A novel linear plasmid mediates flagellar variation in Salmonella Typhi

Unlike the majority of Salmonella enterica serovars, Salmonella Typhi (S. Typhi), the etiological agent of human typhoid, is monophasic. S. Typhi normally harbours only the phase 1 flagellin gene (fliC), which encodes the H:d antigen. However, some S. Typhi strains found in Indonesia express an additional flagellin antigen termed H:z66. Molecular analysis of H:z66+ S. Typhi revealed that the H:z66 flagellin structural gene (fljB(z66)) is encoded on a linear plasmid that we have named pBSSB1. The DNA sequence of pBSSB1 was determined to be just over 27 kbp, and was predicted to encode 33 coding sequences. To our knowledge, pBSSB1 is the first non-bacteriophage-related linear plasmid to be described in the Enterobacteriaceae.     

Identification of common genetic variation that modulates alternative splicing

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron–exon boundary, although the distance between these SNPs and the intron–exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.     

A metabolomics perspective of human brain tumours

During the past decade or so, a wealth of information about metabolites in various human brain tumour preparations (cultured cells, tissue specimens, tumours in vivo) has been accumulated by global profiling tools. Such holistic approaches to cellular biochemistry have been termed metabolomics. Inherent and specific metabolic profiles of major brain tumour cell types, as determined by proton nuclear magnetic resonance spectroscopy ((1)H MRS), have also been used to define metabolite phenotypes in tumours in vivo. This minireview examines the recent advances in the field of human brain tumour metabolomics research, including advances in MRS and mass spectrometry technologies, and data analysis.     

Jumping robots: a biomimetic solution to locomotion across rough terrain

This paper introduces jumping robots as a means to traverse rough terrain; such terrain can pose problems for traditional wheeled, tracked and legged designs. The diversity of jumping mechanisms found in nature is explored to support the theory that jumping is a desirable ability for a robot locomotion system to incorporate, and then the size-related constraints are determined from first principles. A series of existing jumping robots are presented and their performance summarized. The authors present two new biologically inspired jumping robots, Jollbot and Glumper, both of which incorporate additional locomotion techniques of rolling and gliding respectively. Jollbot consists of metal hoop springs forming a 300 mm diameter sphere, and when jumping it raises its centre of gravity by 0.22 m and clears a height of 0.18 m. Glumper is of octahedral shape, with four 'legs' that each comprise two 500 mm lengths of CFRP tube articulating around torsion spring 'knees'. It is able to raise its centre of gravity by 1.60 m and clears a height of 1.17 m. The jumping performance of the jumping robot designs presented is discussed and compared against some specialized jumping animals. Specific power output is thought to be the performance-limiting factor for a jumping robot, which requires the maximization of the amount of energy that can be stored together with a minimization of mass. It is demonstrated that this can be achieved through optimization and careful materials selection.     

Interplay of pathogens, cytokines and other stress signals in the regulation of dendritic cell function

Dendritic cells (DCs) are the only antigen-presenting cell capable of activating na?ve T lymphocytes, and hence they play a crucial role in the induction of adaptive immunity. Immature DCs sample and process antigens, and efficiently sense a large variety of signals from the surrounding environment. Upon activation, they become capable to activate na?ve T cells and to direct the differentiation and polarization of effector T lymphocytes. It is becoming increasingly clear that different signals are able to determine distinct programs of DC differentiation and different forms of immunity and tolerance. In the past few years many advances have been made in addressing the action exerted by pathogen-associated molecular patterns (PAMPs), cytokines, chemokines, and other less characterized stress molecules on the activity of DCs. In this review we focus on the multiplicity of innate signals able to modulate the functional profile of DCs.     

Aquaporins contribute to diarrhoea caused by attaching and effacing bacterial pathogens

Attaching and effacing (A/E) pathogens such as enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) cause serious global health problems. These bacteria colonize the gastrointestinal system, attach to intestinal epithelial cells, efface (collapse) infected cell microvilli and cause overt diarrhoea that may ultimately result in death of the host. Although pathogenically induced diarrhoea is a significant global health issue, the molecular mechanisms that underlie this disease remain largely unknown. A natural murine infection model, employing the A/E pathogen Citrobacter rodentium, has been helpful in studying the diseases in vivo. C. rodentium colonize the colon at high levels, attach to colonocytes, efface microvilli and cause hyperplasia and inflammation in infected mice. As the disease progresses, the mice develop a diarrhoea-like phenotype. Aquaporin (AQP) water channels have been proposed to play a role in the normal dehydration of faecal contents. Here we examine whether C. rodentium infection may alter AQP localization in colonocytes. We demonstrate that during infection, AQP2 and AQP3 are mislocalized from their normal location along cell membranes to the cell cytoplasm. The change in localization of these proteins correlates with the diarrhoea-like phenotype present in infected mice. Mice that recover from the infection at 28-35 days post inoculum regain their normal membrane AQP localization. The altered localization of AQPs is partially dependent on the bacterial type III effector proteins EspF and EspG. We conclude that altered AQP localization may be a contributing factor to diarrhoea during bacterial infection.     

The ATM Signaling Cascade Promotes Recombination-Dependent Pachytene Arrest in Mouse Spermatocytes

Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13^mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative. Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.     

Pat1 protects centromere-specific histone H3 variant Cse4 from Psh1-mediated ubiquitination

Evolutionarily conserved histone H3 variant Cse4 and its homologues are essential components of specialized centromere (CEN)-specific nucleosomes and serve as an epigenetic mark for CEN identity and propagation. Cse4 is a critical determinant for the structure and function of the kinetochore and is required to ensure faithful chromosome segregation. The kinetochore protein Pat1 regulates the levels and spatial distribution of Cse4 at centromeres. Deletion of PAT1 results in altered structure of CEN chromatin and chromosome segregation errors. In this study, we show that Pat1 protects CEN-associated Cse4 from ubiquitination in order to maintain proper structure and function of the kinetochore in budding yeast. PAT1-deletion strains exhibit increased ubiquitination of Cse4 and faster turnover of Cse4 at kinetochores. Psh1, a Cse4-specific E3-ubiquitin ligase, interacts with Pat1 in vivo and contributes to the increased ubiquitination of Cse4 in pat1∆ strains. Consistent with a role of Psh1 in ubiquitination of Cse4, transient induction of PSH1 in a wild-type strain resulted in phenotypes similar to a pat1∆ strain, including a reduction in CEN-associated Cse4, increased Cse4 ubiquitination, defects in spatial distribution of Cse4 at kinetochores, and altered structure of CEN chromatin. Pat1 interacts with Scm3 and is required for its maintenance at kinetochores. In conclusion, our studies provide novel insights into mechanisms by which Pat1 affects the structure of CEN chromatin and protects Cse4 from Psh1-mediated ubiquitination for faithful chromosome segregation.