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Genomic studies of pediatric cancer have primarily focused on specific tumor types or high-risk disease. Here, we used a three-platform sequencing approach, including whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA sequencing (RNA-seq), to examine tumor and germline genomes from 309 prospectively identified children with newly diagnosed (85%) or relapsed/refractory (15%) cancers, unselected for tumor type. Eighty-six percent of patients harbored diagnostic (53%), prognostic (57%), therapeutically relevant (25%), and/or cancer-predisposing (18%) variants. Inclusion of WGS enabled detection of activating gene fusions and enhancer hijacks (36% and 8% of tumors, respectively), small intragenic deletions (15% of tumors), and mutational signatures revealing of pathogenic variant effects. Evaluation of paired tumor–normal data revealed relevance to tumor development for 55% of pathogenic germline variants. This study demonstrates the power of a three-platform approach that incorporates WGS to interrogate and interpret the full range of genomic variants across newly diagnosed as well as relapsed/refractory pediatric cancers. Significance: Pediatric cancers are driven by diverse genomic lesions, and sequencing has proven useful in evaluating high-risk and relapsed/refractory cases. We show that combined WGS, WES, and RNA-seq of tumor and paired normal tissues enables identification and characterization of genetic drivers across the full spectrum of pediatric cancers. This article is highlighted in the In This Issue feature, p. 2945

Cancer genomics has illuminated a wide spectrum of genes and core molecular processes contributing to human malignancy. Still, the genetic and molecular basis of many cancers remains only partially explained. Genetic predisposition accounts for 5-10% of cancer diagnoses(1,2) and genetic events cooperating with known somatic driver events are poorly understood. Analyzing established cancer predisposition genes in medulloblastoma (MB), a malignant childhood brain tumor, we recently identified pathogenic germline variants that account for 5% of all MB patients(3). Here, by extending our previous analysis to include all protein-coding genes, we discovered and replicated rare germline loss-of-function (LoF) variants across Elongator Complex Protein 1 (ELP1) on 9q31.3 in 15% of pediatric MB(SHH) cases, thus implicating ELP1 as the most common MB predisposition gene and increasing genetic predisposition to 40% for pediatric MB(SHH). Inheritance was verified based on parent-offspring and pedigree analysis, which identified two families with a history of pediatric MB. ELP1-associated MBs were restricted to the molecular SHHα subtype(4) and were characterized by universal biallelic inactivation of ELP1 due to somatic loss of chromosome 9q. The majority of ELP1-associated MBs exhibited co-occurring somatic PTCH1 (9q22.32) alterations, suggesting that ELP1-deficiency predisposes to tumor development in combination with constitutive activation of SHH signaling. ELP1 is an essential subunit of the evolutionary conserved Elongator complex, whose primary function is to enable efficient translational elongation through tRNA modifications at the wobble (U(34)) position(5,6). Biochemical, transcriptional, and proteomic analyses revealed that ELP1-associated MB(SHH) are characterized by a destabilized core Elongator complex, loss of Elongator-dependent tRNA wobble modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response (UPR), consistent with deregulation of protein homeostasis due to Elongator-deficiency in model systems(7–9). Our findings suggest that genetic predisposition to proteome instability is a previously underappreciated determinant in the pathogenesis of pediatric brain cancer. These results provide a strong rationale for further investigating the role of protein homeostasis in other cancer types and potential opportunities for novel therapeutic interference.

The XAF1-E134* variant increases the cancer risk for carriers of the TP53-R337H allele., Cancer risk is highly variable in carriers of the common TP53-R337H founder allele, possibly due to the influence of modifier genes. Whole-genome sequencing identified a variant in the tumor suppressor XAF1 (E134*/Glu134Ter/rs146752602) in a subset of R337H carriers. Haplotype-defining variants were verified in 203 patients with cancer, 582 relatives, and 42,438 newborns. The compound mutant haplotype was enriched in patients with cancer, conferring risk for sarcoma (P = 0.003) and subsequent malignancies (P = 0.006). Functional analyses demonstrated that wild-type XAF1 enhances transactivation of wild-type and hypomorphic TP53 variants, whereas XAF1-E134* is markedly attenuated in this activity. We propose that cosegregation of XAF1-E134* and TP53-R337H mutations leads to a more aggressive cancer phenotype than TP53-R337H alone, with implications for genetic counseling and clinical management of hypomorphic TP53 mutant carriers.

