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Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

A key principle of insulation coordination is to compare the stress which could potentially be applied to equipment when it is in service with its withstand level. The withstand level is known based on standard tests. For AC systems an important knowledge exists at international level and numerous reference documents are available. In addition, the IEC 60,071 has standardized the rated impulse withstand voltage to be used for equipment and proposed for each value of highest voltage for equipment preferential values regarding impulse withstand voltage. The situation is not the same for DC systems. Less experience has been shared within the international community and there is no standardization of the withstand voltage. In addition, with regard to AC lines their structure is different. All these elements lead to a careful reassessment of hypotheses, which are usually accepted as valid when performing AC system insulation coordination studies. In this paper, the influence of lightning current shape on overvoltage is examined. A 500 kV bipolar DC overhead line is considered. The overvoltages due to bipolar lightning strokes based on measurements are calculated. Measurements available in literature have been used. These overvoltages are compared with calculations based on the use of the CIGRE wave shape as it is usually the case when performing insulation coordination studies and analyzed from an insulation coordination point of view.

Vaccine-induced high-avidity IgA can protect against bacterial enteropathogens by directly neutralizing virulence factors or by poorly defined mechanisms that physically impede bacterial interactions with the gut tissues ('immune exclusion'). IgA-mediated cross-linking clumps bacteria in the gut lumen and is critical for protection against infection by non-typhoidal Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium). However, classical agglutination, which was thought to drive this process, is efficient only at high pathogen densities (≥108 non-motile bacteria per gram). In typical infections, much lower densities (100-107 colony-forming units per gram) of rapidly dividing bacteria are present in the gut lumen. Here we show that a different physical process drives formation of clumps in vivo: IgA-mediated cross-linking enchains daughter cells, preventing their separation after division, and clumping is therefore dependent on growth. Enchained growth is effective at all realistic pathogen densities, and accelerates pathogen clearance from the gut lumen. Furthermore, IgA enchains plasmid-donor and -recipient clones into separate clumps, impeding conjugative plasmid transfer in vivo. Enchained growth is therefore a mechanism by which IgA can disarm and clear potentially invasive species from the intestinal lumen without requiring high pathogen densities, inflammation or bacterial killing. Furthermore, our results reveal an untapped potential for oral vaccines in combating the spread of antimicrobial resistance.

Mast cells are implicated in the innate proinflammatory immune defence against bacterial insult, but the mechanisms through which mast cells respond to bacterial encounter are poorly defined. Here, we addressed this issue and show that mast cells respond vividly to wild type Streptococcus equi by up‐regulating a panel of proinflammatory genes and by secreting proinflammatory cytokines. However, this response was completely abrogated when the bacteria lacked expression of sagA, whereas the lack of a range of other potential virulence genes (seeH, seeI, seeL, seeM, hasA, seM, aroB, pyrC, and recA) had no effect on the amplitude of the mast cell responses. The sagA gene encodes streptolysin S, a lytic toxin, and we next showed that the wild type strain but not a sagA‐deficient mutant induced lysis of mast cells. To investigate whether host cell membrane perturbation per se could play a role in the activation of the proinflammatory response, we evaluated the effects of detergent‐ and pneumolysin‐dependent lysis on mast cells. Indeed, exposure of mast cells to sublytic concentrations of all these agents resulted in cytokine responses of similar amplitudes as those caused by wild type streptococci. This suggests that sublytic membrane perturbation is sufficient to trigger full‐blown proinflammatory signalling in mast cells. Subsequent analysis showed that the p38 and Erk1/2 signalling pathways had central roles in the proinflammatory response of mast cells challenged by either sagA‐expressing streptococci or detergent. Altogether, these findings suggest that sagA‐dependent mast cell membrane perturbation is a mechanism capable of activating the innate immune response upon bacterial challenge.

The emergence of antibiotic-resistant bacteria through mutations or the acquisition of genetic material such as resistance plasmids represents a major public health issue1,2. Persisters are subpopulations of bacteria that survive antibiotics by reversibly adapting their physiology3–10, and can promote the emergence of antibiotic-resistant mutants11. We investigated whether persisters can also promote the spread of resistance plasmids. In contrast to mutations, the transfer of resistance plasmids requires the co-occurrence of both a donor and a recipient bacterial strain. For our experiments, we chose the facultative intracellular entero-pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) and Escherichia coli, a common member of the microbiota12. S. Typhimurium forms persisters that survive antibiotic therapy in several host tissues. Here we show that tissue-associated S. Typhimurium persisters represent long-lived reservoirs of plasmid donors or recipients. The formation of reservoirs of S. Typhimurium persisters requires Salmonella pathogenicity island (SPI)-1 and/or SPI-2 in gut-associated tissues, or SPI-2 at systemic sites. The re-seeding of these persister bacteria into the gut lumen enables the co-occurrence of donors with gut-resident recipients, and thereby favours plasmid transfer between various strains of Enterobacteriaceae. We observe up to 99% transconjugants within two to three days of re-seeding. Mathematical modelling shows that rare re-seeding events may suffice for a high frequency of conjugation. Vaccination reduces the formation of reservoirs of persisters after oral infection with S. Typhimurium, as well as subsequent plasmid transfer. We conclude that—even without selection for plasmid-encoded resistance genes—small reservoirs of pathogen persisters can foster the spread of promiscuous resistance plasmids in the gut. The re-seeding of antibiotic-resistant persister subpopulations of Salmonella enterica into the gut lumen favours the transfer of resistance plasmids to gut-resident enterobacteria, showing that even small reservoirs of persister bacteria facilitate the spread of antibiotic resistance.

