Evanescent wave and video microscopy methods for directly measuring interactions between surface-immobilized biomolecules

Spatial and temporal tracking of passively diffusing functionalized colloids continues to be an improving and auspicious approach to measuring weak specific and non-specific biomolecular interactions. Evidence of this is given by the recent increase in published studies involving the development and implementation of these methods. The primary aim of the work presented in this dissertation was to modify and optimize video microscopy (VM) and total internal reflection microscopy (TIRM) methods to permit the collection of equilibrium binding and sampling data from interaction of surface-immobilized biomolecules. Supported lipid bilayers were utilized as model systems for functionalizing colloid and wall surfaces. Preliminary results measuring calcium-specific protein-protein interactions between surface immobilized cadherin fragments demonstrate the potential utility of this experimental system and these methods. Additionally, quantum dot-modified colloids were synthesized and evanescent wave-excited luminescence from these particles was used to construct potential energy profiles. Results from this work demonstrate that colloids can be used as ultra-sensitive probes of equilibrium interactions between biomolecules, and specialized probes, such as those modified with quantum dots, could be used in a spectral multiplexing mode to simultaneously monitor multiple interactions

Microfluidic devices for the rapid and automated processing of sample populations

Microfluidic devices for the rapid and automated processing of sample populations are provided. Described are multiplexer microfluidic devices configured to serially deliver a plurality of distinct sample populations to a sample processing element rapidly and automatically, without cross-contaminating the distinct sample populations. Also provided are microfluidic sample processing elements that can be used to rapidly and automatically manipulate and/or interrogate members of a sample population. The microfluidic devices can be used to improve the throughput and quality of experiments involving model organisms, such as C. elegans.Board of Regents, University of Texas Syste

An Automated Microfluidic Multiplexer for Fast Delivery of C. elegans Populations from Multiwells

Automated biosorter platforms, including recently developed microfluidic devices, enable and accelerate high-throughput and/or high-resolution bioassays on small animal models. However, time-consuming delivery of different organism populations to these systems introduces a major bottleneck to executing large-scale screens. Current population delivery strategies rely on suction from conventional well plates through tubing periodically exposed to air, leading to certain disadvantages: 1) bubble introduction to the sample, interfering with analysis in the downstream system, 2) substantial time drain from added bubble-cleaning steps, and 3) the need for complex mechanical systems to manipulate well plate position. To address these concerns, we developed a multiwell-format microfluidic platform that can deliver multiple distinct animal populations from on-chip wells using multiplexed valve control. This Population Delivery Chip could operate autonomously as part of a relatively simple setup that did not require any of the major mechanical moving parts typical of plate-handling systems to address a given well. We demonstrated automatic serial delivery of 16 distinct C. elegans worm populations to a single outlet without introducing any bubbles to the samples, causing cross-contamination, or damaging the animals. The device achieved delivery of more than 90% of the population preloaded into a given well in 4.7 seconds; an order of magnitude faster than delivery modalities in current use. This platform could potentially handle other similarly sized model organisms, such as zebrafish and drosophila larvae or cellular micro-colonies. The device’s architecture and microchannel dimensions allow simple expansion for processing larger numbers of populations.The authors would like to thank the National Institutes of Health (www.nih.gov) for its generous support of this research. Specifically, the grants that made this work possible are the NIH Director's Transformative Award (NIH R01 AG041135), NIH R21 NS067340, and NIH R01 NS060129. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biomedical EngineeringElectrical and Computer EngineeringMechanical Engineerin

Presacral malakoplakia presenting as foot drop: a case report

Background: Malakoplakia is a rare condition characterized by inflammatory masses with specific histological characteristics. These soft tissue masses can mimic tumors and tend to develop in association with chronic or recurrent infections, typically of the urinary tract. A specific defect in innate immunity has been described. In the absence of randomized controlled trials, management is based on an understanding of the biology and on case reports. Case presentation: Here we describe a case of presacral malakoplakia in a British Indian woman in her late 30s, presenting with complex unilateral foot drop. Four years earlier, she had suffered a protracted episode of intrapelvic sepsis following a caesarean delivery. Resection of her presacral soft tissue mass was not possible. She received empiric antibiotics, a cholinergic agonist, and ascorbic acid. She responded well to medical management both when first treated and following a recurrence of symptoms after completing an initial 8 months of therapy. Whole exome sequencing of the patient and her parents was undertaken but no clear causal variant was identified. Conclusions: Malakoplakia is uncommon but the diagnosis should be considered where soft tissue masses develop at the site of chronic or recurrent infections. Obtaining tissue for histological examination is key to making the diagnosis. This case suggests that surgical resection is not always needed to achieve a good clinical and radiological outcome