Interest in planting mixtures of cover crop species has grown in recent years as farmers seek to increase the breadth of ecosystem services cover crops provide. As part of a multidisciplinary project, we quantified the degree to which monocultures and mixtures of cover crops suppress weeds during the fall-to-spring cover crop growing period. Weed-suppressive cover crop stands can limit weed seed rain from summer- and winter-annual species, reducing weed population growth and ultimately weed pressure in future cash crop stands. We established monocultures and mixtures of two legumes (medium red clover and Austrian winter pea), two grasses (cereal rye and oats), and two brassicas (forage radish and canola) in a long fall growing window following winter wheat harvest and in a shorter window following silage corn harvest. In fall of the long window, grass cover crops and mixtures were the most weed suppressive, whereas legume cover crops were the least weed suppressive. All mixtures also effectively suppressed weeds. This was likely primarily due to the presence of fast-growing grass species, which were effective even when they were seeded at only 20% of their monoculture rate. In spring, weed biomass was low in all treatments due to winter kill of summer-annual weeds and low germination of winter annuals. In the short window following silage corn, biomass accumulation by cover crops and weeds in the fall was more than an order of magnitude lower than in the longer window. However, there was substantial weed seed production in the spring in all treatments not containing cereal rye (monoculture or mixture). Our results suggest that cover crop mixtures require only low seeding rates of aggressive grass species to provide weed suppression. This creates an opportunity for other species to deliver additional ecosystem services, though careful species selection may be required to maintain mixture diversity and avoid dominance of winter-hardy cover crop grasses in the spring.Nomenclature: Austrian winter pea, Pisum sativum L.; canola, Brassica napus L.; cereal rye, Secale cereale L., corn, Zea mays L., forage radish, Raphanus sativus L., medium red clover, Trifolium pratense L.; oats, Avena sativa L.; wheat, Triticum aestivum L.

BACKGROUND Our previous analysis of established cancer predisposition genes in medulloblastoma (MB) identified pathogenic germline variants in ~5% of all patients. Here, we extended our analysis to include all protein-coding genes. METHODS Case-control analysis performed on 795 MB patients against >118,000 cancer-free children and adults was performed to identify an association between rare germline variants and MB. RESULTS Germline loss-of-function variants of Elongator Complex Protein 1 (ELP1; 9q31.3) were strongly associated with SHH subgroup (MBSHH). ELP1-associated-MBs accounted for ~15% (29/202) of pediatric MBSHH cases and were restricted to the SHHα subtype. ELP1-associated-MBs demonstrated biallelic inactivation of ELP1 due to somatic chromosome 9q loss and most tumors exhibited co-occurring somatic PTCH1 (9q22.32) alterations. Inheritance was verified by parent-offspring sequencing (n=3) and pedigree analysis identified two families with a history of pediatric MB. ELP1-associated-MBSHH were characterized by desmoplastic/nodular histology (76%; 13/17) and demonstrated a favorable clinical outcome when compared to TP53-associated-MBSHH (5-yr OS 92% vs 20%; p-value=1.3e-6) despite both belonging to the SHHα subtype. ELP1 is a subunit of the Elongator complex, that promotes efficient translational elongation through tRNA modifications at the wobble (U34) position. Biochemical, transcriptional, and proteomic analyses revealed ELP1-associated-MBs exhibit destabilization of the core Elongator complex, loss of tRNA wobble modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response. CONCLUSIONS We identified ELP1 as the most common MB predisposition gene, increasing the total genetic predisposition for pediatric MBSHH to 40%. These results mark MBSHH as an overwhelmingly genetically-predisposed disease and implicate disruption of protein homeostasis in MBSHH development.

The mechanisms involved in anthelmintic resistance (AR) are complex but a greater understanding of AR management is essential for effective and sustainable control of parasitic helminth worms in livestock. Current tests to measure AR are time consuming and can be technically problematic, gold standard diagnostics are therefore urgently required to assist in combatting the threat from drug resistant parasites. For anthelmintics such as ivermectin (IVM), target proteins may be present in the cellular membrane. As proteins usually act in complexes and not in isolation, AR may develop and be measurable in the target associated proteins present in the parasite membrane. The model nematode Caenorhabditis elegans was used to develop a sub-proteomic assay to measure protein expression differences, between IVM resistant and IVM susceptible isolates in the presence and absence of drug challenge. Evaluation of detergents including CHAPS, ASB-14, C7BzO, Triton ×100 and TBP (tributyl phosphine) determined optimal conditions for the resolution of membrane proteins in Two Dimensional Gel Electrophoresis (2DE). These sub-proteomic methodologies were then translated and evaluated using IVM-susceptible and IVM-resistant Haemonchus contortus; a pathogenic blood feeding parasitic nematode which is of global importance in livestock health, welfare and productivity. We have demonstrated the successful resolution of membrane associated proteins from both C. elegans and H. contortus isolates, using a combination of CHAPS and the zwitterionic amphiphilic surfactant ASB-14 to further support the detection of markers for AR.

