HIV-infected individuals have an increased risk of cardiovascular disease (CVD). T-allele carriers of the CD14 C-260T single-nucleotide polymorphism (SNP) have reported increased expression of the LPS-binding receptor, CD14 and inflammation in the general population. Our aim was to explore the relationship of this SNP with monocyte/macrophage activation and inflammation and its association with sub-clinical atherosclerosis in HIV-infected individuals.
Patients with no pre-existing CVD risk factors on suppressive antiretroviral therapy were recruited from University Malaya Medical Centre, Malaysia (n = 84). The CD14 C-260T and TLR4 SNPs, Asp299Gly and Thr399Ile were genotyped and soluble(s) CD14 and sCD163 and high-sensitivity C-reactive protein, hsCRP were measured in plasma. Subclinical atherosclerosis was assessed by measuring carotid intima media thickness (cIMT). The association between CD14 C-260T SNP carriage and cIMT was assessed in a multivariable quantile regression model where a p-value of <0.05 was considered significant.
We found the CD14 C-260T T-allele in 56% of the cohort and evidence of subclinical atherosclerosis in 27%. TT genotype was associated with higher sCD163 (p = 0.009) but only marginally higher sCD14 (p = 0.209) and no difference in hsCRP (p = 0.296) compared to CC/CT. In multivariable analysis, only Framingham risk score was independently associated with higher cIMT while lower sCD163 was trending towards significance. No association was found in TT-genotype carriers and cIMT measurements.
The CD14 C-260T SNP was associated with increased monocyte activation but not systemic inflammation or cIMT in this HIV-infected cohort with low CVD risk profile.
HIV; Lipopolysaccharide; CD12 C-260T; Soluble CD14; Soluble CD163; Monocyte activation; C-reactive protein; Atherosclerosis; Carotid intima media thickness
The long bones of the vertebrate body are built by the initial formation of a cartilage template that is later replaced by mineralized bone. The proliferation and maturation of the skeletal precursor cells (chondrocytes) within the cartilage template and their replacement by bone is a highly coordinated process which, if misregulated, can lead to a number of defects including dwarfism and other skeletal deformities. This is exemplified by the fact that abnormal bone development is one of the most common types of human birth defects. Yet, many of the factors that initiate and regulate chondrocyte maturation are not known. We identified a recessive dwarf mouse mutant (pug) from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. pug mutant skeletal elements are patterned normally during development, but display a ~20% length reduction compared to wild-type embryos. We show that the pug mutation does not lead to changes in chondrocyte proliferation but instead promotes premature maturation and early ossification, which ultimately leads to disproportionate dwarfism. Using sequence capture and high-throughput sequencing, we identified a missense mutation in the Xylosyltransferase 1 (Xylt1) gene in pug mutants. Xylosyltransferases catalyze the initial step in glycosaminoglycan (GAG) chain addition to proteoglycan core proteins, and these modifications are essential for normal proteoglycan function. We show that the pug mutation disrupts Xylt1 activity and subcellular localization, leading to a reduction in GAG chains in pug mutants. The pug mutant serves as a novel model for mammalian dwarfism and identifies a key role for proteoglycan modification in the initiation of chondrocyte maturation.
forward genetics; Xylt1; dwarfism; chondrocytes; skeletal development; proteoglycans; CSPGs; HSPGs; glycosaminoglycans
Cell fate decisions during embryogenesis and adult life govern tissue formation, homeostasis and repair. Two key decisions that must be tightly coordinated are proliferation and differentiation. Overproliferation can lead to hyperplasia or tumor formation while premature differentiation can result in a depletion of proliferating cells and organ failure. Maintaining this balance is especially important in tissues that undergo rapid turnover like skin however, despite recent advances, the genetic mechanisms that balance cell differentiation and proliferation are still unclear. In an unbiased genetic screen to identify genes affecting early development, we identified an essential regulator of the proliferation-differentiation balance in epidermal progenitor cells, the Keratinocyte differentiation factor 1 (Kdf1; 1810019J16Rik) gene. Kdf1 is expressed in epidermal cells from early stages of epidermis formation through adulthood. Specifically, Kdf1 is expressed both in epidermal progenitor cells where it acts to curb the rate of proliferation as well as in their progeny where it is required to block proliferation and promote differentiation. Consequently, Kdf1 mutants display both uncontrolled cell proliferation in the epidermis and failure to develop terminal fates. Our findings reveal a dual role for the novel gene Kdf1 both as a repressive signal for progenitor cell proliferation through its inhibition of p63 and a strong inductive signal for terminal differentiation through its interaction with the cell cycle regulator Stratifin.