Salmonella Typhimurium (S.Tm) causes acute enteropathy resolving after 4-7 days. Strikingly, antibiotic therapy does not accelerate disease resolution. We screened for factors blocking remission using a S.Tm enterocolitis model. The antibiotic ciprofloxacin clears pathogen stool loads within 3-24 hr, while gut pathology resolves more slowly (ψ50: ∼48 hr, remission: 6-9 days). This delayed resolution is mediated by an interferon-γ (IFN-γ)-dependent response that is triggered during acute infection and continues throughout therapy. Specifically, IFN-γ production by mucosal T and NK cells retards disease resolution by maintaining signaling through the transcriptional regulator STAT1 and boosting expression of inflammatory mediators like IL-1β, TNF, and iNOS. Additionally, sustained IFN-γ fosters phagocyte accumulation and hampers antimicrobial defense mediated by IL-22 and the lectin REGIIIβ. These findings reveal a role for IFN-γ in delaying resolution of intestinal inflammation and may inform therapies for acute Salmonella enteropathy, chronic inflammatory bowel diseases, or disease resolution during antibiotic treatment.

The emergence of antibiotic-resistant bacteria through mutations or the acquisition of genetic material such as resistance plasmids represents a major public health issue

Summary Mucus separates gut-luminal microbes from the tissue. It is unclear how pathogens like Salmonella Typhimurium (S.Tm) can overcome this obstacle. Using live microscopy, we monitored S.Tm interactions with native murine gut explants and studied how mucus affects the infection. A dense inner mucus layer covers the distal colon tissue, limiting direct tissue access. S.Tm performs near-surface swimming on this mucus layer, which allows probing for colon mucus heterogeneities, but can also entrap the bacterium in the dense inner colon mucus layer. In the cecum, dense mucus fills only the bottom of the intestinal crypts, leaving the epithelium between crypts unshielded and prone to access by motile and non-motile bacteria alike. This explains why the cecum is highly infection permissive and represents the primary site of S.Tm enterocolitis in the streptomycin mouse model. Our findings highlight the importance of mucus in intestinal defense and homeostasis., Graphical Abstract, Highlights • Live imaging of Salmonella near-surface swimming on mouse colon inner mucus layer • Colon inner mucus layer traversal requires mucus breaches and flagellar propulsion • The mouse cecum lacks a continuous mucus layer, leaving epithelium tips uncovered • Exposed cecum epithelium tips are a hotspot for Salmonella infection, Using live microscopy, Furter et al. describe how the enteropathogen Salmonella Typhimurium crosses the protective intestinal mucus layer of its murine host. Flagella-driven motility, the mucus architecture, and its distribution determine where the pathogen preferentially infects the gut epithelium.

While the functional importance of inflammasomes in blood-derived cell types is well established, it remains poorly understood how inflammasomes in nonhematopoietic cells contribute to mucosal immunity. Recent studies have revealed functional roles of inflammasomes - particularly NAIP/NLRC4, NLRP6, and noncanonical caspase-4 (caspase-11) - within epithelial cells of the gut in mucosal immune defense, inflammation, and tumorigenesis. Here, we review and discuss these findings in the broader context of tissue compartment-specific mucosal immunity. We propose several models whereby activities of the intestinal epithelial inflammasomes converge on mechanisms to remove compromised epithelial cells, maintain host-microbiota mutualism, and communicate with immune cells of the underlying lamina propria.

The parasite of my parasite is my friend? Virulence factors of pathogenic bacteria can be swapped by means of bacterial viruses called phages. In turn, the pathogenic bacteria are under attack by the hosts' immune responses. Diard et al. discovered that SopEϕ, a phage parasite of pathogenic Salmonella species, is encouraged to spread between bacteria by the mouse host's inflammatory responses. Conversely, mucosal vaccination against Salmonella reduced inflammatory responses and curbed the transfer of SopEϕ to naïve bacteria. Science , this issue p. 1211

Salmonella enterica serovar Typhimurium (S. Tm) is a facultative intracellular pathogen that induces entry into non-phagocytic cells by a Type III secretion system (TTSS) and cognate effector prote ...