Patients harboring germline pathogenic biallelic variants in genes involved in the recognition and repair of DNA damage are known to have a substantially increased cancer risk. Emerging evidence suggests that individuals harboring heterozygous variants in these same genes may also be at heightened, albeit lesser, risk for cancer. Herein, we sought to determine whether heterozygous variants in RECQL4, the gene encoding an essential DNA helicase that is defective in children with the autosomal recessive cancer-predisposing condition Rothmund–Thomson syndrome (RTS), are associated with increased risk for childhood cancer. To address this question, we interrogated germline sequence data from 4435 pediatric cancer patients at St. Jude Children's Research Hospital and 1127 from the National Cancer Institute Therapeutically Applicable Research to Generate Effective Treatment (TARGET) database and identified 24 (0.43%) who harbored loss-of-function (LOF) RECQL4 variants, including five of 249 (2.0%) with osteosarcoma (OS). These RECQL4 variants were significantly overrepresented in children with OS, the cancer most frequently observed in patients with RTS, as compared to 134,187 noncancer controls in the Genome Aggregation Database (gnomAD v2.1; P = 0.00087, odds ratio [OR] = 7.1, 95% CI, 2.9–17). Nine of the 24 (38%) individuals possessed the same c.1573delT (p.Cys525Alafs) variant located in the highly conserved DNA helicase domain, suggesting that disruption of this domain is central to oncogenesis. Altogether these data expand our understanding of the genetic factors predisposing to childhood cancer and reveal a novel association between heterozygous RECQL4 LOF variants and development of pediatric OS.

RECQL4 encodes an essential helicase that repairs DNA damage and maintains genomic stability. Biallelic pathogenic germline mutations in RECQL4 cause the autosomal recessive (AR) Rothmund-Thomson, Baller-Gerold, and RAPADILINO syndromes. Predisposition to cancer has been observed in all three syndromes, with osteosarcoma (OS) representing the greatest risk. Monoallelic pathogenic variants in DNA damage response genes (e.g., ATM, NBN) are associated with a moderate increase in cancer risk. Building upon this notion, we sought to determine whether monoallelic RECQL4 loss of function (LOF) variants contribute to childhood cancer, particularly OS. Here, LOF variants were defined as nonsense, frameshift, or canonical splice altering variants that are classified as pathogenic or likely pathogenic based on the 2015 ACMG Guidelines. Among 4,436 pediatric cancer patients at St. Jude and 1,127 in the National Cancer Institute TARGET database (total: 5,563 patients), we identified 20 individuals (0.36%; tumor types in Table) harboring germline monoallelic RECQL4 LOF variants. Compared to reference controls in the Genome Aggregation Database (gnomAD), we observed an enrichment of RECQL4 LOF variants in pediatric cancer patients (P = 0.046, prevalence ratio [PR] = 1.51). We next assessed for enrichment of RECQL4 LOF variants across tumor types (Table). This examination revealed a significant association between monoallelic RECQL4 mutations and leukemia (P = 0.032, PR = 1.91) and more notably, with OS (P = 0.0028, PR = 7.03) where 1.7% of pediatric OS patients carried monoallelic RECQL4 LOF variants. No evidence of association was observed for the other tumor types examined. Our data provide the first evidence of an association linking germline monoallelic RECQL4 LOF variants to childhood cancer, especially OS. Examination of larger cohorts are warranted to elucidate the extent to which these mutations increase the risk for leukemia and OS and the mechanisms by which they promote tumor formation. Pediatric Cancer PatientsgnomAD ControlsCancer Risk (Fisher’s Exact Test)Average age at cancer diagnosis (range)Carriers, RECQL4mut/wtTotal Patients, RECQL4wt/wtCarriers, RECQL4mut/wtTotal Controls, RECQL4wt/wtPrevalence Ratio (95% CI)P ValueAll cancers7 (0.3 - 18)2055632811206591.51 (0.99, 2.30)0.046OS10 (6 - 16)42432811206597.03 (2.65, 18.69)0.0028CNS tumor515792811206590.74 (0.10, 5.26)0.74GCT0.31752811206595.74 (0.81, 40.88)0.16Leukemia4 (3 - 11)1124312811206591.91 (1.07, 3.41)0.032Lymphoma10 (7 - 18)35862811206592.19 (0.71, 6.76)0.16OS = osteosarcoma; CNS tumor = central nervous system tumor; GCT = germ cell tumor Citation Format: Jamie L. Maciaszek, Gang Wu, Kayla Hamilton, Rose B. McGee, Zhaoming Wang, Regina Nuccio, Stacy Hines-Dowell, Lynn Harrison, Elsie L. Gerhardt, Annastasia Ouma, Scott Newman, Aman Patel, Joy Nakitandwe, Elizabeth Azzato, Alberto S. Pappo, Sheila A. Shurtleff, David W. Ellison, James R. Downing, Melissa M. Hudson, Leslie L. Robison, Victor Santana, Jinghui Zhang, Kim E. Nichols, Chimene A. Kesserwan. Increased prevalence of germline monoallelic RECQL4 mutations in children with cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3651.