progenitor cells; epidermis; development; keratinocyte; cell fate decision; mouse; forward genetics
Clickers might own a bright future in China if properly introduced although they have not been widely acknowledged as an effective tool to facilitate English learning and teaching in Chinese contexts. By randomly selecting participants from undergraduates in a university in China over four academic years, this study aims to identify the impact of clickers on college English listening and speaking skills, and differences in cognitive loads between clickers and traditional multimedia assisted instruction modes. It was concluded that in China's college English class, compared with multimedia assisted instruction, (1) clickers could improve college English listening skills; (2) clickers could improve college English speaking skills; and (3) clickers could reduce undergraduates' cognitive loads in College English Class. Reasons for the results and defects in this study were also explored and discussed, based on learning, teaching and cognitive load theories. Some Suggestions for future research were also raised.
The purpose of this study was to investigate and summarize the surgical procedure and efficacy of transnasal endoscopic repair of cerebrospinal fluid (CSF) rhinorrhea. This is a retrospective study including 22 patients with CSF rhinorrhea who underwent transnasal endoscopic repair between January 2003 and August 2010. The location of the lesion was determined by both HRCT scan and intranasal endoscopy, and the lesion was repaired with a graft from autologous thigh muscle fascia. The grafts were placed and fixed above (inlay) or beneath (onlay) the lesions or defects. The causes of CSF rhinorrhea in our study included trauma, surgical injury, and meningoencephalocele, or idiopathic etiology. There were 15 of 17 patients with histories of trauma or surgical injury who were found to have defects in skull base by HRCT examination. The other 5 patients had an intact skull base, minor local osteoporosis, or bone resorption. All 22 patients had no reoccurrence of CSF rhinorrhea during the follow-up period, which ranges from 4 to 72 months. The transnasal endoscopic approach is a safe and effective procedure, with minimal invasion, for the repair of CSF rhinorrhea.
Cerebrospinal fluid rhinorrhea; Repair; Endoscopy
RNA splicing is the cellular process that has only recently been found to
be an important target for various cancers. Among the spliceosome genes that are
involved in cancers, SF3B1 is most frequently mutated. Recurrent mutation in
codon 625 has been found in uveal melanoma, but this mutation has not been
identified in cutaneous melanoma. We used whole-exome sequencing to explore the
mutational landscape of 295 melanoma samples, 231 of which are cutaneous
melanoma. Out of these cutaneous melanoma samples, we found 2 samples with R625
mutation in SF3B1 gene. The results were validated by Sanger sequencing. We
conclude that SF3B1 R625 mutation does occur in cutaneous melanoma, although
with a low frequency (~1%).
Melanoma; SF3B1; Cancer genetics; Whole-exome sequencing
The ability to three-dimensionally interweave biological tissue with functional electronics could enable the creation of bionic organs possessing enhanced functionalities over their human counterparts. Conventional electronic devices are inherently two-dimensional, preventing seamless multidimensional integration with synthetic biology, as the processes and materials are very different. Here, we present a novel strategy for overcoming these difficulties via additive manufacturing of biological cells with structural and nanoparticle derived electronic elements. As a proof of concept, we generated a bionic ear via 3D printing of a cell-seeded hydrogel matrix in the precise anatomic geometry of a human ear, along with an intertwined conducting polymer consisting of infused silver nanoparticles. This allowed for in vitro culturing of cartilage tissue around an inductive coil antenna in the ear, which subsequently enables readout of inductively-coupled signals from cochlea-shaped electrodes. The printed ear exhibits enhanced auditory sensing for radio frequency reception, and complementary left and right ears can listen to stereo audio music. Overall, our approach suggests a means to intricately merge biologic and nanoelectronic functionalities via 3D printing.