Bacteriophage transfer (lysogenic conversion) promotes bacterial virulence evolution. There is limited understanding of the factors that determine lysogenic conversion dynamics within infected hosts. A murine

Vertebrates express 9–17 septin family members known to oligomerize into diverse structures, but their native assembly states have remained elusive. The results presented suggest a generic model for how the temporal order of septin assembly directs the subunit arrangement within distinct pools of six- to eight-subunit core heteromers., Septins are conserved GTP-binding proteins that assemble into lateral diffusion barriers and molecular scaffolds. Vertebrate genomes contain 9–17 septin genes that encode both ubiquitous and tissue-specific septins. Expressed septins may assemble in various combinations through both heterotypic and homotypic G-domain interactions. However, little is known regarding assembly states of mammalian septins and mechanisms directing ordered assembly of individual septins into heteromeric units, which is the focus of this study. Our analysis of the septin system in cells lacking or overexpressing selected septins reveals interdependencies coinciding with previously described homology subgroups. Hydrodynamic and single-particle data show that individual septins exist solely in the context of stable six- to eight-subunit core heteromers, all of which contain SEPT2 and SEPT6 subgroup members and SEPT7, while heteromers comprising more than six subunits also contain SEPT9. The combined data suggest a generic model for how the temporal order of septin assembly is homology subgroup-directed, which in turn determines the subunit arrangement of native heteromers. Because mammalian cells normally express multiple members and/or isoforms of some septin subgroups, our data also suggest that only a minor fraction of native heteromers are arranged as perfect palindromes.

Septin assemblies during the interphase of animal cells remain poorly defined and are the topic of this report. The data point to a general model for assembly of higher-order septin arrangements at locations providing the greatest opportunity for binding cooperativity, which depends on both the cell type and external cues., Septin family proteins oligomerize through guanosine 5′-triphosphate–binding domains into core heteromers, which in turn polymerize at the cleavage furrow of dividing fungal and animal cells. Septin assemblies during the interphase of animal cells remain poorly defined and are the topic of this report. In this study, we developed protocols for visualization of authentic higher-order assemblies using tagged septins to effectively replace the endogenous gene product within septin core heteromers in human cells. Our analysis revealed that septins assemble into microtubule-supported, disk-like structures at the plasma membrane. In the absence of cell substrate adhesion, this is the predominant higher-order arrangement in interphase cells and each of the seven to eight septin family members expressed by the two analyzed cell types appears equally represented. However, studies of myeloid and lymphoid cell model systems revealed cell type–specific alterations of higher-order septin arrangements in response to substrate adhesion. Live-cell observations suggested that all higher-order septin assemblies are mutually exclusive with plasma membrane regions undergoing remodeling. The combined data point to a mechanism by which densely arranged cortical microtubules, which are typical for nonadhered spherical cells, support plasma membrane–bound, disk-like septin assemblies.

The microtubule-system organizes the cytoplasm during interphase and segregates condensed chromosomes during mitosis. Four unrelated conserved proteins, XMAP215/Dis1/TOGp, MCAK, MAP4 and Op18/stathmin, have all been implicated as predominant regulators of tubulin monomer-polymer partitioning in animal cells. However, while studies employing the Xenopus egg extract model system indicate that the partitioning is largely governed by the counteractive activities of XMAP215 and MCAK, studies of human cell lines indicate that MAP4 and Op18 are the predominant regulators of the interphase microtubule-array. Here, we review functional interplay of these proteins during interphase and mitosis in various cell model systems. We also review the evidence that MAP4 and Op18 have interphase-specific, counteractive and phosphorylation-inactivated activities that govern tubulin subunit partitioning in many mammalian cell types. Finally, we discuss evidence indicating that partitioning regulation by MAP4 and Op18 may be of significance to establish cell polarity.

Op18/stathmin (Op18), a conserved microtubule-depolymerizing and tubulin heterodimer-binding protein, is a major interphase regulator of tubulin monomer–polymer partitioning in diverse cell types in which Op18 is abundant. Here, we addressed the question of whether the microtubule regulatory function of Op18 includes regulation of tubulin heterodimer synthesis. We used two human cell model systems, K562 and Jurkat, combined with strategies for regulatable overexpression or depletion of Op18. Although Op18 depletion caused extensive overpolymerization and increased microtubule content in both cell types, we did not detect any alteration in polymer stability. Interestingly, however, we found that Op18 mediates positive regulation of tubulin heterodimer content in Jurkat cells, which was not observed in K562 cells. By analysis of cells treated with microtubule-poisoning drugs, we found that Jurkat cells regulate tubulin mRNA levels by a posttranscriptional mechanism similarly to normal primary cells, whereas this mechanism is nonfunctional in K562 cells. We present evidence that Op18 mediates posttranscriptional regulation of tubulin mRNA in Jurkat cells through the same basic autoregulatory mechanism as microtubule-poisoning drugs. This, combined with potent regulation of tubulin monomer–polymer partitioning, enables Op18 to exert global regulation of the microtubule system.

