exons and encoding proteins of identical length. Both msp4 and msp5 encode proteins that contain hydrophobic signal sequences, apparent glycosylphosphatidylinositol (GPI) attachment signals and a single epidermal growth factor-like (EGF-like) domain at their carboxyl termini. Nevertheless, the remainder of their protein coding regions are quite dissimilar. It appears that one of these genes arose as a result of a relatively ancient gene duplication event and both genes have subsequently diverged considerably. This study shows that msp5 is transcribed in asexual stages and its encoded product is a 40 kDa protein that appears to be located on the merozoite surface as determined by immunofluorescence assays.

A second merozoite surface protein (MSP-4) of Plasmodium falciparum that contains an epidermal growth factor-like domain.

Merozoite surface proteins of Plasmodium falciparum play a critical role in the invasion of human erythrocytes by the malaria parasite. Here we describe the identification of a novel protein with a molecular mass of 40 kDa that is found on the merozoite surface of P. falciparum. We call this protein merozoite surface protein 4 (MSP-4). Evidence for the surface location of MSP-4 includes (i) a staining pattern that is consistent with merozoite surface location in indirect immunofluorescent studies of cultured parasites, (ii) localization of MSP-4 in the detergent phase in Triton X-114 partitioning studies, and (iii) nucleotide sequencing studies which predict the presence of an N-terminal signal sequence and a hydrophobic C-terminal sequence in the protein. Immunoprecipitation studies of biosynthetically labelled parasites with [3H] myristic acid indicated that MSP-4 is anchored on the merozoite surface by a glycosylphosphatidylinositol moiety. Of considerable interest is the presence of a single epidermal growth factor-like domain at the C terminus of the MSP-4 protein, making it the second protein with such a structure to be found on the merozoite surface.

Molecular variation in a novel polymorphic antigen associated with Plasmodium falciparum merozoites.

A cDNA clone encoding part of a novel polymorphic merozoite antigen from Plasmodium falciparum was isolated by screening a cDNA library with human immune serum from Papua New Guinea. Immunofluorescence microscopy and immunoblotting with affinity-purified antibodies recognized a highly polymorphic antigen, Ag956, present in schizonts and merozoites. Biosynthetic labeling and immunoprecipitation experiments demonstrated that Ag956 is proteolytically cleaved during merozoite maturation. The complete genomic sequence of Ag956 from the D10 clone of P. falciparum isolate FC27 encodes a secreted protein of calculated molecular mass 43,243 that is very hydrophilic and contains a region of unusual heptad repeats of the general structure AXXAXXX. This antigen has been named the secreted polymorphic antigen associated with merozoites (SPAM). The sequence of a second SPAM allele from the 3D7 clone of isolate NF54 reveals that the alanine heptad repeats and the hydrophilic C-terminal half of the protein are conserved. Variation among SPAM alleles is the result of deletions and amino acid substitutions in non-repetitive sequences within and flanking the alanine heptad-repeat domain. Heptad repeats in which the a and d position contain hydrophobic residues generate amphipathic alpha-helices which give rise to helical bundles or coiled-coil structures in proteins. Thus, SPAM is the first example of a P. falciparum antigen in which a repetitive sequence has features characteristic of a well-defined structural element.

Allelic variants of the Plasmodium falciparum merozoite surface antigen 2 (MSA-2) in a geographically restricted area of Irian Jaya.

Blood samples were collected from 12 residents of 4 villages in the Oksibil area of Irian Jaya. Eleven patients were positive for Plasmodium falciparum infection as evidenced by successful amplification of the MSA-2 gene by the polymerase chain reaction. Two patients showed evidence of infection by 2 strains of Plasmodium falciparum. All MSA-2 genes were completely sequenced and all could be assigned to one of the two major allelic families of MSA-2, however all MSA-2 gene sequences differed from previously described alleles. Five new allelic forms were identified, one of which was present in 8 of the 11 patients. Within small natural populations of P. falciparum, it appears that variation in MSA-2 approximates that seen world-wide. All samples were also analysed by hybridisation of amplified DNA to family specific probes and all samples hybridised to known probes. Our results demonstrate that there is a degree of microheterogeneity of MSA-2 that is undetectable by hybridisation studies alone.