The major immunopathological consequences of infection with Schistosoma mansoni, a T helper type 2 response and granuloma formation leading to fibrotic tissue damage, are caused by the egg stage of the parasite. Three antigens of S. mansoni eggs, termed IPSE/alpha-1, omega-1 and kappa-5, have been found to be the primary targets of the egg-directed antibody response of the host. Here, we report on the isolation, cloning and characterisation of kappa-5. Apart from an uncharacterised mRNA sequence in S. japonicum, no significant similarities of kappa-5 to known sequences from other species were found. In contrast to IPSE/alpha-1 and omega-1, which have been found only in eggs, kappa-5 was present in miracidia as well as in eggs at the mRNA and protein levels. In eggs, isoforms of kappa-5 were observed with both three and four fully occupied N-glycosylation sites, while in miracidia only one isoform with four N-glycans could be detected. Interestingly, in Western blots sera from S. mansoni-infected Africans were reactive against kappa-5 with IgE and IgG isotype antibodies, but against IPSE/alpha-1 and omega-1 only with IgG antibodies. The further characterisation of kappa-5 as one of the three major egg antigens should help to better understand the immunology and immunopathology of schistosomiasis.

The rapid growth of proteomics has been made possible by the development of reproducible 2D gels and biological mass spectrometry. However, despite technical improvements 2D gels are still less than perfectly reproducible and gels have to be aligned so spots for identical proteins appear in the same place. Gels can be warped by a variety of techniques to make them concordant. When gels are manipulated to improve registration, information is lost, so direct methods for gel registration which make use of all available data for spot matching are preferable to indirect ones. In order to identify proteins from gel spots a property or combination of properties that are unique to that protein are required. These can then be used to search databases for possible matches. Molecular mass, pI, amino acid composition and short sequence tags can all be used in database searches. Currently the method of choice for protein identification is mass spectrometry. Proteins are eluted from the gels and cleaved with specific endoproteases to produce a series of peptides of different molecular mass. In peptide mass fingerprinting, the peptide profile of the unknown protein is compared with theoretical peptide libraries generated from sequences in the different databases. Tandem mass spectroscopy (MS/MS) generates short amino acid sequence tags for the individual peptides. These partial sequences combined with the original peptide masses are then used for database searching, greatly improving specificity. Increasingly protein identification from MS/MS data is being fully or partially automated. When working with organisms, which do not have sequenced genomes (the case with most helminths), protein identification by database searching becomes problematical. A number of approaches to cross species protein identification have been suggested, but if the organism being studied is only distantly related to any organism with a sequenced genome then the likelihood of protein identification remains small. The dynamic nature of the proteome means that there really is no such thing as a single representative proteome and a complete set of metadata (data about the data) is going to be required if the full potential of database mining is to be realised in the future.

Hamilton, J. V., Lehane, M. J. (2005). Tsetse midgut immunity - DiGE-ESTing for clues into African sleeping sickness. Outlooks on Pest Management, 16, (1), 19-22.