Op18/stathmin (Op18) is a phosphorylation-regulated and differentially expressed microtubule-destabilizing protein in animal cells. Op18 regulates tubulin monomer-polymer partitioning of the interphase microtubule system and forms complexes with tubulin heterodimers. Recent reports have shown that specific tubulin-folding cofactors and related proteins may disrupt tubulin heterodimers. We therefore investigated whether Op18 protects unpolymerized tubulin from such disruptive activities. Our approach was based on inducible overexpression of two tubulin-disrupting proteins, namely TBCE, which is required for tubulin biogenesis, and E-like, which has been proposed to regulate tubulin turnover and microtubule stability. Expression of either of these proteins was found to cause a rapid degradation of both alpha-tubulin and beta-tubulin subunits of unpolymerized, but not polymeric, tubulin heterodimers. We found that depletion of Op18 by means of RNA interference increased the susceptibility of tubulin to TBCE or E-like mediated disruption, while overexpressed Op18 exerted a tubulin-protective effect. Tubulin protection was shown to depend on Op18 levels, binding affinity, and the partitioning between tubulin monomers and polymers. Hence, the present study reveals that Op18 at physiologically relevant levels functions to preserve the integrity of tubulin heterodimers, which may serve to regulate tubulin turnover rates.

A combined school- and community-based campaign targeting the entire school-age population of Burkina Faso with drugs against schistosomiasis (praziquantel) and soil-transmitted helminthiasis (albendazole) was implemented in 2004-2005. In total, 3,322,564 children from 5 to 15 years of age were treated, equivalent to a 90.8% coverage of the total school-age population of the country. The total costs of the campaign were estimated to be US 1,067,284 dollars, of which 69.4% was spent on the drugs. Delivery costs per child treated were US 0.098 dollar, in the same range as school-based only interventions implemented in other countries; total costs per child treated (including drugs) were US 0.32 dollar. We conclude that a combined school- and community-based strategy is effective in attaining a high coverage among school-age children in countries where school enrolment is low and where primary schools cannot serve as the exclusive drug distribution points. The challenge for Burkina Faso will now be to ensure the sustainability of these disease control activities.

Inflammasome signaling impinges on the activation of inflammatory caspases (i.e., caspase-1 and caspase-4/5/11) and endows host cells with a sentinel system to sense microbial intrusion and thereby initiate appropriate immune responses. Lately, it has become evident that mammalian inflammasome-dependent responses to infection are not confined solely to cells of hematopoietic origin. Epithelial cells that line the body's mucosal surfaces use inflammasome signaling to sense and counteract pathogenic microorganisms that compromise barrier integrity. Many of the molecular mechanisms of epithelial inflammasome signaling remain unexplored. However, it now seems clear that epithelial inflammasome activation has a profound impact both on the infected cell itself and on its ability to communicate with other cell types of the mucosa. Here, we summarize current knowledge regarding the output of epithelial inflammasome activation during bacterial infection. Well-established downstream effects include epithelial cell death, release of soluble mediators, and subsequent recruitment of effector cell types, including NK cells, mast cells, and neutrophils, to sites of mucosal infection. We discuss the implications of recent findings for antibacterial defense in the mucosa and sketch out areas for future exploration.

SummaryThe gut mucosal epithelium separates the host from the microbiota, but enteropathogens such as Salmonella Typhimurium (S.Tm) can invade and breach this barrier. Defenses against such acute insults remain incompletely understood. Using a murine model of Salmonella enterocolitis, we analyzed mechanisms limiting pathogen loads in the epithelium during early infection. Although the epithelium-invading S.Tm replicate initially, this intraepithelial replicative niche is restricted by expulsion of infected enterocytes into the lumen. This mechanism is compromised if inflammasome components (NAIP1-6, NLRC4, caspase-1/-11) are deleted, or ablated specifically in the epithelium, resulting in ∼100-fold higher intraepithelial loads and accelerated lymph node colonization. Interestingly, the cytokines downstream of inflammasome activation, interleukin (IL)-1α/β and IL-18, appear dispensable for epithelial restriction of early infection. These data establish the role of an epithelium-intrinsic inflammasome, which drives expulsion of infected cells to restrict the pathogen’s intraepithelial proliferation. This may represent a general defense mechanism against mucosal infections.

© 2014 Elsevier Ltd. Antibiotics are powerful therapeutics but are not equally effective against all cells in bacterial populations. Bacteria that express an antibiotic tolerant phenotype ("persisters") can evade treatment [1]. Persisters can cause relapses of the infection after the end of the therapy [2]. It is still poorly understood whether persistence affects the evolution of bacterial virulence. During infections persisters have been found preferentially at particular sites within the host [3 4]. If bacterial virulence factors are required to reach such sites treatment with antibiotics could impose selection on the expression of virulence genes in addition to their well established effects on bacterial resistance. Here we report that treatment with antibiotics selects for virulence and fosters transmissibility of Salmonella Typhimurium. In a mouse model for Salmonella diarrhea treatment with the broadspectrum antibiotic ciprofloxacin reverses the outcome of competition between wild type bacteria and avirulent mutants that can spontaneously arise during within host evolution [5]. While avirulent mutants take over the gut lumen and abolish disease transmission in untreated mice ciprofloxacin tilts the balance in favor of virulent wild type bacteria. This is explained by the need for virulence factors to invade gut tissues and form a persistent reservoir. Avirulent mutants remain in the gut lumen and are eradicated. Upon cessation of antibiotic treatment tissue lodged wild type pathogens reseed the gut lumen and thereby facilitate disease transmissibility to new hosts. Our results suggest a general principle by which antibiotic treatment can promote cooperative virulence during within host evolution increase duration of transmissibility and thereby enhance the spread of an infectious disease.

