Characterization of the core and accessory genomes of Pseudomonas aeruginosa using bioinformatic tools Spine and AGEnt

Background

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for many infections in hospitalized and immunocompromised patients. Previous reports estimated that approximately 10% of its 6.6 Mbp genome varies from strain to strain and is therefore referred to as “accessory genome”. Elements within the accessory genome of P. aeruginosa have been associated with differences in virulence and antibiotic resistance. As whole genome sequencing of bacterial strains becomes more widespread and cost-effective, methods to quickly and reliably identify accessory genomic elements in newly sequenced P. aeruginosa genomes will be needed.

Results

We developed a bioinformatic method for identifying the accessory genome of P. aeruginosa. First, the core genome was determined based on sequence conserved among the completed genomes of twelve reference strains using Spine, a software program developed for this purpose. The core genome was 5.84 Mbp in size and contained 5,316 coding sequences. We then developed an in silico genome subtraction program named AGEnt to filter out core genomic sequences from P. aeruginosa whole genomes to identify accessory genomic sequences of these reference strains. This analysis determined that the accessory genome of P. aeruginosa ranged from 6.9-18.0% of the total genome, was enriched for genes associated with mobile elements, and was comprised of a majority of genes with unknown or unclear function. Using these genomes, we showed that AGEnt performed well compared to other publically available programs designed to detect accessory genomic elements. We then demonstrated the utility of the AGEnt program by applying it to the draft genomes of two previously unsequenced P. aeruginosa strains, PA99 and PA103.

Conclusions

The P. aeruginosa genome is rich in accessory genetic material. The AGEnt program accurately identified the accessory genomes of newly sequenced P. aeruginosa strains, even when draft genomes were used. As P. aeruginosa genomes become available at an increasingly rapid pace, this program will be useful in cataloging the expanding accessory genome of this bacterium and in discerning correlations between phenotype and accessory genome makeup. The combination of Spine and AGEnt should be useful in defining the accessory genomes of other bacterial species as well.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-737) contains supplementary material, which is available to authorized users.     

Elevation and orientation of external loads influence trunk neuromuscular response and spinal forces despite identical moments at the L5–S1 level

A wide range of loading conditions involving external forces with varying magnitudes, orientations and locations are encountered in daily activities. Here we computed the effect on trunk biomechanics of changes in force location (two levels) and orientation (5 values) in 4 subjects in upright standing while maintaining identical external moment of 15 Nm, 30 N m or 45 Nm at the L5–S1. Driven by measured kinematics and gravity/external loads, the finite element models yielded substantially different trunk neuromuscular response with moderate alterations (up to 24% under 45 Nm moment) in spinal loads as the load orientation varied. Under identical moments, compression and shear forces at the L5–S1 as well as forces in extensor thoracic muscles progressively decreased as orientation of external forces varied from downward gravity (90°) all the way to upward (−25°) orientation. In contrast, forces in local lumbar muscles followed reverse trends. Under larger horizontal forces at a lower elevation, lumbar muscles were much more active whereas extensor thoracic muscle forces were greater under smaller forces at a higher elevation. Despite such differences in activity pattern, the spinal forces remained nearly identical (<6% under 45 Nm moment). The published recorded surface EMG data of extensor muscles trend-wise agreed with computed local muscle forces as horizontal load elevation varied but were overall different from results in both local and global muscles when load orientation altered. Predictions demonstrate the marked effect of external force orientation and elevation on the trunk neuromuscular response and spinal forces and questions attempts to estimate spinal loads based only on consideration of moments at a spinal level.     

CT影像和X线平片在脊柱爆裂型骨折诊断中的对比分析

目的分析X线平片与CT影像在脊柱爆裂骨折诊断中的应用价值。方法选取2015年7月~2016年7月我院收治的脊柱爆裂骨折患者70例为研究对象,按随机数字表法分为对照组和观察组,对照组患者予以X线平片进行诊断,观察组患者予以CT影像进行诊断,对比分析两组患者的检出率、检出时间以及检查费用情况。结果对照组患者的检出率为65.71%,检出时间(9.21±0.13)min,检查费用(115±25.20)元;观察组患者的检出率为88.57%,检出时间(2.32±0.30)min,检查费用(628±52.6)元。两组比较差异均有统计学意义(均P0.05)。结论脊柱爆裂型骨折患者行CT影像学诊断的临床价值明显优于X线平片,值得作为脊柱爆裂型骨折的首选检查方法。     