Circulating IgG antibody reactivity and excreted egg counts were investigated in 489 Kenyans given chemotherapy for schistosomiasis mansoni. Antibody reactivity was measured in ELISA, using either unfractionated aqueous soluble constituents of Schistosoma mansoni eggs (SEA) or CEF6 (a soluble fraction of S. mansoni eggs containing two cationic antigens) as the antigen source. Antibody reactivity for each antigen source was strongly associated with egg counts, both pre- and post-treatment. Approximately 6 months after chemotherapy, egg counts were zero in 84% of the subjects. The mean optical densities (OD) measured in the post-treatment ELISA were 60% (CEF6) or 45% (SEA) lower than the pre-treatment values, the reduction in the OD with CEF6 as antigen source being significantly greater than that observed with SEA (P

The gut epithelium is an essential interface in insects that transmit parasites. We investigated the role that local innate immunity might have on vector competence, taking Stomoxys calcitrans as a model. S. calcitrans is sympatric with tsetse flies, feeds on many of the same vertebrate hosts, and is thus regularly exposed to the trypanosomes that cause African sleeping sickness and nagana. Despite this, S. calcitrans is not a cyclical vector of these trypanosomes. Trypanosomes develop exclusively in the lumen of digestive organs, and so epithelial immune mechanisms, and in particular antimicrobial peptides (AMPs), may be the prime determinants of the fate of an infection. To investigate why S. calcitrans is not a cyclical vector of trypanosomes, we have looked in its midgut for AMPs with trypanolytic activity. We have identified a new AMP of 42 amino acids, which we named stomoxyn, constitutively expressed and secreted exclusively in the anterior midgut of S. calcitrans. It displays an amphipathic helical structure and exhibits a broad activity spectrum affecting the growth of microorganisms. Interestingly, this AMP exhibits trypanolytic activity to Trypanosoma brucei rhodesiense. We argue that stomoxyn may help to explain why S. calcitrans is not a vector of trypanosomes causing African sleeping sickness and nagana.

Using ELISA we provide direct evidence that the midgut defensins of the blood-sucking fly Stomoxys calcitrans are secreted into the gut lumen. We show that midgut defensin peptide levels increase up to fortyfold in response to a blood meal but not to a sugar meal. The data suggests the midgut defensin genes are post-transcriptionally regulated and that their function is protection of the stored blood meal from bacterial attack while it awaits digestion. Using recombinant defensins produced in Pichia pastoris we demonstrate that while in the gut cells the midgut defensins are bound in an SDS-stable complex to proteins with an apparent molecular weight of > 26 kDa from which they are released when secreted into the gut lumen. This > 26 kDa protein (Ssp3) has been cloned and sequenced and is a member of the serine protease S1 family with homologies to multiple insect proteases and to vertebrate trypsins and elastases.

Schistosomiasis remains a serious world-wide public health problem with a still unfulfilled need for routine cost-effective methods of diagnosis. Such methods are required not only for people in endemic areas, but increasingly for tourists who may have become infected during visits to such places. This article reviews the wide range of immunoassays and antigenic preparations that have been shown to have potential for diagnosis of schistosomiasis by the indirect method of antibody detection. Antigens in native form derived from cercariae, adult worms and eggs are considered, as well as schistosome antigens produced by recombinant DNA technology and the schistosome cross-reactive antigen, keyhole limpet haemocyanin (KLH). Respective advantages and disadvantages of antibody detection, circulating antigen detection and parasitological methods of diagnosis are analysed. It is suggested that due to the relative insensitivity of both parasitology and antigen detection, antibody detection methods could find increasing use in situations of low infection intensity.

Both CEF6, a cation-exchange fraction of soluble Schistosoma mansoni egg antigens (SEA), composed of the 2 antigens, alpha-1 and omega-1, and haemocyanin from the keyhole limpet, Megathura crenulata, have shown potential for immunodiagnosis of human schistosomiasis. Possible cross-reactivity between antigens in SEA and keyhole limpet haemocyanin (KLH) was explored by Western immunoblotting and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with KLH, SEA, CEF6, alpha-1, omega-1, or egg antigen k5. Both immunoassays revealed a high degree of serological cross-reactivity between the schistosome egg antigens and KLH, much of it due to sodium periodate- sensitive epitopes. Cross-reactivity with schistosome antigens with proven diagnostic efficacy may thus, in part, explain the usefulness of KLH for the diagnosis of human schistosomiasis mansoni.

the brighter coloured, more contrasted and sunflecked background of foliage. Mangrove ellobiaceans may be worth further study with respect to substrate and shell coloration. Pythia pLcata is unusual both in being associated with leaves and in its polymorphism. 1 am grateful to Professor M Shahadat All, University of Dhaka, for making the visit possible, to Professor M Salai Khan for information on the plants and to the Bntish Council for financial support Dr David Reid (Natural History Museum) very kindly identified the species recorded and provided helpful information on nomenclature and comments on the paper