Septins are filament-forming proteins important for organizing the cortex of animal and fungal cells. In mammals, 13 septin paralogues were recently shown to assemble into core heterohexamer and heterooctamer complexes, which serve as building blocks for apolar filamentous structures that differ among cell types. To determine how tissue-specific septin paralogue expression may shape core heteromer repertoires and thereby modulate properties of septin filaments, we devised protocols to analyze native septin heteromers with distinct numbers of subunits. Our evidence based on genetically manipulated human cells supports and extends recent concepts of homology subgroup–restricted assembly into distinct categories of apolar heterohexamers and heterooctamers. We also identify a category of tetramers that have a subunit composition equivalent to an octameric building block. These atypical tetramers are prevalent in lymphocytes and neural tissues, in which octamers are abundant but hexamers are rare. Our results can be explained by tissue-specific expression of SEPT3 subgroup members: SEPT3, SEPT9, and SEPT12. These serve as cognate subunits in either heterooctamers or atypical tetramers but exhibit different preferences in various tissues. The identified tissue-specific repertoires of septin heteromers provide insights into how higher-order septin structures with differential properties and stabilities may form in diverse animal cell types., Molecular Biology of the Cell, 25 (10), ISSN:1939-4586, ISSN:1059-1524

Salmonella enterica serovar Typhimurium (S. Tm) is a facultative intracellular pathogen that induces entry into non-phagocytic cells by a Type III secretion system (TTSS) and cognate effector proteins. Upon host cell entry, S. Tm expresses a second TTSS and subverts intracellular trafficking to create a replicative niche - the Salmonella-containing vacuole (SCV). SopE, a guanidyl exchange factor (GEF) for Rac1 and Cdc42, is translocated by the TTSS-1 upon host cell contact and promotes entry through triggering of actin-dependent ruffles. After host cell entry, the bulk of SopE undergoes proteasomal degradation. Here we show that a subfraction is however detectable on the nascent SCV membrane up to ∼ 6 h post infection. Membrane localization of SopE and the closely related SopE2 differentially depend on the Rho-GTPase-binding GEF domain, and to some extent involves also the unstructured N-terminus. SopE localizes transiently to the early SCV, dependent on continuous synthesis and secretion by the TTSS-1 during the intracellular state. Mutant strains lacking SopE or SopE2 are attenuated in early intracellular replication, while complementation restores this defect. Hence, the present study reveals an unanticipated role for SopE and SopE2 in establishing the Salmonella replicative niche, and further emphasizes the importance of entry effectors in later stages of host-cell manipulation.

Recent characterization of nuclear ribosomal small subunit (SSU) genes has shown that variant nucleotides within this region could be useful for species and species group identification within the genus Lymnaea (Gastropoda: Lymnaeidae). This study aimed to characterize a range of populations of Lymnaea natalensis Krauss, 1848 on Madagascar, and addressed two related questions. First, is there any evidence of intraspecific variation of the SSU and, if so, what might be its significance? Secondly, might this variation jeopardize the use of SSU for lymnaeid taxonomy and phylogeny? Lymnaea natalensis (n = 212) was collected from 17 sampling localities, spanning the northern and southern ends of the island. Variation within a selected region of the SSU known to vary between species, the V1 and V2, was assayed by polymerase chain reaction (PCR) linked restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) analysis. The PCR-RFLP profiles indicated a striking dimorphism across populations at two restriction site loci (CfoI & MspI) within the E10-1 helix of the V2 region. The observed RFLP variation was confirmed by direct sequencing and by genomic digestion with subsequent hybridization. Putative heterozygotes were also encountered and in these individuals the SSU arrays composed of two distinct types approximately 1% divergent. A severe departure from Hardy–Weinberg equilibrium with a highly statistically significant (P < 10-5) heterozygote deficiency was found and genetic variation among populations was highly structured (Fst = 0.53). The geographic distribution of the variants was mapped, revealing that one variant was restricted to higher, predominately colder environments and was thought to be an adaptation. The molecular basis of the SSU variation was caused by single nucleotide polymorphisms (SNPs). To test for the possibility of cryptic taxa, an analysis of individuals representative of the SSU variant types with isoenzyme analysis (ISA), randomly amplified polymorphic DNA (RAPDs) and PCR-RFLP analysis of the ribosomal Internal Transcribed Spacer (ITS) was performed. Little variation was revealed and none that correlated to the groups suggested by SSU, confirming that the SSU variation was intraspecific. The levels of intraspecific divergence of the V1 and V2 within Lymnaea were not appreciably different (1%) from interspecific and would therefore question the validity of these data for lymnaeid taxonomy and phylogeny.

