Immunity to schistosomiasis: glycans are potential antigenic targets for immune intervention

The major humoral immune responses in animals infected with Schistosoma mansoni are directed toward carbohydrate antigens. Among these antigens are complex-type N-glycans expressing LDN [GalNAcbeta1-4GlcNAc-R], LDNF [GalNAcbeta1-4(Fucalpha1-3)GlcNAc-R], and polymeric Lewis x (Lex) [Galbeta1-4(Fucalpha1-3)GlcNAc]n-R epitopes. We have now evaluated the potential of the three glycan antigens as targets for immune-mediated intervention of infections and serodiagnosis. A variety of approaches were employed, including ELISA, Western blot, immunohistology, and in vitro complement lysis assays, to determine the immunogenicity of the glycans in infected humans, their localization on the parasites and their efficacy as targets for parasite lysis. Our results show that S. mansoni-infected patients, with either intestinal or hepatosplenic disease, generate predominantly IgM, but also IgG and IgA, antibodies to LDN, LDNF, and Lex. However, immune responses to Lex are generally lower than responses to LDN and LDNF and less specific to schistosome infections. Western blot analysis with monoclonal antibodies (mAb) to LDN, LDNF, and Lex determinants show that the glycan antigens occur on multiple glycoproteins from cercariae, 3-h, 48-h, and lung stage schistosomula, as well as adults and eggs. Immunohistological studies demonstrate that LDN, LDNF, and Lex are expressed on the parasite surface at all stages of development in the vertebrate host. Importantly, a mAb to LDN in the presence of complement efficiently kills schistosomula in vitro, as demonstrated by flow-cytometric assays that quantify cytolysis by propidium iodide uptake into damaged parasites. These findings raise the possibility that LDN and LDNF may be targets for vaccination and/or serodiagnosis of chronic schistosomiasis in humans.     

Overexpression of the CT GalNAc transferase inhibits muscular dystrophy in a cleavage-resistant dystroglycan mutant mouse

Transgenic mice that express dystroglycan containing a serine to alanine point mutation at the normal site of cleavage (DG(S654A)) in their skeletal muscles fail to express endogenously cleaved dystroglycan and have muscular dystrophy [Neuromusc. Disord., in press]. Dystrophic DG(S654A) muscles have reduced binding of antibodies, including VIA4-1, that recognize carbohydrate antigens on alpha dystroglycan, a finding similar to muscles in some forms of congenital muscular dystrophy. Here we describe one DG(S654A) transgenic line where VIA4-1 antibody binding is absent in skeletal muscle. In theory, the absence of this carbohydrate antigen should inhibit later glycosylation events that would occur on the structure or structures this antibody binds to. One such modification is likely to be the CT carbohydrate antigen, which is present on alpha dystroglycan in muscles overexpressing the CT GalNAc transferase [Dev. Biol. 242 (2002) 58]. To test the relationship between the VIA4-1 and CT carbohydrate antigens, we made DG(S654A)/CT GalNAc transferase (DG(S654A)/CT) transgenic mice. Surprisingly, dystroglycan was cleaved, and alpha dystroglycan was glycosylated with the VIA4-1 antigen, in DG(S654A)/CT muscles. In addition, muscles in DG(S654A)/CT transgenic mice had little or no evidence of muscular dystrophy when compared to DG(S654A) littermates. These experiments demonstrate that the CT GalNAc transferase can affect the post-translational processing of dystroglycan and the extent of muscular dystrophy even in muscles where the VIA4-1 antigen is not present.     

Some new micromycetes from New Zealand

New collections of micromycetes from New Zealand are recorded, including several new species and a new variety of the genera Cercospora, Entylomella, Gonatophragmium, Pseudocercospora, Ramularia and Subramaniomyces as well as some new combinations and a new name. Based on a new generic concept of the Pseudocercospora/Cercostigmina complex, which is supported by new molecular examinations, all species of Cercostigmina are re-allocated to Pseudocercospora.     