The insulin/insulin-like growth factor-1 (IGF-1) signaling system is a public regulator of aging in the model animals Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus. For the first time, proteomic analyses of the environmentally resistant and 'nonaging' C. elegans dauer stage and long-lived daf-2 mutants has provided a unique insight into the protein changes which mediate survival against endogenously produced toxins. These changes support a diversion of energy consumption away from anabolic processes toward enhanced cellular maintenance and detoxification processes as previously described by the 'Green Theory of Aging'. Important components of this enhanced longevity system identified in this proteomics study include the alpha-crystallin family of small heat shock proteins, anti-ROS defense systems and cellular phase II detoxification (in daf-2 only). Among those proteins involved in phase II cellular detoxification that were significantly upregulated was a Pi-class glutathione transferase (GST) CE00302. Targeting this GST with RNAi revealed compensatory regulation within the Pi-class GSTs. Furthermore, a recombinant form of the GST protein was found to detoxify and/or bind short-chain aldehydic natural toxic products of lipid peroxidation and long-chained fatty-acids at physiologically relevant concentrations, which may indicate a role in longevity.

Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. Unlike the well characterized schistosomes, only the adult and juvenile stages of F. hepatica are implicated in disease, whereas the freely voided egg is not thought to contribute to host–parasite interactions. We investigated specific immune responses to soluble F. hepatica egg proteins (SFHEP), during a 14-week experimental infection, demonstrating significant increases in anti-SFHEP IgG1 (P = 0·001), transforming growth factor beta-1 (P = 0·008) and IL-10 (P < 0·001) titres at the onset of egg production. Western blot analysis of soluble SFHEP demonstrates that protein bands migrating at 61·6, 54·8 and 44 kDa become sero-reactive before the appearance of eggs within host faeces. Therefore, expression of some egg-associated proteins indicates progression to chronic disease. Antigenic bands were investigated through mass spectrometry, identifying a protein disulphide isomerase (PDI) (61·6 kDa), an enolase and ferritin-related proteins (54·8 kDa), and a cocktail of dehydrogenases (44 kDa). Biochemical analysis of egg secretions reveals proteolytic activity, which increases over time, indicating that proteases may be continually secreted during the course of egg maturation. The implications of egg-specific immune responses and proteolytic secretions are further discussed.

Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. We report the first post-genomic investigation of cellular proteins expressed by embryonic F. hepatica via two-dimensional electrophoresis, image analysis and tandem mass spectrometry. Antioxidant proteins and protein chaperones are prominently expressed by embryonic F. hepatica. Molecular differences between the egg and other characterized F. hepatica lifecycle stages were noted. Furthermore, proteins expressed within liver fluke eggs differ to those isolated from the well-characterized eggs of the human blood flatworm Schistosoma mansoni were revealed. Plasticity in expression of major proteins, particularly a prominently expressed 65kDa protein cluster was seen between natural populations of embryonating F. hepatica eggs suggesting that liver fluke embryogenisis is a plastic process. Immunoblotting revealed that the abundant 65kDa protein cluster is recognised by infection sera from three F. hepatica challenged host species. Mass spectrometry and BLAST analyses demonstrated that the 65kDa antigen shows homology to egg antigens of other flatworm parasites, and is represented in a F. hepatica EST database constructed from adult fluke transcripts. EST clones encoding the egg antigen were re-sequenced, predicting two forms of the protein. Four clones predict a 312 aa polypeptide, three clones encode a putative 110 amino acid extension at the N-terminus which may be involved in protein secretion, although this extension was not expressed by natively extracted proteins. Consistent expression of alpha crystallin domains confirmed the protein to be a member of the alpha crystallin containing small heat shock protein (AC/sHSP) superfamily. AC/sHSPs are ubiquitous in nature, however, this is the first time a member of this protein superfamily has been described from F. hepatica. The antigenic AC/sHSP was named Fh-HSP35alpha based on predictions of molecular weight. Production of recombinant Fh-HSP35alpha reveals considerable mass discrepancy between native and recombinant proteins, although descriptions of other characterized flatworm AC/sHSPs, suggest that the native form is a dimer. Immunoblot analyses confirm that the recombinant protein is recognised by F. hepatica challenged hosts, but does not react with sera from non-infected animals. We discuss the potential of recombinant Fh-HSP35alpha as an egg-based diagnostic marker for liver fluke infection.