The Telecommunications Information Networking Architecture (TINA) Consortium is designing a common software architecture which allows providers to build, deliver and manage new multimedia services quickly and easily. The article concentrates on the task carried out by a European project of implementing a pan-European TINA-based service management platform. While simple services were created at the beginning of the project, more advanced multimedia services are now being implemented, such as audio/video conferencing. The status of the implementation and interoperability issues are presented.

The ability to carry stream information over a range of network types in a managed way will become an essential requirement for telecommunications network operators as future services evolve to include the transmission of audio/video and bulk data streams. This paper details the architecture of a working pan-European demonstrator offering multimedia services through the integration of stream control, as specified in the Object Management Group‘s (OMG‘s) control and management of audio/video streams, with a TINA-compliant service management environment. This CORBA-based demonstrator, known as the EURESCOM services platform (ESP), has been developed by six European telecommunications companies within EURESCOM project P715. Various modelling concepts, as defined by TINA, ODP and OMG, have been used and verified in a first prototype implementation, with connectivity provided by commercially available multimedia technology such as H.320/323 products. This EURESCOM services platform prototype is one of the first demonstrators world-wide to implement OMG‘s control and management of audio/video streams specification.

Reorganization of the host cell actin cytoskeleton is crucial during pathogen invasion. We established micropatterned cells as a standardized infection model for cell invasion to quantitatively stu ...

Septin-family proteins assemble into rod-shaped heteromeric complexes that form higher-order arrangements at the cell cortex, where they serve apparently conserved functions as diffusion barriers and molecular scaffolds. There are 13 confirmed septin paralogues in mammals, which may be ubiquitous or tissue specific. Septin hetero-oligomerization appears homology subgroup directed, which in turn determines the subunit arrangement of six- to eight-subunit core heteromers. Here we address functional properties of human SEPT9, which, due to variable mRNA splicing, exists as multiple isoforms that differ between tissues. Myeloid K562 cells express three SEPT9 isoforms, all of which have an equal propensity to hetero-oligomerize with SEPT7-containing hexamers to generate octameric heteromers. However, due to limiting amounts of SEPT9, K562 cells contain both hexameric and octameric heteromers. To generate cell lines with controllable hexamer-to-octamer ratios and that express single SEPT9 isoforms, we developed a gene product replacement strategy. By this means we identified SEPT9 isoform–specific properties that either facilitate septin heteromer polymerization along microtubules or modulate the size range of submembranous septin disks—a prevalent septin structure in nonadhered cells. Our findings show that the SEPT9 expression level directs the hexamer-to-octamer ratio, and that the isoform composition and expression level together determine higher-order arrangements of septins., Molecular Biology of the Cell, 23 (21), ISSN:1939-4586, ISSN:1059-1524

SUMMARY We assayed the vaccine potentialities of a recombinant S. bovis-derived glutathione S-transferase (rSb28GST), member of a molecular family already shown to have protective capacities in the S. mansoni and S. japonicum models. Injection of the rSb28GST in Freund's Complete Adjuvant resulted in good specific IgG responses allowing all the animals to display high antibody titres on the day of experimental challenge with S. bovis cercariae. No statistically significant differences were observed in the faecal egg output. Although tissue egg counts in vaccinated animals were lower than in controls, the difference was not statistically significant, apart from the number of eggs trapped in the liver (P

Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18-->E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.

The Sm16/SmSLP/SmSPO-1 (Sm16) protein is secreted by the parasite Schistosoma mansoni during skin penetration and has been ascribed immunosuppressive activities. Here we describe the strategy behind the design of a modified Sm16 protein with a decreased aggregation propensity, thus facilitating the expression and purification of an Sm16 protein that is soluble in physiological buffers. The Stokes radii and sedimentation coefficients of recombinant and native proteins indicate that Sm16 is an approximately nine-subunit oligomer. Analysis of truncated Sm16 derivatives showed that both oligomerization and binding to the plasma membrane of human cells depend on multiple C-terminal regions. For analysis of immunomodulatory activities, Sm16 was expressed in Pichia pastoris to facilitate the preparation of a pyrogen/endotoxin-free purified protein. Recombinant Sm16 was found to have no effect on T-lymphocyte activation, cell proliferation, or the basal level of cytokine production by whole human blood or monocytic cells. However, Sm16 exerts potent inhibition of the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and poly(I:C) while being less efficient at inhibiting the response to the TLR ligand peptidoglycan or a synthetic lipopeptide. Since Sm16 specifically inhibits the degradation of the IRAK1 signaling protein in LPS-stimulated monocytes, our findings indicate that inhibition is exerted proximal to the TLR complex.

