The hidden perils of read mapping as a quality assessment tool in genome sequencing

This article provides a comparative analysis of the various methods of genome sequencing focusing on verification of the assembly quality. The results of a comparative assessment of various de novo assembly tools, as well as sequencing technologies, are presented using a recently completed sequence of the genome of Lactobacillus fermentum 3872. In particular, quality of assemblies is assessed by using CLC Genomics Workbench read mapping and Optical mapping developed by OpGen. Over-extension of contigs without prior knowledge of contig location can lead to misassembled contigs, even when commonly used quality indicators such as read mapping suggest that a contig is well assembled. Precautions must also be undertaken when using long read sequencing technology, which may also lead to misassembled contigs

Horizontal DNA transfer mechanisms of bacteria as weapons of intragenomic conflict

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated

Prolactin receptor is a negative prognostic factor in patients with squamous cell carcinoma of the head and neck

Background: The influence of human prolactin (hPRL) on the development of breast and other types of cancer is well established. Little information, however, exists on the effects of hPRL on squamous cell carcinomas of the head and neck (SCCHNs). Methods: In this study, we evaluated prolactin receptor (PRLR) expression in SCCHN cell lines and assessed by immunohistochemistry the expression in 89 patients with SCCHNs. The PRLR expression was correlated with clinicopathological characteristics as well as clinical outcome. The effect of hPRL treatment on tumour cell growth was evaluated in vitro. Results: Immunoreactivity for PRLR was observed in 85 out of 89 (95%) tumours. Multivariate COX regression analysis confirmed high levels of PRLR expression (>25% of tumour cells) to be an independent prognostic factor with respect to overall survival (HR=3.70, 95% CI: 1.14–12.01; P=0.029) and disease-free survival (P=0.017). Growth of PRLR-positive cancer cells increased in response to hPRL treatment. Conclusion: Our data indicate that hPRL is an important growth factor for SCCHN. Because of PRLR expression in a vast majority of tumour specimens and its negative impact on overall survival, the receptor represents a novel prognosticator and a promising drug target for patients with SCCHNs

The effects of varying protein and energy intakes on the growth and body composition of very low birth weight infants

<p>Abstract</p> <p>Objective</p> <p>To determine the effects of high dietary protein and energy intake on the growth and body composition of very low birth weight (VLBW) infants.</p> <p>Study design</p> <p>Thirty-eight VLBW infants whose weights were appropriate for their gestational ages were assessed for when they could tolerate oral intake for all their nutritional needs. Thirty-two infants were included in a longitudinal, randomized clinical trial over an approximate 28-day period. One control diet (standard preterm formula, group A, n = 8, 3.7 g/kg/d of protein and 129 kcal/kg/d) and two high-energy and high-protein diets (group B, n = 12, 4.2 g/kg/d and 150 kcal/kg/d; group C, n = 12, 4.7 g/kg/d and 150 kcal/kg/d) were compared. Differences among groups in anthropometry and body composition (measured with bioelectrical impedance analysis) were determined. An enriched breast milk group (n = 6) served as a descriptive reference group.</p> <p>Results</p> <p>Groups B and C displayed greater weight gains and higher increases in fat-free mass than group A.</p> <p>Conclusion</p> <p>An intake of 150 kcal/kg/d of energy and 4.2 g/kg/d of protein increases fat-free mass accretion in VLBW infants.</p

Medical students’ preparedness for professional activities in early clerkships

Background Sufficient preparedness is important for transitions to workplace participation and learning in clinical settings. This study aims to analyse medical students’ preparedness for early clerkships using a three-dimensional, socio-cognitive, theory-based model of preparedness anchored in specific professional activities and their supervision level. Methods Medical students from a competency-based undergraduate curriculum were surveyed about preparedness for 21 professional activities and level of perceived supervision during their early clerkships via an online questionnaire. Preparedness was operationalized by the three dimensions of confidence to carry out clerkship activities, being prepared through university teaching and coping with failure by seeking support. Factors influencing preparedness and perceived stress as outcomes were analysed through step-wise regression. Results Professional activities carried out by the students (n = 147; 19.0%) and their supervision levels varied. While most students reported high confidence to perform the tasks, the activity-specific analysis revealed important gaps in preparation through university teaching. Students regularly searched for support in case of difficulty. One quarter of the variance of each preparedness dimension was explained by self-efficacy, supervision quality, amount of prior clerkship experience and nature of professional activities. Preparedness contributed to predicting perceived stress. Conclusions The applied three-dimensional concept of preparedness and the task-specific approach provided a detailed and meaningful view on medical students’ workplace participation and experiences in early clerkships

The impact of disease progression on perceived health status and quality of life of long-term cancer survivors

Introduction The number of cancer survivors experiencing disease progression (DP) is increasing with the number of cancer survivors. However, little is known whether DP affects health-related quality of life (HRQL) of long-term cancer survivors. We aimed therefore to compare the health status (HS) and HRQL of DP and disease-free (DF) survivors up to 15 years after initial diagnosis. Methods 232 cancer survivors with DP identified through the Eindhoven Cancer Registry were matched with 232 DF survivors of similar demographic and clinical characteristics. Patients completed generic HS (SF-36) and cancer-specific HRQL (QOL-CS) questionnaires 5-15 years after diagnosis. Results Compared with DF survivors, DP survivors exhibited significantly lower scores on all SF-36 and QOL-CS (except spiritual well-being) dimensions. DF survivors had better scores than the normative population on all SF-36 dimensions. Among survivors with DP, those with short survival (<5 years) had significantly poorer HS scores on all dimensions except bodily pain compared with the normative population. Comparatively, the long survival (≥5 years) DP group had better HRQL than the short DP group but poorer HRQL than the normative population. In multivariate analyses, DP and DF survival time were independently associated with aspects of HS and HRQL in cancer survivors. Discussions/Conclusions DP cancer survivors have poorer long-term HS and HRQL compared with DF survivors. However, there is suggestion that HS and HRQL does improve over time following DP. Implication for Cancer Survivors Although DP survivors report poorer long-term HRQL compared with DF cancer survivors, results suggest that time can attenuate the distress of DP on HRQL. Psycho-educational programs could help to increase patients' sense of empowerment and personal control should DP occur

Conservation of the S10-spc-α Locus within Otherwise Highly Plastic Genomes Provides Phylogenetic Insight into the Genus Leptospira

S10-spc-α is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-α locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-α locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-α locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously

The identification of genes important in pseudomonas syringae pv. phaseolicola plant colonisation using in vitro screening of transposon libraries

The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant’s natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interactio

Psychological and Environmental Correlates of Well-being Among Undergraduate University Students

This study explored whether the university environment provides similar well-being enhancing elements to those that have been found in the workplace and school contexts. Whether psychological inflexibility accounts for well-being over and above personality and environmental influences was also explored. A representative sample of 163 undergraduate university students in an Australian university completed an online survey measuring the key constructs. Environmental influences assessed included financial resources, physical security, opportunity to use new skills, externally generated goals, variety, environmental clarity, interpersonal contact, and valued social position. Hierarchical multiple linear regression analyses were then conducted to test for predictors of three domains of subjective well-being: positive affect, negative affect, and life satisfaction. The results suggested that university context contributes significantly to undergraduate students’ well-being by providing a valued social role, externally generated goals, and variety. Students’ perception of their physical security was also an important influence on their well-being. These results are consistent with the literature on well-being and employment. Neuroticism significantly predicted negative affect, while psychological inflexibility accounted for unique variance in life satisfaction and negative affect even when personality and environmental influences were taken into account. The implications of these findings for enhancing undergraduate university students’ well-being are discussed

Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors

Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species