The soluble global proteome of adult nematode Heligmosomoides polygyrus (H. p.) bakeri, a hookworm laboratory model was compared for the first time in the intestines of a slow-responder mouse host strain (C57/BL10) that is known to support a primary parasite infection for many months, and rapid-responder mouse host (SWR) that is known to eliminate the nematode infection by week 6 postinfection. At week 4 postinfection, major adult nematode proteins selectively produced following establishment of infection in C57/BL10 hosts include several globin forms, calreticulin and a phosphatidylethanolamine-binding protein. The increased synthesis of forms of myosin, actin and troponin in the nematode living in the rapid-responder SWR host may relate to the attempted reorganisation or repair of the cytoskeleton and/or muscle layer in the host immune initiated, increased mucus production and smooth muscle activity within intestinal environment. Initial evidence suggests weakly antigenic forms of globins dominant in the cytosol of H. p. bakeri adults in the intestinal environment compared to their low production in a related free-living nematode. The demonstration of considerable plasticity within a parasitic nematode proteome provides a molecular basis for the previously observed phenotypic plasticity within different host environments. Proteome plasticity has relevance to the efficiency of future vaccine and drug therapy, and the continued failure of defined antigen vaccines in mammalian populations.

A simple, cheap, sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. This review considers some of the advantages and disadvantages of parasitological microscopy, circulating-antigen detection and antibody-detection methods. Immunoassays for the detection of antibodies reactive against schistosome egg antigens have the potential to be useful in both clinical and epidemiological settings.

n-Hexane, dichloromethane and methanol extracts of the seeds of Agrimonia eupatoria have been assessed for antibacterial and free radical scavenging activity.

SUMMARYThe reduction in worm burden obtained by treatment of Schistosoma mansoni with praziquantel and oxamniquine was greater in mice with heavy infections than in relatively lightly infected animals. The reduction in worm burden achieved by each drug correlated with the size of the pre-treatment worm burden (r2 = 0·82 and 0·81 for praziquantel and oxamniquine, respectively). Intensity of infection did not affect the degree of tegumental damage and drug-induced antigen exposure on worms recovered soon after treatment with praziquantel. However, praziquantel-treated worms from mice with heavy infections had significantly more murine antibody attached to the treated-worm surface than worms from praziquantel-treated lightly infected mice. Heavily infected mice had greater levels of circulating anti-worm antibodies than lighter infected mice. The correlation between infection intensity and cure rates achieved by praziquantel and oxamniquine may thus be a reflection of the higher litres of relevant antibody in heavily infected mice mediating death of drug-treated worms.

Hamilton, J. V., Lehane, M. J., Braig, H. R. (2003). Letter. Isolation of Enterobacter sakazakii from midgut of Stomoxys calcitrans. Emerging Infectious Diseases, 9, (10), 1355-1356.

Background: Tsetse flies transmit African trypanosomiasis leading to half a million cases annually. Trypanosomiasis in animals (nagana) remains a massive brake on African agricultural development. While trypanosome biology is widely studied, knowledge of tsetse flies is very limited, particularly at the molecular level. This is a serious impediment to investigations of tsetse-trypanosome interactions. We have undertaken an expressed sequence tag (EST) project on the adult tsetse midgut, the major organ system for establishment and early development of trypanosomes. Results: A total of 21,427 ESTs were produced from the midgut of adult Glossina morsitans morsitans and grouped into 8,876 clusters or singletons potentially representing unique genes. Putative functions were ascribed to 4,035 of these by homology. Of these, a remarkable 3,884 had their most significant matches in the Drosophila protein database. We selected 68 genes with putative immune-related functions, macroarrayed them and determined their expression profiles following bacterial or trypanosome challenge. In both infections many genes are downregulated, suggesting a malaise response in the midgut. Trypanosome and bacterial challenge result in upregulation of different genes, suggesting that different recognition pathways are involved in the two responses. The most notable block of genes upregulated in response to trypanosome challenge are a series of Toll and Imd genes and a series of genes involved in oxidative stress responses. Conclusions: The project increases the number of known Glossina genes by two orders of magnitude. Identification of putative immunity genes and their preliminary characterization provides a resource for the experimental dissection of tsetse-trypanosome interactions.