Sm16/SmSLP/SPO-1 (Sm16) has been identified as a developmentally regulated protein that is released from specific glands of the Schistosoma mansoni parasite during skin penetration. Sm16 has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. Here we used a cell line to confirm signal peptide-dependent secretion and to define the secreted form of Sm16 for production in E. coli. We present evidence from both in vitro experiments and studies on transfected human cells that refute any functional similarity with stathmin/Op18. Instead of an Op18-like activity, we found that targeting of Sm16 to the cytosol of human cells, which was achieved by ectopic expression of Sm16 lacking the signal peptide, results in a caspase-dependent apoptotic response. Interestingly, by analysis of recombinant preparations we found that the secreted form of Sm16 is a lipid bilayer-binding protein that efficiently binds to the surface of diverse cell types by a polyanion-independent mechanism, which results in uptake by endocytosis. While the significance of the pro-apoptotic activity exerted by cytosolic Sm16 remains unclear, the present findings on cell-surface-binding properties of Sm16 seems likely to be of functional relevance during skin penetration of the parasite.

Of the four species of Bulinus found on Madagascar, three species: B. obtusispira, B. liratus and B. bavayi are endemic while the fourth, B. forskalii, is probably a recent introduction from the African mainland. The evolutionary relationships of these species with Bulinus species from Africa were studied by phylogenetic analysis of DNA sequence variation at two mitochondrial loci: cytochrome oxidase subunit I (COI) and large ribosomal subunit (LSU) or 16S. The observed levels of nucleotide divergence within Bulinus were substantial but may underestimate the true levels as there was evidence of ‘saturation' of transitional substitutions at both loci. A putative secondary structure model for the sequenced segment of the 16S was developed. Subsequent phylogenetic analysis using transversional changes only for both loci, showed that there were contrasting levels of divergence within the four species groups. B. obtusispira was consistently placed within the B. africanus group, appearing ancestral to this group and was closest to the basal node within Bulinus. Together with B. bavayi, the two species appear to have been isolated on Madagascar for a long time, contrasting with both B. liratus and B. forskalii that appear more recent colonisers; however, estimate of exact times of divergence is problematic. A PCR-RFLP assay was developed to enable identification and discrimination of B. obtusispira and B. liratus using discriminatory variation within the COI. To enable population genetic analysis within B. obtusispira, microsatellite markers were developed using an enrichment method and 8 primer pairs are reported. Laboratory infection experiments using Madasgacan S. haematobium from the Mahabo area showed that certain populations of B. obtusispira, B. liratus and B. bavayi were compatible.

Antibodies that preferentially and specifically target pathological oligomeric protein and peptide assemblies, as opposed to their monomeric and amyloid counterparts, provide therapeutic and diagnostic opportunities for protein misfolding diseases. Unfortunately, the molecular properties associated with oligomer-specific antibodies are not well understood, and this limits targeted design and development. We present here a generic method that enables the design and optimisation of oligomer-specific antibodies. The method takes a two-step approach where discrimination between oligomers and fibrils is first accomplished through identification of cryptic epitopes exclusively buried within the structure of the fibrillar form. The second step discriminates between monomers and oligomers based on differences in avidity. We show here that a simple divalent mode of interaction, as within e. g. the IgG isotype, can increase the binding strength of the antibody up to 1500 times compared to its monovalent counterpart. We expose how the ability to bind oligomers is affected by the monovalent affinity and the turnover rate of the binding and, importantly, also how oligomer specificity is only valid within a specific concentration range. We provide an example of the method by creating and characterising a spectrum of different monoclonal antibodies against both the A beta peptide and alpha-synuclein that are associated with Alzheimer's and Parkinson's diseases, respectively. The approach is however generic, does not require identification of oligomer-specific architectures, and is, in essence, applicable to all polypeptides that form oligomeric and fibrillar assemblies.

Cercarial emergence patterns were used to analyse the intraspecific variability within and between nine populations of Schistosoma haematobium collected along a transect line from the north to the South of the Ivory Coast (Africa) and using Bulinus truncatus or Bulinus globosus as intermediate snail hosts. Statistical comparison demonstrated the existence of a chronobiological polymorphism and the existence of three homogeneous groups of S. haematobium isolates with mean shedding times of the cercariae decreasing from the North to the South. The chronobiological variability observed was not correlated with the species of Bulinus (B. truncatus vs. B. globosus) implicated in the parasite transmission but with the climatic and vegetal features of the transmission area. S. haematobium from shaded sites of the forest zone (South) showed cercarial emergence patterns significantly earliest than that of S. haematobium from open sites of the savanna zone (North). Differences in sensitivity to light intensity could characterize the existence of eco-geographical races of S. haematobium one of the forest, the other from the savanna.