Recombination in Streptococcus pneumoniae Lineages Increase with Carriage Duration and Size of the Polysaccharide Capsule

Streptococcus pneumoniae causes a high burden of invasive pneumococcal disease (IPD) globally, especially in children from resource-poor settings. Like many bacteria, the pneumococcus can import DNA from other strains or even species by transformation and homologous recombination, which has allowed the pneumococcus to evade clinical interventions such as antibiotics and pneumococcal conjugate vaccines (PCVs). Pneumococci are enclosed in a complex polysaccharide capsule that determines the serotype; the capsule varies in size and is associated with properties including carriage prevalence and virulence. We determined and quantified the association between capsule and recombination events using genomic data from a diverse collection of serotypes sampled in Malawi. We determined both the amount of variation introduced by recombination relative to mutation (the relative rate) and how many individual recombination events occur per isolate (the frequency). Using univariate analyses, we found an association between both recombination measures and multiple factors associated with the capsule, including duration and prevalence of carriage. Because many capsular factors are correlated, we used multivariate analysis to correct for collinearity. Capsule size and carriage duration remained positively associated with recombination, although with a reduced P value, and this effect may be mediated through some unassayed additional property associated with larger capsules. This work describes an important impact of serotype on recombination that has been previously overlooked. While the details of how this effect is achieved remain to be determined, it may have important consequences for the serotype-specific response to vaccines and other interventions

Early signals of vaccine driven perturbation seen in pneumococcal carriage population genomic data

BACKGROUND: Pneumococcal conjugate vaccines (PCV) have reduced pneumococcal diseases globally. Pneumococcal genomic surveys elucidate PCV effects on population structure but are rarely conducted in low-income settings despite the high disease burden. METHODS:We undertook whole genome sequencing of 660 pneumococcal isolates collected through surveys from healthy carriers two years from PCV14 introduction and one-year post-rollout in northern Malawi. We investigated changes in population structure, within-lineage serotype dynamics, serotype diversity, and frequency of antibiotic resistance (ABR) and accessory genes. RESULTS:In the under-fives, frequency and diversity of vaccine serotypes (VT) decreased significantly post-PCV but no significant changes occurred in over-fives. Clearance of VT serotypes was consistent across different genetic backgrounds (lineages). There was an increase of non-vaccine serotypes (NVT) namely 7C, 15B/C, 23A in under-fives but 28F increased in both age groups. While carriage rates have been recently shown to remain stable post-PCV due replacement serotypes, there was no change in diversity of NVTs. Additionally, frequency of intermediate-penicillin-resistant lineages decreased post-PCV. While frequency of ABR genes remained stable, other accessory genes especially those associated with MGEs and bacteriocins showed changes in frequency post-PCV. CONCLUSIONS:We demonstrate evidence of significant population restructuring post-PCV driven by decreasing frequency of vaccine serotypes and increasing frequency of few NVTs mainly in under-fives. Continued surveillance with WGS remains crucial to fully understand dynamics of the residual VTs and replacement NVT serotypes post-PCV

Population genetic structure, antibiotic resistance, capsule switching and evolution of invasive pneumococci before conjugate vaccination in Malawi

INTRODUCTION: Pneumococcal infections cause a high death toll in Sub Saharan Africa (SSA) but the recently rolled out pneumococcal conjugate vaccines (PCV) will reduce the disease burden. To better understand the population impact of these vaccines, comprehensive analysis of large collections of pneumococcal isolates sampled prior to vaccination is required. Here we present a population genomic study of the invasive pneumococcal isolates sampled before the implementation of PCV13 in Malawi. MATERIALS AND METHODS: We retrospectively sampled and whole genome sequenced 585 invasive isolates from 2004 to 2010. We determine the pneumococcal population genetic structure and assessed serotype prevalence, antibiotic resistance rates, and the occurrence of serotype switching. RESULTS: Population structure analysis revealed 22 genetically distinct sequence clusters (SCs), which consisted of closely related isolates. Serotype 1 (ST217), a vaccine-associated serotype in clade SC2, showed highest prevalence (19.3%), and was associated with the highest MDR rate (81.9%) followed by serotype 12F, a non-vaccine serotype in clade SC10 with an MDR rate of 57.9%. Prevalence of serotypes was stable prior to vaccination although there was an increase in the PMEN19 clone, serotype 5 ST289, in clade SC1 in 2010 suggesting a potential undetected local outbreak. Coalescent analysis revealed recent emergence of the SCs and there was evidence of natural capsule switching in the absence of vaccine induced selection pressure. Furthermore, majority of the highly prevalent capsule-switched isolates were associated with acquisition of vaccine-targeted capsules. CONCLUSIONS: This study provides descriptions of capsule-switched serotypes and serotypes with potential to cause serotype replacement post-vaccination such as 12F. Continued surveillance is critical to monitor these serotypes and antibiotic resistance in order to design better infection prevention and control measures such as inclusion of emerging replacement serotypes in future conjugate vaccines

