KGraph: a system for visualizing and evaluating complex genetic associations

The KGraph is a data visualization system that has been developed to display the complex relationships between the univariate and bivariate associations among an outcome of interest, a set of covariates, and a set of genetic factors, such as single nucleotide polymorphisms (SNPs). It allows for easy viewing and interpretation of genetic associations, correlations among covariates and SNPs, and information about the replication and cross-validation of the associations. The KGraph allows the user to more easily investigate multicollinearity and confounding through visualization of the multidimensional correlation structure underlying genetic associations. It emphasizes gene-environment and gene-gene interaction, both important components of any genetic system that are often overlooked in association frameworks. AVAILABILITY: http://www.epidkardia.sph.umich.edu/software/kgrapher     

Diurnal variation in temperature, mental and physical performance, and tasks specifically related to football (soccer)

Football (soccer) training and matches are scheduled at different times throughout the day. Association football involves a variety of fitness components as well as psychomotor and game-related cognitive skills. The purpose of the present research, consisting of two separate studies, was to determine whether game-related skills varied with time of day in phase with global markers of both performance and the body clock. In the first study, eight diurnally active male association football players (19.1+/-1.9 yrs of age; mean+/-SD) with 10.8+/-2.1 yrs playing experience participated. Measurements were made on different days at 08:00, 12:00, 16:00, and 20:00 h in a counterbalanced manner. Time-of-day changes in intra-aural temperature (used as a marker of the body clock), grip strength, reaction times, flexibility (markers of aspects of performance), juggling and dribbling tasks, and wall-volley test (football-specific skills) were compared. Significant (repeated measures analysis of variance, ANOVA) diurnal variations were found for body temperature (p<0.0005), choice reaction time (p<0.05), self-rated alertness (p<0.0005), fatigue (p<0.05), forward (sit-and-reach) flexibility (p<0.02), and right-hand grip strength (p<0.02), but not left-hand grip strength (p=0.40) nor whole-body (stand-and-reach) flexibility (p=0.07). Alertness was highest and fatigue lowest at 20:00 h. Football-specific skills of juggling performance showed significant diurnal variation (p<0.05, peak at 16:00 h), whereas performance on the wall-volley test tended to peak at 20:00 h and dribbling showed no time-of-day effect (p=0.55). In a second study, eight diurnally active subjects (23.0+/-0.7 yrs of age) completed five test sessions, at the same times as in the first study but with a second session at 08:00 h. Test-re-test comparisons at 08:00 h for all components indicated good reliability. Intra-aural temperature showed a significant time-of-day effect (p<0.001) with mean temperature at 16:00 h (36.4 degrees C) higher than at 08:00 h (35.4 degrees C). There was no significant effect of chronotype on the temperature acrophase (peak time) (p>0.05). Diurnal variation was found for performance tests, including sit-and-reach flexibility (p<0.01) and spinal hyper-extension (p<0.05). Peaks occurred between 16:00 and 20:00 h and the daytime changes paralleled the temperature rhythm. Diurnal variation was also found for football-specific tests, including dribbling time (p<0.001, peak at 20:00 h) and chip test performance (p<0.01), being more accurate at 16:00 h (mean error=0.75 m) than at 08:00 h (mean error=1.01 m). Results indicate football players perform at an optimum between 16:00 and 20:00 h when not only football-specific skills but also measures of physical performance are at their peak. Body temperature peaked at a similar time, but positive mood states seemed to peak slightly earlier. While causal links cannot be established in these experiments, the results indicate that the diurnal variation of some aspects of football performance is affected by factor(s) other than body temperature alone.     

Growth of Campylobacter jejuni on nitrate and nitrite: electron transport to NapA and NrfA via NrfH and distinct roles for NrfA and the globin Cgb in protection against nitrosative stress

Pathways of electron transport to periplasmic nitrate (NapA) and nitrite (NrfA) reductases have been investigated in Campylobacter jejuni, a microaerophilic food-borne pathogen. The nap operon is unusual in lacking napC (encoding a tetra-haem c-type cytochrome) and napF, but contains a novel gene of unknown function, napL. The iron-sulphur protein NapG has a major role in electron transfer to the NapAB complex, but we show that slow nitrate-dependent growth of a napG mutant can be sustained by electron transfer from NrfH, the electron donor to the nitrite reductase NrfA. A napL mutant possessed approximately 50% lower NapA activity than the wild type but showed normal growth with nitrate as the electron acceptor. NrfA was constitutive and was shown to play a role in protection against nitrosative stress in addition to the previously identified NO-inducible single domain globin, Cgb. However, nitrite also induced cgb expression in an NssR-dependent manner, suggesting that growth of C. jejuni with nitrite causes nitrosative stress. This was confirmed by lack of growth of cgb and nssR mutants, and slow growth of the nrfA mutant, in media containing nitrite. Thus, NrfA and Cgb together provide C. jejuni with constitutive and inducible components of a robust defence against nitrosative stress.     