Aggregated and monomeric alpha-synuclein bind to the S6' proteasomal protein and inhibit proteasomal function

The accumulation of aggregated alpha-synuclein is thought to contribute to the pathophysiology of Parkinson's disease, but the mechanism of toxicity is poorly understood. Recent studies suggest that aggregated proteins cause toxicity by inhibiting the ubiquitin-dependent proteasomal system. In the present study, we explore how alpha-synuclein interacts with the proteasome. The proteasome exists as a 26 S and a 20 S species. The 26 S proteasome is composed of the 19 S cap and the 20 S core. Aggregated alpha-synuclein strongly inhibited the function of the 26 S proteasome. The IC(50) of aggregated alpha-synuclein for ubiquitin-independent 26 S proteasomal activity was 1 nm. Aggregated alpha-synuclein also inhibited 26 S ubiquitin-dependent proteasomal activity at a dose of 500 nm. In contrast, the IC(50) of aggregated alpha-synuclein for 20 S proteasomal activity was > 1 microm. This suggests that aggregated alpha-synuclein selectively interacts with the 19 S cap. Monomeric alpha-synuclein also inhibited proteasomal activity but with lower affinity and less potency. Recombinant monomeric alpha-synuclein inhibited the activity of the 20 S proteasomal core with an IC(50) > 10 microm, exhibited no inhibition of 26 S ubiquitin-dependent proteasomal activity at doses up to 5 microm, and exhibited only partial inhibition (50%) of the 26 S ubiquitin-independent proteasomal activity at doses up to 10 mm. Binding studies demonstrate that both aggregated and monomeric alpha-synuclein selectively bind to the proteasomal protein S6', a subunit of the 19 S cap. These studies suggest that proteasomal inhibition by aggregated alpha-synuclein could be mediated by interaction with S6'.     

Increased coverage obtained by combination of methods for protein sequence database searching

MOTIVATION: Sequence alignment methods that compare two sequences (pairwise methods) are important tools for the detection of biological sequence relationships. In genome annotation, multiple methods are often run and agreement between methods taken as confirmation. In this paper, we assess the advantages of combining search methods by comparing seven pairwise alignment methods, including three local dynamic programming algorithms (PRSS, SSEARCH and SCANPS), two global dynamic programming algorithms (GSRCH and AMPS) and two heuristic approximations (BLAST and FASTA), individually and by pairwise intersection and union of their result lists at equal p-value cut-offs. RESULTS: When applied singly, the dynamic programming methods SCANPS and SSEARCH gave significantly better coverage (p=0.01) compared to AMPS, GSRCH, PRSS, BLAST and FASTA. Results ranked by BLAST p-values gave significantly better coverage compared to ranking by BLAST e-values. Of 56 combinations of eight methods considered, 19 gave significant increases in coverage at low error compared to the parent methods at an equal p-value cutoff. The union of results by BLAST (p-value) and FASTA at an equal p-value cutoff gave significantly better coverage than either method individually. The best overall performance was obtained from the intersection of the results from SSEARCH and the GSRCH62 global alignment method. At an error level of five false positives, this combination found 444 true positives, a significant 12.4% increase over SSEARCH applied alone.     

Surface membrane proteins of Biomphalaria glabrata embryonic cells bind fucosyl determinants on the tegumental surface of Schistosoma mansoni primary sporocysts