Cybernetics; tissue engineering; bioelectronics; cyborg organs; electronic implants; additive manufacturing
A common goal of tumor sequencing projects is finding genes whose
mutations are selected for during tumor development. This is accomplished by
choosing genes that have more non-synonymous mutations than expected from an
estimated background mutation frequency. While this background frequency is
unknown, it can be estimated using both the observed synonymous mutation
frequency and the non-synonymous to synonymous mutation ratio. The synonymous
mutation frequency can be determined across all genes or in a gene-specific
manner. This choice introduces an interesting trade-off. A gene-specific
frequency adjusts for an underlying mutation bias, but is difficult to estimate
given missing synonymous mutation counts. Using a genome-wide synonymous
frequency is more robust, but is less suited for adjusting biases. Studying four
evaluation criteria for identifying genes with high non-synonymous mutation
burden (reflecting preferential selection of expressed genes, genes with
mutations in conserved bases, genes with many protein interactions, and genes
that show loss of heterozygosity), we find that the gene-specific synonymous
frequency is superior in the gene expression and protein interaction tests. In
conclusion, the use of the gene-specific synonymous mutation frequency is well
suited for assessing a gene’s non-synonymous mutation burden.
cancer; sequencing; melanoma
There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance.
A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization.
The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.
Interpreting variants, especially noncoding ones, in the increasing
number of personal genomes is challenging. We used patterns of polymorphisms in
functionally annotated regions in 1092 humans to identify deleterious variants;
then we experimentally validated candidates. We analyzed both coding and
noncoding regions, with the former corroborating the latter. We found regions
particularly sensitive to mutations (“ultrasensitive”) and
variants that are disruptive because of mechanistic effects on
transcription-factor binding (that is, “motif-breakers”). We also
found variants in regions with higher network centrality tend to be deleterious.
Insertions and deletions followed a similar pattern to single-nucleotide
variants, with some notable exceptions (e.g., certain deletions and enhancers).
On the basis of these patterns, we developed a computational tool (FunSeq),
whose application to ~90 cancer genomes reveals nearly a hundred
candidate noncoding drivers.
[Purpose] The purpose of this study was to examine the effects of different types of
bridging exercises on the activities of the trunk muscles. [Methods] Twenty-four students
participated in this experiment. The activities of the internal oblique (IO), external
oblique (EO), rectus abdominis (RA), and erector spinae (ES) muscles were measured in four
different bridging exercises. [Results] There were significant differences in the IO, EO,
RA, and ES among the four kinds of bridging exercise. The activities of IO, EO and RA were
the highest in prone bridging (exercise 4), followed by unilateral bridging (exercise 3),
and supine bridging on balance pads (exercise 2). In conventional bridging (exercise 1),
the activities of IO, EO, and RA were the lowest. The activity of ES was the highest in
exercise 3 followed by exercises 2 and 1. The activity of ES was the lowest than in
exercise 1. [Conclusions] Bridging exercise in the prone position may be a more effective
method of enhancing trunk muscle activities exercises in other positions.
Bridge exercise; Muscle activity; EMG
Cilia are dynamic organelles that are essential for a vast array of developmental patterning events, including left-right specification, skeletal formation, neural development, and organogenesis. Despite recent advances in understanding cilia form and function, many key ciliogenesis components have yet to be identified. By using a forward genetics approach, we isolated a novel mutant allele (schlei) of the mouse Transmembrane protein 107 (Tmem107) gene, which we show here is critical for cilia formation and embryonic patterning. Tmem107 is required for normal Sonic hedgehog (Shh) signaling in the neural tube and acts in combination with Gli2 and Gli3 to pattern ventral and intermediate neuronal cell types. schlei mutants also form extra digits, and we demonstrate that Tmem107 acts in the Shh pathway to determine digit number, but not identity, by regulating a subset of Shh target genes. Phenotypically, schlei mutants share several features with other cilia mutants; however, spatial restriction of mutant phenotypes and lack of left-right patterning defects in schlei animals suggest differential requirements for Tmem107 in cilia formation in distinct tissues. Also, in contrast to mutants with complete loss of cilia, schlei mutants retain some function of both Gli activator and repressor forms. Together, these studies identify a previously unknown regulator of ciliogenesis and provide insight into how ciliary factors affect Shh signaling and cilia biogenesis in distinct tissues.
cilia; Sonic hedgehog (Shh); limb; neural tube; mouse; forward genetics; Tmem107
We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1P29S) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1P29S showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.