Effectiveness of pedicle screw inclusion at the fracture level in short-segment fixation constructs for the treatment of thoracolumbar burst fractures: a computational biomechanics analysis

When treating thoracolumbar burst fractures (BF), short-segment posterior fixation (SSPF) represents a less invasive alternative to the traditional long-segment posterior fixation (LSPF) approach. However, hardware failure and loss of sagittal alignment have been reported in patients treated with SSPF. Including pedicle screws at the fracture level in SSPF constructs has been proposed to improve stiffness and reliability of the construct. Accordingly, the biomechanical performance of the proposed construct was compared to LSPF via a computational analysis. Pedicle screws at fracture level improved the performance of the short-segment construct. However, LSPF still represent a biomechanically superior option for treating thoracolumbar BF.     

Spinal loads and trunk muscles forces during level walking – A combined in vivo and in silico study on six subjects

During level walking, lumbar spine is subjected to cyclic movements and intricate loading of the spinal discs and trunk musculature. This study aimed to estimate the spinal loads (T12–S1) and trunk muscles forces during a complete gait cycle.Six men, 24–33 years walk barefoot at self-selected speed (4–5 km/h). 3D kinematics and ground reaction forces were recorded using a motion capturing system and two force plates, implemented in an inverse dynamic musculoskeletal model to predict the spinal loads and trunk muscles forces. Additionally, the sensitivity of the intra-abdominal pressure and lumbar segment rotational stiffness was investigated.Peak spinal loads and trunk muscle forces were between the gait instances of heel strike and toe off. In L4–L5 segment, sensitivity analysis showed that average peak compressive, antero-posterior and medio-lateral shear forces were 130–179%, 2–15% and 1–6%, with max standard deviation (±STD) of 40%, 6% and 3% of the body weight. Average peak global muscles forces were 24–55% (longissimus thoracis), 11–23% (iliocostalis thoracis), 12–16% (external oblique), 17–25% (internal oblique) and 0–8% (rectus abdominus) of body weight whereas, the average peak local muscles forces were 11–19% (longissimus lumborum), 14–31% (iliocostalis lumborum) and 12–17% (multifidus). Maximum ± STD of the global and local muscles forces were 13% and 8% of the body weight.Large inter-individual differences were found in peak compressive and trunk muscles forces whereas the sensitivity analysis also showed a substantial variation.     

δ-Catenin Regulates Spine Architecture via Cadherin and PDZ-dependent Interactions

The ability of neurons to maintain spine architecture and modulate it in response to synaptic activity is a crucial component of the cellular machinery that underlies information storage in pyramidal neurons of the hippocampus. Here we show a critical role for δ-catenin, a component of the cadherin-catenin cell adhesion complex, in regulating spine head width and length in pyramidal neurons of the hippocampus. The loss of Ctnnd2, the gene encoding δ-catenin, has been associated with the intellectual disability observed in the cri du chat syndrome, suggesting that the functional roles of δ-catenin are vital for neuronal integrity and higher order functions. We demonstrate that loss of δ-catenin in a mouse model or knockdown of δ-catenin in pyramidal neurons compromises spine head width and length, without altering spine dynamics. This is accompanied by a reduction in the levels of synaptic N-cadherin. The ability of δ-catenin to modulate spine architecture is critically dependent on its ability to interact with cadherin and PDZ domain-containing proteins. We propose that loss of δ-catenin during development perturbs synaptic architecture leading to developmental aberrations in neural circuit formation that contribute to the learning disabilities in a mouse model and humans with cri du chat syndrome.     

Effects of eight different ligament property datasets on biomechanics of a lumbar L4-L5 finite element model

