Choice of Reference Sequence and Assembler for Alignment of Listeria monocytogenes Short-Read Sequence Data Greatly Influences Rates of Error in SNP Analyses

The wide availability of whole-genome sequencing (WGS) and an abundance of open-source software have made detection of single-nucleotide polymorphisms (SNPs) in bacterial genomes an increasingly accessible and effective tool for comparative analyses. Thus, ensuring that real nucleotide differences between genomes (i.e., true SNPs) are detected at high rates and that the influences of errors (such as false positive SNPs, ambiguously called sites, and gaps) are mitigated is of utmost importance. The choices researchers make regarding the generation and analysis of WGS data can greatly influence the accuracy of short-read sequence alignments and, therefore, the efficacy of such experiments. We studied the effects of some of these choices, including: i) depth of sequencing coverage, ii) choice of reference-guided short-read sequence assembler, iii) choice of reference genome, and iv) whether to perform read-quality filtering and trimming, on our ability to detect true SNPs and on the frequencies of errors. We performed benchmarking experiments, during which we assembled simulated and real Listeria monocytogenes strain 08-5578 short-read sequence datasets of varying quality with four commonly used assemblers (BWA, MOSAIK, Novoalign, and SMALT), using reference genomes of varying genetic distances, and with or without read pre-processing (i.e., quality filtering and trimming). We found that assemblies of at least 50-fold coverage provided the most accurate results. In addition, MOSAIK yielded the fewest errors when reads were aligned to a nearly identical reference genome, while using SMALT to align reads against a reference sequence that is ∼0.82% distant from 08-5578 at the nucleotide level resulted in the detection of the greatest numbers of true SNPs and the fewest errors. Finally, we show that whether read pre-processing improves SNP detection depends upon the choice of reference sequence and assembler. In total, this study demonstrates that researchers should test a variety of conditions to achieve optimal results.     

Fatty Chains of Different Lipid Classes of Semliki Forest Virus and Host Cell Membranes

Semliki Forest virus was grown in BHK-21 cells. The major classes of phospho-and glycolipids of the virus were analyzed for the compositions of fatty acids, aldehydes, and sphingosine bases, and the major glycerophospholipids were analyzed for the relative proportions of alkenyl-acyl, alkyl-acyl, and diacyl forms. All viral lipid classes proved to be mixtures of several molecular species. Each class contained a characteristic mixture of fatty chains, which was different in all other classes. All viral lipid classes resembled their counterparts of the host plasma membrane and also those of the endoplasmic reticulum. The gangliosides of the virus and the plasma membrane proved to be similar even at the level of individual molecular species. The number of certain lipid molecules in an average virion was less than the number of the protein molecules.     

Kinetic modeling of hyperpolarized 13C label exchange between pyruvate and lactate in tumor cells

Measurements of the kinetics of hyperpolarized (13)C label exchange between [1-(13)C]pyruvate and lactate in suspensions of intact and lysed murine lymphoma cells, and in cells in which lactate dehydrogenase expression had been modulated by inhibition of the PI3K pathway, were used to determine quantitatively the role of enzyme activity and membrane transport in controlling isotope flux. Both steps were shown to share in the control of isotope flux in these cells. The kinetics of label exchange were well described by a kinetic model that employed rate constants for the lactate dehydrogenase reaction that had been determined previously from steady state kinetic studies. The enzyme showed pyruvate inhibition in steady state kinetic measurements, which the kinetic model predicted should also be observed in the isotope exchange measurements. However, no such pyruvate inhibition was observed in either intact cells or cell lysates and this could be explained by the much higher enzyme concentrations present in the isotope exchange experiments. The kinetic analysis presented here shows how lactate dehydrogenase activity can be determined from the isotope exchange measurements. The kinetic model should be useful for modeling the exchange reaction in vivo, particularly as this technique progresses to the clinic.     