The composition of the upper respiratory tract microbial community may influence the risk for colonization by the acute otitis media (AOM) pathogens Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. We used culture-independent methods to describe upper respiratory tract microbial communities in healthy children and children with upper respiratory tract infection with and without concurrent AOM. Nasal swabs and data were collected in a cross-sectional study of 240 children between 6 months and 3 years of age. Swabs were cultured for S. pneumoniae, and real-time PCR was used to identify S. pneumoniae, H. influenzae, and M. catarrhalis. The V1-V2 16S rRNA gene regions were sequenced using 454 pyrosequencing. Microbial communities were described using a taxon-based approach. Colonization by S. pneumoniae, H. influenzae, and M. catarrhalis was associated with lower levels of diversity in upper respiratory tract flora. We identified commensal taxa that were negatively associated with colonization by each AOM bacterial pathogen and with AOM. The balance of these relationships differed according to the colonizing AOM pathogen and history of antibiotic use. Children with antibiotic use in the past 6 months and a greater abundance of taxa, including Lactococcus and Propionibacterium, were less likely to have AOM than healthy children (odds ratio [OR], 0.46; 95% confidence interval [CI], 0.25 to 0.85). Children with no antibiotic use in the past 6 months, a low abundance of Streptococcus and Haemophilus, and a high abundance of Corynebacterium and Dolosigranulum were less likely to have AOM (OR, 0.51; 95% CI, 0.31 to 0.83). An increased understanding of polymicrobial interactions will facilitate the development of effective AOM prevention strategies.
A 42-year-old man from Ghana presented with bilateral painful corneal perforations following ingestion of a sulphur-based antibiotic. Emergency bilateral penetrating keratoplasty was performed, with restoration of globe integrity. However, surgical complications arose such as non-healing epithelial defect, secondary infection, graft dehiscence, and mounting intraocular pressure. This case illustrates the challenges faced in managing corneal grafts in patients with already compromised ocular surfaces.
corneal perforation; epithelial defect; graft dehiscence; Stevens-Johnson syndrome; penetrating keratoplasty
Exome sequencing has transformed human genetic analysis and may do the same for other vertebrate model systems. However, a major challenge is sifting through the large number of sequence variants to identify the causative mutation for a given phenotype. In models like Xenopus tropicalis, an incomplete and occasionally incorrect genome assembly compounds this problem. To facilitate cloning of X. tropicalis mutants identified in forward genetic screens, we sought to combine bulk segregant analysis and exome sequencing into a single step.
Here we report the first use of exon capture sequencing to identify mutations in a non-mammalian, vertebrate model. We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping.
Exon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens. With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.
Exon capture sequencing; Forward genetics; Xenopus tropicalis; Bulk segregant analysis; Cilia; Kidney development; SNP discovery; Genome assembly; ccdc40; pax8
To report on the suturing techniques and aspects of postoperative management in penetrating keratoplasty in the United Kingdom.
A postal questionnaire was sent to 137 ophthalmic consultants identified from a Royal College of Ophthalmology database as having a special interest in anterior segment surgery. The questionnaire surveyed surgeon preferences for surgical and suturing technique for penetrating keratoplasty surgery, and the postoperative care of corneal grafts.
In all, 68% of questionnaires were completed and returned: 73% of respondents used a Flieringa ring or equivalent, 94% routinely used cardinal sutures, with 50.5% removing them at the end of the procedure. The most common suturing technique for routine penetrating keratoplasty was a single continuous suture (35%). In these cases, a 10/0 nylon suture was used by 89%. Sixty-six percent changed their technique in high-risk cases, 52% used a 3-1-1 knot, and 75% made a distinction between a reef and granny knot, with 76% using a reef. Thirty percent buried the knots within the donor material, and 29% within the host tissue. Twenty-five percent had no routine time for graft suture removal, but 41% removed them between 1 and 2 years post-surgery. After suture removal, 98% used steroids and 88% used topical antibiotics. Thirty-four percent stopped topical steroids before suture removal, with 38% stopping topical steroids more than 3 months prior to suture removal.