The gross structure and optical properties of the eye of the marsh periwinkle,Littorina irrorata, are described. Each eye is about 270 × 240 μm in adults and is located on a short protuberance lateral to a cephalic tentacle. Light passes through a transparent cornea, a large fixed pupil, a spherical lens, and a large vitreous body, before striking a non-reflective retina. The lens is at least partially corrected for spherical aberration and has a short focal length (f/r = 2.71 for a 170-μm diameter lens in saline), indicating that its refractive index decreases from core to periphery. Rays parallel to the optic axis converge on the central retina's surface in a 1.1-μm diameter blur circle. The Airy disc on the central retina is less than 0.83 μm in diameter, indicating that image quality is nearly diffraction limited. The average adult retina contains about 7,450 photoreceptors whose average diameter is 3.44 μm, and whose average separation distance is 4.37 μm. On the central retina a single average photoreceptor subtends an angle of 1.3 ° from the posterior nodal point, while the angular separation of neighboring average photoreceptors is 1.7 °. Each eye has a potential field of view of 240 °, and the two eyes could provide 170 ° of forward-directed binocular vision. The depth of focus is estimated to be from 2.3 mm to infinity. The 5-mm long cephalic tentacles permit physical contact with objects too close to be in focus.

The daily movements of Littorina irrorata were examined. These snails move about on the substrate at low tide, and ascend plant stems on the advancing tide, thus avoiding predators. Snails released on the substrate amid plant stems, just prior to the release area being inundated by the advancing tide, moved in a relatively straight path and usually (67 %) ascended the closest plant stem. The propensity of these snails to ascend the closest stem was shown not to result from collisions resulting from snails travelling a straight path in a random direction. Field experiments involving transparent and black rods of equal size showed a significant tendency for snails to move towards black rods. These results suggest vision mediates the oriented movements of L. irrorata to plant stems in their natural habitat.

The distribution of Littorina irrorata Say on a low-energy barrier beach on the northern Gulf of Mexico is described, and correlated with the presence of Spartina alterniflora and other plants in the upper intertidal zone. The movements of 66 individually tagged snails were followed in the S. alterniflora zone for an average of 226 days, during which time an average of 10.6 positions were recorded per snail. The snails travelled an average total path distance of at least 995 cm, but due to contorted paths, ended up an average resultant distance of only 399 cm away from their original positions. They moved an estimated resultant distance of approximately 20 to 25 cm per activity period. Despite a slight offshore movement during the fall and winter, the snails moved more parallel to the shoreline than perpendicular to it.

Littorina irrorata displaced from their natural location amid plant stems in the upper inter‐tidal zone to areas of intertidal zone devoid of vegetation, oriented onshore under all sky and light conditions tested. The responses of snails tested in experimental arenas in the upper intertidal zone indicated that the natural substrate slope of 3 degrees was ignored in favor of movement toward a black paper rectangle. Subsequent releases on natural substrate supported the conclusion that the onshore‐oriented response resulted from visual perception of a dark area (bushes and trees near the high tide line). Where vegetation was equally tall in all directions, snails oriented toward the closest vegetation regardless of the actual onshore direction.

Blue crabs from five locations on the Gulf of Mexico and Atlantic Ocean coasts of Florida, USA, and juvenile crabs reared in the laboratory, were examined for the incidence of cheliped laterality (location of the crusher and cutter chelipeds). The proportion of crabs possessing the crusher cheliped on the right decreased with size (age) from 100% in the smallest crabs to about 74% in the largest crabs. Crabs with two cutter chelipeds were not uncommon and apparently resulted from regeneration of a cutter regardless of which cheliped was autotomized. Only 0.4% of all crabs possessed two crusher chelipeds.

Induction of Epstein-Barr virus (EBV) in the human lymphoma lines P 3 HR-1, and Daudi by TPA (phorbol-12-myristate-13-acetate), n -butyrate, and superinfection with P 3 HR-1 virus is associated with an increase in the number of cells bearing Fc receptor (Fc-R). The number of Fc-R per cell, as measured by flow cytometry, is also increased in both cell lines after effective induction of EBV antigens. The increase in cells bearing Fc-R correlates with increased expression of the membrane antigen complex (MA). A high proportion of induced cells concomitantly express both Fc-R and MA in double fluorescence studies. Fc-R activity was not induced by these treatments in EBV-negative lines. After induction of B-95-8 cells (an EBV-transformed marmoset cell line), there is an increase both in MA and whole virus production; however, no Fc-R activity has been detected, suggesting that the B-95-8 EBV strain is incapable of inducing an Fc-R at the cell surface.

Summary The case is described of a 15-year-old patient who developed acute pneumomediastinum and bilateral pneumothoraces whilst playing cricket. He made a rapid and uncomplicated recovery.