Biomarker Profiling by Nuclear Magnetic Resonance Spectroscopy for the Prediction of All-Cause Mortality: An Observational Study of 17,345 Persons

Background

Early identification of ambulatory persons at high short-term risk of death could benefit targeted prevention. To identify biomarkers for all-cause mortality and enhance risk prediction, we conducted high-throughput profiling of blood specimens in two large population-based cohorts.

Methods and Findings

106 candidate biomarkers were quantified by nuclear magnetic resonance spectroscopy of non-fasting plasma samples from a random subset of the Estonian Biobank (n = 9,842; age range 18–103 y; 508 deaths during a median of 5.4 y of follow-up). Biomarkers for all-cause mortality were examined using stepwise proportional hazards models. Significant biomarkers were validated and incremental predictive utility assessed in a population-based cohort from Finland (n = 7,503; 176 deaths during 5 y of follow-up). Four circulating biomarkers predicted the risk of all-cause mortality among participants from the Estonian Biobank after adjusting for conventional risk factors: alpha-1-acid glycoprotein (hazard ratio [HR] 1.67 per 1–standard deviation increment, 95% CI 1.53–1.82, p = 5×10⁻³¹), albumin (HR 0.70, 95% CI 0.65–0.76, p = 2×10⁻¹⁸), very-low-density lipoprotein particle size (HR 0.69, 95% CI 0.62–0.77, p = 3×10⁻¹²), and citrate (HR 1.33, 95% CI 1.21–1.45, p = 5×10⁻¹⁰). All four biomarkers were predictive of cardiovascular mortality, as well as death from cancer and other nonvascular diseases. One in five participants in the Estonian Biobank cohort with a biomarker summary score within the highest percentile died during the first year of follow-up, indicating prominent systemic reflections of frailty. The biomarker associations all replicated in the Finnish validation cohort. Including the four biomarkers in a risk prediction score improved risk assessment for 5-y mortality (increase in C-statistics 0.031, p = 0.01; continuous reclassification improvement 26.3%, p = 0.001).

Conclusions

Biomarker associations with cardiovascular, nonvascular, and cancer mortality suggest novel systemic connectivities across seemingly disparate morbidities. The biomarker profiling improved prediction of the short-term risk of death from all causes above established risk factors. Further investigations are needed to clarify the biological mechanisms and the utility of these biomarkers for guiding screening and prevention. Please see later in the article for the Editors'' Summary     

Host controlled restriction and modification of bacteriophage Mu and Mu-promoted chromosome mobilization in Citrobacter freundii

Summary Bacteriophage Mu grown on Escherichia coli K12 (Mu. K) is restricted by wild type Citrobacter freundii. In two C. freundii mutants, where the restriction of foreign F factors is absent (de Graaff and Stouthamer, 1971), the restriction for Mu. K, although at a lower level, still exists. Consequently two host specificity systems exist in C. freundii, one affecting mainly the acceptance of foreign plasmidal and chromosomal DNA and one affecting foreign DNA of bacteriophage Mu. Mu is able to lysogenize C. freundii and to induce mutations at random in its chromosome. Furthermore Mu is able to promote the mobilization of the C. freundii chromosome in strains carrying F factors. Mu promoted integration of F ts 114 lac ⁺ into the C. freundii chromosome was observed, resulting in the formation of stable Hfr strains. In this way it is possible to devise a method for chromosome transfer in other genera than E. coli to which plasmids of E. coli can be transferred, but in which no chromosome mobilization is possible because of poor DNA homology between the foreign plasmid and the host chromosome.     