Ligaments assist trunk muscles in balancing external moments and providing spinal stability. In absence of the personalized material properties for ligaments, finite element (FE) models use dispersed data from the literature. This study aims to investigate the relative effects of eight different ligament property datasets on FE model responses. Eight L4-L5 models distinct only in ligament properties were constructed and loaded under moment (15 N m) alone or combined with a compressive follower load (FL). Range of motions (RoM) of the disc-alone model matched well in vitro data. Ligament properties significantly affected only sagittal RoMs (∼3.0–7.1° in flexion and ∼3.8–5.8° in extension at 10 N m). Sequential removal of ligaments shifted sagittal RoMs in and out of the corresponding in vitro ranges. When moment was combined with FL, center of rotation matched in vivo data for all models (3.8 ± 0.9 mm and 4.3 ± 1.8 mm posterior to the disc center in flexion and extension, respectively). Under 15 N m sagittal moments, ligament strains were often smaller or within the in vitro range in flexion whereas some posterior ligament forces approached their failure forces in some models. Ligament forces varied substantially within the models and affected the moment-sharing and internal forces on the disc and facet joints. Intradiscal pressure (IDP) had the greatest variation between models in extension. None of the datasets yielded results in agreement with all reported measurements. Results emphasized the important role of ligaments especially under larger moments and the need for their accurate representation in search for valid spinal models.     

Cervical spine morphology and ligament property variations: A finite element study of their influence on sagittal bending characteristics

Cervical spine finite element models reported in biomechanical literature usually represent a static morphology. Not considering morphology as a model parameter limits the predictive capabilities for applications in personalized medicine, a growing trend in modern clinical practice. The objective of the study was to investigate the influence of variations in spinal morphology on the flexion-extension responses, utilizing mesh-morphing-based parametrization and metamodel-based sensitivity analysis. A C5-C6 segment was used as the baseline model. Variations of intervertebral disc height, facet joint slope, facet joint articular processes height, vertebral body anterior-posterior depth, and segment size were parametrized. In addition, material property variations of ligaments were considered for sensitivity analysis. The influence of these variations on vertebral rotation and forces in the ligaments were analyzed. The disc height, segmental size, and body depth were found to be the most influential (in the cited order) morphology variations; while among the ligament material property variations, capsular ligament and ligamentum flavum influenced vertebral rotation the most. Changes in disc height influenced forces in the posterior ligaments, indicating that changes in the anterior load-bearing column of the spine could have consequences on the posterior column. A method to identify influential morphology variations is presented in this work, which will help automation efforts in modeling to focus on variations that matter. This study underscores the importance of incorporating influential morphology parameters, easily obtained through computed tomography/magnetic resonance images, to better predict subject-specific biomechanical responses for applications in personalized medicine.     

Evaluation of the concentration of selected elements in serum patients with intervertebral disc degeneration

Quantitative analysis of the trace element content of human intervertebral discs (IVDs) is essential because it can identify specific enzymes or metabolites that may be related to human intervertebral disc degeneration (IVDD). The goal of this study was to assess the concentrations of copper (Cu), iron (Fe), manganese (Mn), lead (Pb), zinc (Zn), sodium (Na), magnesium (Mg), potassium (K), phosphorus (P), and calcium (Ca) in serum samples obtained from patients with IVDD in comparison to healthy volunteers (a control group). The study group consisted of 113 Caucasian patients qualified by a specialist neurosurgeon for microdiscectomy. The control group consisted of 113 healthy volunteers who met the eligibility criteria for blood donors. The examined clinical material was the serum samples obtained from both groups.Based on the quantitative analysis of selected elements, there were statistically significantly (p 0.05) higher concentrations of Cu (1180 μg/L±800 μg/L vs. 1230 μg/L±750 μg/L), Zn (790 μg/L±300 μg/L vs. 850 μg/L±200 μg/L), and Mg (21730 μg/L±4360 μg/L vs. 23820 μg/L±4990 μg/L) in the serum of healthy volunteers compared to those in the study group. In addition, statistically significant changes were not detected in the concentrations of any elements among either sex in either the study or control group or in their body mass index (BMI) values (p > 0.05). In the serum samples from the study group, the strongest relationships were noted between the concentrations of Zn and Pb (r = 0.61), Zn and P (r = 0.69), Zn and Ca (r = 0.84), Zn and Cu (r = 0.83), Mg and Ca (r = 0.74), and Ca and P (r = 0.98).It has been indicated that, above all, the concentrations of Cu, Zn, Ca, and Mg depend on the advancement of radiological changes, according to the Pfirrmann scale. However, no influence on pain intensity was found, depending on the concentration of the assessed elements.The analysis indicates that the determination of serum Cu, Zn, Ca, and Mg concentrations may have diagnostic significance in predicting the onset of lumbosacral IVDD. The predictive evaluation of changes in the concentrations of selected elements in patients with degenerative lumbar IVD lesions appears to be a promising, cost-effective strategy.