The capacity of a recombinant glutathione S-transferase from Schistosoma mansoni (rSm28GST) to vaccinate primates (Erythrocebus patas) against a heterologous infection with Schistosoma haematobium has been tested. Two injections of the purified molecule with Muramyl-Di-Peptide (MDP) as adjuvant resulted in a high level antibody response in the five immunized animals and in a significant reduction in worm fecundity compared to the controls which received adjuvant alone. Mean levels of daily egg excretion in urine an faeces were reduced by respectively 55% and 74% although perfusion revealed that worm burdens were similar in both groups. The protective effect was long lasting since it was maintained up to the end of the experiment, 42 weeks after infection. Hatching rates and the numbers of intra-uterine eggs were also significantly affected by the vaccination. Tissue eggs were also drastically diminished in the urogenital system (-80%) but the reduction was not statistically significant. One animal was not protected by the immunization. There was a good correlation between parasitological data and the intensity of bladder lesions assessed by microscopic examination. Polypoid formations together with an intense exudation of the lamina propria were frequently seen in the controls but rarely in the vaccinated group where formation of scar tissue was predominant. These results underline the vaccine potential of the recombinant Sm28GST as a possible valuable prophylactic tool for the control of egg-induced pathology and transmission of African schistosomes.

Many Gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated beta1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.

Characterization of schistosome populations from human in eastern Niger, through intra-uterine egg morphology of female parasites and phenotopic analyses of worms observed for acid phosphatase using electrophoretic separation, renders results that suggest an intogression of S. haematobium from man by genes of S. bovis, a parasite of domestic livestock. The origin of this introgression, that could implicate S. curassoni as well, another livestock parasite that hybridize with S. bovis, is discussed.

The emergence pattern of Schistosoma curassoni cercariae from Bulinus umbilicatus, whose adult worms parasitize bovine, caprine, and ovine ungulates in Niger, is of a circadian type with a mean emission time at 0855 hr +/- 1 hr 6 min, characteristic of the schistosome species parasitizing domestic or wild cattle. The comparison of this cercarial emergence pattern with those of the other 3 sympatric species of schistosomes (Schistosoma haematobium, Schistosoma bovis, and Schistosoma mansoni) shows a significant difference between the chronobiology of the cercariae infective for human and those infective for bovine hosts. This difference may improve epidemiological surveys based on snail prevalences by allowing the distinction between bulinids infected with human and bovine parasites.

L’efficacite d’un agent de surface, la laurylamidopropylbetaine a ete testee dans des conditions de terrain sur les miracidiums de Schistosoma mansoni. Ces experiences ont eu lieu dans des mares artificielles de 20 m3 reproduisant le plus fidelement possible certains types de sites de transmission. La degradation de la laurylamidopropylbetaine est assez lente et son efficacite sur les miracidiums de Schistosoma mansoni est tres nette : a la concentration de 2 ppm, le pourcentage de Mollusques infectes est egal a zero. Il est suggere d’incorporer le produit actif dans les savons afin de limiter la transmission des schistosomes dans les lieux de lavage et de baignade.

In a dry savanna village in Upper-Volta, after mass treatment with metrifonate (four doses of 7,5 mg/kg at three weeks intervals) checking of the population was carried out for the three following years. In children and adolescents, results show a progressive decline in the cure rate (69.0% one year after treatment, 38.1% three years after treatment) and in egg-output reduction (69.9% one year after treatment, 75.4% three years after treatment). In adults, these two parameters still remain high three years after treatment (90.0% for cure rate and 87.3% for egg-output reduction). The authors point out that in a dry savanna village with a sedentary population and a transmission limited to a small collection of water, chemotherapy even employed alone is an appreciable control method to relieve populations.

The laurylamidopropylbétaïne, a surface active substance, has been tested in field conditions, in Niger, against Schistosoma mansoni miracidia. Experiments were carried out in artificial ponds of 20 m3, in which conditions were similar, as far as possible, to conditions found in transmission sites. The product showed a satisfactory remanence and the percentage of infected molluscs dropped down to zero with a low (2 ppm) concentration. It is suggested that laurylamidopropylbetaïne could be used into soaps in order to renew constantly the product in washing and bathing places and reduce schistosome transmission.

This study of S. haematobium infection in communities associated with irrigated schemes shows that prevalence rates are unusually high in adults, especially in men; and distribution disease is probably associated with at least the following three factors: (i) distance of the place of residence from the main transmission sites (ii) the dynamics of transmission in the waterbody and (iii) the human/water contact and contamination patterns. During this chemotherapy trial, at the first survey, only a little more than half (57.47%) of the people recorded in the census was present. Five visits of the survey team were necessary to obtain a compliance rate of 80.51% of the entire population. The indifference of the local people to seek diagnosis and treatment increased with succeeding surveys. It is only, during a subsequent treatment survey, carried out during the season of low agricultural activity and following an official written convocation, that a compliance rate similar to that of the first survey was recorded. If treatment is not given immediately after diagnosis as many as a fifth of positive cases do not appear for treatment. Oltipraz and praziquantel were administered. Their effectiveness could not be truly compared as there was a time lag of six months separating the administration of the treatments. However, based on the results of this trial both drugs gave good results especially in egg output intensity. But, a decrease of this parameter was also observed in non treated people. Seasonal variations of egg output or decrease of transmission could be involved.