Inhibition of TRPC6 by protein kinase C isoforms in cultured human podocytes

Transient receptor potential canonical‐6 (TRPC6) ion channels, expressed at high levels in podocytes of the filtration barrier, are recently implicated in the pathogenesis of various forms of proteinuric kidney diseases. Indeed, inherited or acquired up‐regulation of TRPC6 activities are suggested to play a role in podocytopathies. Yet, we possess limited information about the regulation of TRPC6 in human podocytes. Therefore, in this study, we aimed at defining how the protein kinase C (PKC) system, one of the key intracellular signalling pathways, regulates TRPC6 function and expression. On human differentiated podocytes, we identified the molecular expressions of both TRPC6 and several PKC isoforms. We also showed that TRPC6 channels are functional since the TRPC6 activator 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) induced Ca²⁺‐influx to the cells. By assessing the regulatory roles of the PKCs, we found that inhibitors of the endogenous activities of classical and novel PKC isoforms markedly augmented TRPC6 activities. In contrast, activation of the PKC system by phorbol 12‐myristate 13‐acetate (PMA) exerted inhibitory actions on TRPC6 and suppressed its expression. Importantly, PMA treatment markedly down‐regulated the expression levels of PKCα, PKCβ, and PKCη reflecting their activation. Taken together, these results indicate that the PKC system exhibits a ‘tonic’ inhibition on TRPC6 activity in human podocytes suggesting that pathological conditions altering the expression and/or activation patterns of podocyte‐expressed PKCs may influence TRPC6 activity and hence podocyte functions. Therefore, it is proposed that targeted manipulation of certain PKC isoforms might be beneficial in certain proteinuric kidney diseases with altered TRPC6 functions.     

<Emphasis Type="Italic">Acremonium strictum</Emphasis> Fungaemia in a Paediatric Immunocompromised Patient: Diagnosis and Treatment Difficulties

During the past two decades, an increasing number of unusual moulds has been reported as responsible for septicaemia and systemic or disseminated infections in immunocompromised patients. Investigation of fever in a 10-year-old boy with acute myeloblastic leukaemia, including blood cultures on selective media, allowed the diagnosis of a fungaemia due to the slow-growing fungus Acremonium strictum. The patient recovered with liposomal amphotericin B (AmB) and voriconazole, followed by voriconazole alone due to AmB resistance. Facing a neutropenic patient with fever, clinicians usually suspect bacterial or viral aetiologies. This case, however, illustrates the need for mycological analysis of blood samples in febrile neutropenic patients and for antifungal susceptibility testing.     

Helicobacter pylori infection and childhood

Pediatric-based Helicobacter?pylori research continues to contribute significantly to our understanding of both clinical and pathophysiological aspects of this infection. Here, we review the published pediatric H.?pylori literature from April 2009-March 2010. Analysis of pediatric H.?pylori strains continues to suggest that cagA(+) and cagPAI competent strains are less prevalent than in adult isolates. Studies from the Middle East report a high H.?pylori prevalence and intrafamilial transmission. Data continue to show a lack of association between H.?pylori and recurrent abdominal pain of childhood, gastroesophageal reflux disease, and growth retardation. Recent probiotic trials have not shown a benefit on H.?pylori eradication in children, while sequential therapy remains an attractive therapeutic eradication strategy in children, which requires validation in different geographic regions.     

Jumping height in former elite athletes

To evaluate lower-limb explosive strength with respect to lifetime athletic activity, we measured vertical jumping height on a contact mat in former male runners (n = 28). soccer players (n = 31), weightlifters (n = 29) and shooters (n = 29) (age range 45 68 years). There were no statistically significant age-adjusted sport-group differences in jumping height, but differences by sport were evident among the subgroup of athletes without hip or knee osteoarthritis (n = 65) (P < 0.05). Thus, sports that increased jumping height also predisposed to lower-limb osteoarthritis. After adjustment for age and sport, the subjects without osteoarthritis jumped higher than those with osteoarthritis (n = 33) (P < 0.01). In a multiple linear regression analysis, age, reported hip and knee disability, and knee pain reduced jumping height. Hours spent in team-training during the past 12 months and the hours spent during their lifetime in power training were associated with improved vertical jumping height and together explained 41% of the difference among the subjects. The ability to jump even among athletes with hip or knee osteoarthritis would suggest that former elite athletes possess advanced lower limb muscle function.