This survey demonstrates that there is considerable variation in suturing techniques and postoperative care for penetrating keratoplasty. These significant variations in practice need to be considered when interpreting outcomes and research.
corneal graft; penetrating keratoplasty; anterior segment surgery; cornea; corneal surgery; corneal transplantation
Genetic selection from libraries expressing proteins with randomized amino acid segments is a powerful approach to identify proteins with novel biological activities. Here, we assessed the utility of deep DNA sequencing to characterize the composition, diversity, size and stability of such randomized libraries. We used 454 pyrosequencing to sequence a retroviral library expressing small proteins with randomized transmembrane domains. Despite the potential for unintended random mutagenesis during its construction, the overall hydrophobic composition and diversity of the proteins encoded by the sequenced library conformed well to its design. In addition, our sequencing results allowed us to calculate a more accurate estimate of the number of different proteins encoded by the library and suggested that the traditional methods for estimating the size of randomized libraries may overestimate their true size. Our results further demonstrated that no significant genetic bottlenecks exist in the methods used to express complex retrovirus libraries in mammalian cells and recover library sequences from these cells. These findings suggest that deep sequencing can be used to determine the quality and content of other libraries with randomized segments and to follow individual sequences during selection.
bovine papillomavirus; E5 protein; randomization; sequencing; traptamers
The Notch signaling pathway plays an important role in the proliferation and differentiation of cells. Although recent studies have shown that Notch plays a role in the mechanisms of cisplatin resistance, the mechanism by which Notch plays roles in intrinsic or acquired cisplatin resistance remains unclear. In the present study, poorly differentiated nasopharyngeal carcinoma cells were treated with a γ-secretase inhibitor (DAPT), which led to a decrease in the Notch intracellular domain and inhibition of Notch signaling. Treatment was not sufficient to induce pronounced apoptosis of CNE-2 cells, but did result in the down-regulation of the P-glycoprotein and ERCC1 protein. In contrast, the combined treatment of DAPT and cisplatin induced substantial cell apoptosis compared to cisplatin treatment alone.
Notch signaling; cisplatin resistance; nasopharyngeal carcinoma
A computational pipeline for constructing a personal diploid genome and determining sites of allele-specific activity is developed. Using a regulatory network framework, allele-specific binding and expression are found to be significantly coordinated across the genome.
Software was developed for building a personal diploid genome sequence, and determining sites of allele-specific binding and expression (AlleleSeq).This computational pipeline was used to analyze variation data, and deeply sequenced RNA-Seq and ChIP-Seq datasets, for individual NA12878 from the 1000 Genomes Project.The interaction between allele-specific binding and allele-specific expression are investigated, revealing clear coordination.
To study allele-specific expression (ASE) and binding (ASB), that is, differences between the maternally and paternally derived alleles, we have developed a computational pipeline (AlleleSeq). Our pipeline initially constructs a diploid personal genome sequence (and corresponding personalized gene annotation) using genomic sequence variants (SNPs, indels, and structural variants), and then identifies allele-specific events with significant differences in the number of mapped reads between maternal and paternal alleles. There are many technical challenges in the construction and alignment of reads to a personal diploid genome sequence that we address, for example, bias of reads mapping to the reference allele. We have applied AlleleSeq to variation data for NA12878 from the 1000 Genomes Project as well as matched, deeply sequenced RNA-Seq and ChIP-Seq data sets generated for this purpose. In addition to observing fairly widespread allele-specific behavior within individual functional genomic data sets (including results consistent with X-chromosome inactivation), we can study the interaction between ASE and ASB. Furthermore, we investigate the coordination between ASE and ASB from multiple transcription factors events using a regulatory network framework. Correlation analyses and network motifs show mostly coordinated ASB and ASE.
allele-specific; ChIP-Seq; networks; RNA-Seq
The clinical features, autofluorescence, B-scan ultrasonography, optical coherence tomography and fluorescein angiography of the lesion were described. Multiple investigation modalities are needed to confirm the benign nature of the lesion. Careful evaluation and follow-up is crucial to avoid misdiagnosis and erroneous management.
melanocytoma; choroidal melanoma; autofluorescence; fluorescein angiography; B-scan ultrasonography; optical coherence tomography.