The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress

We have examined the contribution of metacaspases to the growth and stress response of the opportunistic human mould pathogen, Aspergillus fumigatus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic-like programmed cell death. Single metacaspase-deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase-deficient mutant, DeltacasA/DeltacasB, was constructed by disrupting both genes. Stationary phase cultures of wild-type A. fumigatus were associated with the appearance of typical markers of apoptosis, including elevated proteolytic activity against caspase substrates, phosphatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phosphatidylserine exposure was not observed in stationary phase cultures of the DeltacasA/DeltacasB mutant, although caspase activity and viability was indistinguishable from wild type. The mutant retained wild-type virulence and showed no difference in sensitivity to a range of pro-apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the DeltacasA/DeltacasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contributes to the apoptotic-like loss of membrane phospholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress.     

FliK regulates flagellar hook length as an internal ruler

The mechanism of length control of the flagellar hook is under debate between two theories. One claims that the FliK directly measures the hook length as a molecular ruler, while the other claims that the cytoplasmic substructure measures the amount of hook subunits to determine the hook length. Both agree that the FliK C-terminal domain catalyses the substrate-specificity switch to terminate hook elongation. In this study, we systematically created fliK mutants with deletions and insertions at various sites within the FliK N-terminal domain and analysed their effects on the final hook length. Insertions of peptide fragments from the Yersinia YscP into FliK gave rise to hooks with defined lengths, which was proportional to the molecular size of the FliK-YscP chimeras. Among fliK deletion mutants, only those with small truncations in three specific sites of FliK produced hooks of a defined, shortened length. For the majority of deletion mutants, FliK was secreted, but hook length was not controlled. On the other hand, for some deletion mutants FliK was not secreted, but the hook length was controlled, indicating that FliK secretion is not necessary for hook-length control. We conclude that FliK regulates hook length as an internal molecular ruler.     

Analysis of zebrafish sidetracked mutants reveals a novel role for Plexin A3 in intraspinal motor axon guidance

One of the earliest guidance decisions for spinal cord motoneurons occurs when pools of motoneurons orient their growth cones towards a common, segmental exit point. In contrast to later events, remarkably little is known about the molecular mechanisms underlying intraspinal motor axon guidance. In zebrafish sidetracked (set) mutants, motor axons exit from the spinal cord at ectopic positions. By single-cell labeling and time-lapse analysis we show that motoneurons with cell bodies adjacent to the segmental exit point properly exit from the spinal cord, whereas those farther away display pathfinding errors. Misguided growth cones either orient away from the endogenous exit point, extend towards the endogenous exit point but bypass it or exit at non-segmental, ectopic locations. Furthermore, we show that sidetracked acts cell autonomously in motoneurons. Positional cloning reveals that sidetracked encodes Plexin A3, a semaphorin guidance receptor for repulsive guidance. Finally, we show that sidetracked (plexin A3) plays an additional role in motor axonal morphogenesis. Together, our data genetically identify the first guidance receptor required for intraspinal migration of pioneering motor axons and implicate the well-described semaphorin/plexin signaling pathway in this poorly understood process. We propose that axonal repulsion via Plexin A3 is a major driving force for intraspinal motor growth cone guidance.     

Development of intracellular bacterial communities of uropathogenic Escherichia coli depends on type 1 pili

Uropathogenic Escherichia coli, the predominant causative agent of urinary tract infections, use type 1 pili to bind and invade bladder epithelial cells. Upon entry, the bacteria rapidly replicate and enter a complex developmental pathway ultimately forming intracellular bacterial communities (IBCs), a niche with biofilm-like properties protected from innate defences and antibiotics. Paradoxically, bacteria within IBCs produce type 1 pili, an organelle thought only to be an extracellular colonization factor. Thus, we investigated the function of type 1 pili in IBC development. The cystitis isolate, UTI89, was genetically manipulated for conditional fim expression under control of the tet promoter. In this strain, UTI89-tetR/P(tet) fim, piliation is constitutively inhibited by the tetracycline repressor, TetR. Repression is relieved by anhydrotetracycline (AHT) treatment. UTI89-tetR/P(tet) fim and the isogenic control strain, UTI89-tetR, grown in the presence of AHT, colonized the bladder and invaded the superficial umbrella cells at similar levels at early times in a murine model of infection. However, after invasion UTI89-tetR/P(tet) fim became non-piliated and was unable to form typical IBCs comprised of tightly packed, coccoid-shaped bacteria in contrast to the control strain, UTI89-tetR. Thus, this work changes the extracellular colonization functional paradigm of pili by demonstrating their intracellular role in biofilm formation.     

Gene conversion is a convergent strategy for pathogen antigenic variation

Recent studies on three unrelated vector-borne pathogens, Anaplasma marginale, Borrelia hermsii and Trypanosoma brucei, illustrate the central importance of gene conversion as a mechanism for antigenic variation, which results in subsequent evasion of the immune response and persistence in the reservoir host. The combination of genome sequence data and in vivo studies tracking variant emergence not only provides insight into the genetic mechanisms for variant generation and hierarchy in variant expression but also highlights gaps in our knowledge regarding variant capacity and usage in vivo.