Previous observations that in vitro adherence of Biomphalaria glabrata embryonic (Bge) cells to sporocyst larval stages of Schistosoma mansoni was strongly inhibited by fucoidan, a sulfated polymer of L-fucose, suggested a role for lectinlike Bge cell receptors in sporocyst binding interactions. In the present investigation, monoclonal antibodies with specificities to 3 major glycan determinants found on schistosomes, LacdiNAc, fucosylated LacdiNAc (LDNF), and the Lewis X antigen, were used in adhesion blocking studies to further analyze the molecular interactions at the host-parasite interface. Results showed that only the anti-LDNF antibody significantly reduced snail Bge cell adhesion to the surface of sporocysts, suggesting that fucosyl determinants may be important in larval-host cell interactions. Affinity chromatographic separation of fucosyl-reactive Bge cell proteins from fucoidan-bound Sepharose 4B revealed the presence of polypeptides ranging from 6 to 200 kDa after elution with fucoidan-containing buffer. Pre-elution of the Bge protein-bound affinity column with dextran (Dex) and dextran sulfate (DexS) before introduction of the fucoidan buffer served as controls for protein binding based on nonspecific sugar or negative charge interactions. A subset of polypeptides (approximately 35-150 kDa) released by fucoidan elution was identified as Bge surface membrane proteins, representing putative fucosyl-binding proteins. Far-western blot analysis also demonstrated binding reactivity between Bge cell and sporocyst tegumental proteins. The finding that several of these parasite-binding Bge cell proteins were also fucoidan-reactive suggests the possible involvement of these molecules in mediating cellular interactions with sporocyst tegumental carbohydrates. It is concluded that Bge cells have surface protein(s) that may be playing a role in facilitating host cell adhesion to the surface of schistosome primary sporocysts through larval fucosylated glycoconjugates.     

Assessing structural variation in a personal genome—towards a human reference diploid genome

Background

Characterizing large genomic variants is essential to expanding the research and clinical applications of genome sequencing. While multiple data types and methods are available to detect these structural variants (SVs), they remain less characterized than smaller variants because of SV diversity, complexity, and size. These challenges are exacerbated by the experimental and computational demands of SV analysis. Here, we characterize the SV content of a personal genome with Parliament, a publicly available consensus SV-calling infrastructure that merges multiple data types and SV detection methods.

Results

We demonstrate Parliament’s efficacy via integrated analyses of data from whole-genome array comparative genomic hybridization, short-read next-generation sequencing, long-read (Pacific BioSciences RSII), long-insert (Illumina Nextera), and whole-genome architecture (BioNano Irys) data from the personal genome of a single subject (HS1011). From this genome, Parliament identified 31,007 genomic loci between 100 bp and 1 Mbp that are inconsistent with the hg19 reference assembly. Of these loci, 9,777 are supported as putative SVs by hybrid local assembly, long-read PacBio data, or multi-source heuristics. These SVs span 59 Mbp of the reference genome (1.8%) and include 3,801 events identified only with long-read data. The HS1011 data and complete Parliament infrastructure, including a BAM-to-SV workflow, are available on the cloud-based service DNAnexus.

Conclusions

HS1011 SV analysis reveals the limits and advantages of multiple sequencing technologies, specifically the impact of long-read SV discovery. With the full Parliament infrastructure, the HS1011 data constitute a public resource for novel SV discovery, software calibration, and personal genome structural variation analysis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1479-3) contains supplementary material, which is available to authorized users.     

Lineage Tracing in Humans Enabled by Mitochondrial Mutations and Single-Cell Genomics

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Incidence of psychotic disorders among first-generation immigrants and refugees in Ontario

Background:

Evidence suggests that migrant groups have an increased risk of psychotic disorders and that the level of risk varies by country of origin and host country. Canadian evidence is lacking on the incidence of psychotic disorders among migrants. We sought to examine the incidence of schizophrenia and schizoaffective disorders in first-generation immigrants and refugees in the province of Ontario, relative to the general population.

Methods:

We constructed a retrospective cohort that included people aged 14–40 years residing in Ontario as of Apr. 1, 1999. Population-based administrative data from physician billings and hospital admissions were linked to data from Citizenship and Immigration Canada. We used Poisson regression models to calculate age- and sex-adjusted incidence rate ratios (IRRs) and 95% confidence intervals (CIs) for immigrant and refugee groups over a 10-year period.