In the human genome, it has been estimated that considerably more sequence is under natural selection in non-coding regions [such as transcription-factor binding sites (TF-binding sites) and non-coding RNAs (ncRNAs)] compared to protein-coding ones. However, less attention has been paid to them. To study selective pressure on non-coding elements, we use next-generation sequencing data from the recently completed pilot phase of the 1000 Genomes Project, which, compared to traditional methods, allows for the characterization of a full spectrum of genomic variations, including single-nucleotide polymorphisms (SNPs), short insertions and deletions (indels) and structural variations (SVs). We develop a framework for combining these variation data with non-coding elements, calculating various population-based metrics to compare classes and subclasses of elements, and developing element-aware aggregation procedures to probe the internal structure of an element. Overall, we find that TF-binding sites and ncRNAs are less selectively constrained for SNPs than coding sequences (CDSs), but more constrained than a neutral reference. We also determine that the relative amounts of constraint for the three types of variations are, in general, correlated, but there are some differences: counter-intuitively, TF-binding sites and ncRNAs are more selectively constrained for indels than for SNPs, compared to CDSs. After inspecting the overall properties of a class of elements, we analyze selective pressure on subclasses within an element class, and show that the extent of selection is associated with the genomic properties of each subclass. We find, for instance, that ncRNAs with higher expression levels tend to be under stronger purifying selection, and the actual regions of TF-binding motifs are under stronger selective pressure than the corresponding peak regions. Further, we develop element-aware aggregation plots to analyze selective pressure across the linear structure of an element, with the confidence intervals evaluated using both simple bootstrapping and block bootstrapping techniques. We find, for example, that both micro-RNAs (particularly the seed regions) and their binding targets are under stronger selective pressure for SNPs than their immediate genomic surroundings. In addition, we demonstrate that substitutions in TF-binding motifs inversely correlate with site conservation, and SNPs unfavorable for motifs are under more selective constraints than favorable SNPs. Finally, to further investigate intra-element differences, we show that SVs have the tendency to use distinctive modes and mechanisms when they interact with genomic elements, such as enveloping whole gene(s) rather than disrupting them partially, as well as duplicating TF motifs in tandem.
Streptococcus pneumoniae is an important cause of otitis media and invasive disease. Since introduction of the heptavalent pneumococcal conjugate vaccine, there has been an increase in replacement disease due to serotype 19A clonal complex (CC)199 isolates. The goals of this study were to 1) describe genetic diversity among nineteen CC199 isolates from carriage, middle ear, blood, and cerebrospinal fluid, 2) compare CC199 19A (n = 3) and 15B/C (n = 2) isolates in the chinchilla model for pneumococcal disease, and 3) identify accessory genes associated with tissue-specific disease among a larger collection of S. pneumoniae isolates. CC199 isolates were analyzed by comparative genome hybridization. One hundred and twenty-seven genes were variably present. The CC199 phylogeny split into two main clades, one comprised predominantly of carriage isolates and another of disease isolates. Ability to colonize and cause disease did not differ by serotype in the chinchilla model. However, isolates from the disease clade were associated with faster time to bacteremia compared to carriage clade isolates. One 19A isolate exhibited hypervirulence. Twelve tissue-specific genes/regions were identified by correspondence analysis. After screening a diverse collection of 326 isolates, spr0282 was associated with carriage. Four genes/regions, SP0163, SP0463, SPN05002 and RD8a were associated with middle ear isolates. SPN05002 also associated with blood and CSF, while RD8a associated with blood isolates. The hypervirulent isolate's genome was sequenced using the Solexa paired-end sequencing platform and compared to that of a reference serotype 19A isolate, revealing the presence of a novel 20 kb region with sequence similarity to bacteriophage genes. Genetic factors other than serotype may modulate virulence potential in CC199. These studies have implications for the long-term effectiveness of conjugate vaccines. Ideally, future vaccines would target common proteins to effectively reduce carriage and disease in the vaccinated population.
chemokine; crystal structure; CXCL12; phylogenetic analysis; dimerization; SDF-1α; CXCR4