Results:

In our cohort (n = 4 284 694), we found higher rates of psychotic disorders among immigrants from the Caribbean and Bermuda (IRR 1.60, 95% CI 1.29–1.98). Lower rates were found among immigrants from northern Europe (IRR 0.50, 95% CI 0.28–0.91), southern Europe (IRR 0.60, 95% CI 0.41–0.90) and East Asia (IRR 0.56, 95% CI 0.41–0.78). Refugee status was an independent predictor of risk among all migrants (IRR 1.27, 95% CI 1.04–1.56), and higher rates were found specifically for refugees from East Africa (IRR 1.95, 95% CI 1.44–2.65) and South Asia (IRR 1.51, 95% CI 1.08–2.12).

Interpretation:

The differential pattern of risk across ethnic subgroups in Ontario suggests that psychosocial and cultural factors associated with migration may contribute to the risk of psychotic disorders. Some groups may be more at risk, whereas others are protected.Meta-analytic reviews suggest that international migrants have a two- to threefold increased risk of psychosis compared with the host population, and the level of risk varies by country of origin and host country.^1,2 This increased risk may persist into the second and third generations.^2,3 Incidence rates are not typically found to be elevated in the country of origin;^4–7 therefore, it is believed that the migratory or postmigration experience may play a role in the etiology.The migration-related emergence of psychotic disorders is a potential concern in Canada, which receives about 250 000 new immigrants and refugees each year.⁸ However, there is a notable lack of current epidemiological information on the incidence of psychosis among these groups.⁹ Hospital admission data from the early 1900s suggest that European migrants to British Columbia had a higher incidence of schizophrenia than the general population,¹⁰ and more recent data from Ontario suggest higher rates of hospital admission for psychotic disorders in areas with a large proportion of first-generation migrants.¹¹ The fact that a large and increasing proportion of Canada’s population are migrants has been cited as a potential explanation for the higher prevalence of schizophrenia compared with international estimates.¹²The province of Ontario is home to the largest number of migrants in Canada, with first-generation migrants constituting nearly 30% of the population. Canada operates on a human capital model of immigration, using a points-based system that favours younger age, higher education, and proficiency in English or French. Nearly 60% of all newcomers to Canada are economic migrants, 27% are sponsored by a relative living in Canada, and 13% are refugees or temporary workers.⁸ Canada also requires a prearrival medical examination, but less than 0.001% of all applications are denied on the basis of medical grounds, and exemptions may be granted for refugees and some family-reunification applicants.¹³The Canadian migration process differs from that of many countries where the association between migration and psychotic disorders has been previously investigated.^1,2 In most of these countries, migrants generally originate from a smaller number of countries that have historic ties to the host country, and there tends to be a low proportion of refugees, although these processes have changed in recent years. In Canada, migrants come from a wide array of countries, admission policies focus on migrants with professional skills and there is a larger proportion of refugees. Few studies to date have examined the role of refugee status in the risk of psychotic disorders¹⁴ or have assessed all of the migrant groups within a country, because most studies focus on particular groups considered to be at high risk.¹ An examination of migrants to Canada offers a unique opportunity to investigate the risk of psychotic disorders in a group with diverse geographical origins, and the larger proportion of refugees also allows us to investigate their risk separately from immigrant groups. Thus, the breadth, scope and scale of migration to Canada over time offers a diverse and deep population for advancing our understanding of why some groups may have a higher risk of psychotic disorders.Our primary objective was to examine the incidence of schizophrenia and schizoaffective disorders over a 10-year period in first-generation immigrants and refugees in Ontario, relative to the general population. We also compared the incidence among specific migrant groups, stratified by country of birth and refugee status, because research suggests differences in the degree and direction of risk.^1,2 We restricted the sample to first-generation migrants to estimate the extent to which sociodemographic factors had an impact on the risk of schizophrenia and schizoaffective disorders among all migrants.