Understanding pneumococcal serotype 1 biology through population genomic analysis

Background: Pneumococcus kills over one million children annually and over 90 % of these deaths occur in low-income countries especially in Sub-Saharan Africa (SSA) where HIV exacerbates the disease burden. In SSA, serotype 1 pneumococci particularly the endemic ST217 clone, causes majority of the pneumococcal disease burden. To understand the evolution of the virulent ST217 clone, we analysed ST217 whole genomes from isolates sampled from African and Asian countries. Methods: We analysed 226 whole genome sequences from the ST217 lineage sampled from 9 African and 4 Asian countries. We constructed a whole genome alignment and used it for phylogenetic and coalescent analyses. We also screened the genomes to determine presence of antibiotic resistance conferring genes. Results: Population structure analysis grouped the ST217 isolates into five sequence clusters (SCs), which were highly associated with different geographical regions and showed limited intracontinental and intercontinental spread. The SCs showed lower than expected genomic sequence, which suggested strong purifying selection and small population sizes caused by bottlenecks. Recombination rates varied between the SCs but were lower than in other successful clones such as PMEN1. African isolates showed higher prevalence of antibiotic resistance genes than Asian isolates. Interestingly, certain West African isolates harbored a defective chloramphenicol and tetracycline resistance-conferring element (Tn5253) with a deletion in the loci encoding the chloramphenicol resistance gene (cat(pC194)), which caused lower chloramphenicol than tetracycline resistance. Furthermore, certain genes that promote colonisation were absent in the isolates, which may contribute to serotype 1's rarity in carriage and consequently its lower recombination rates. Conclusions: The high phylogeographic diversity of the ST217 clone shows that this clone has been in circulation globally for a long time, which allowed its diversification and adaptation in different geographical regions. Such geographic adaptation reflects local variations in selection pressures in different locales. Further studies will be required to fully understand the biological mechanisms which makes the ST217 clone highly invasive but unable to successfully colonise the human nasopharynx for long durations which results in lower recombination rates.Peer reviewe

Common Genetic Polymorphisms Influence Blood Biomarker Measurements in COPD

Implementing precision medicine for complex diseases such as chronic obstructive lung disease (COPD) will require extensive use of biomarkers and an in-depth understanding of how genetic, epigenetic, and environmental variations contribute to phenotypic diversity and disease progression. A meta-analysis from two large cohorts of current and former smokers with and without COPD [SPIROMICS (N = 750); COPDGene (N = 590)] was used to identify single nucleotide polymorphisms (SNPs) associated with measurement of 88 blood proteins (protein quantitative trait loci; pQTLs). PQTLs consistently replicated between the two cohorts. Features of pQTLs were compared to previously reported expression QTLs (eQTLs). Inference of causal relations of pQTL genotypes, biomarker measurements, and four clinical COPD phenotypes (airflow obstruction, emphysema, exacerbation history, and chronic bronchitis) were explored using conditional independence tests. We identified 527 highly significant (p 10% of measured variation in 13 protein biomarkers, with a single SNP (rs7041; p = 10−392) explaining 71%-75% of the measured variation in vitamin D binding protein (gene = GC). Some of these pQTLs [e.g., pQTLs for VDBP, sRAGE (gene = AGER), surfactant protein D (gene = SFTPD), and TNFRSF10C] have been previously associated with COPD phenotypes. Most pQTLs were local (cis), but distant (trans) pQTL SNPs in the ABO blood group locus were the top pQTL SNPs for five proteins. The inclusion of pQTL SNPs improved the clinical predictive value for the established association of sRAGE and emphysema, and the explanation of variance (R2) for emphysema improved from 0.3 to 0.4 when the pQTL SNP was included in the model along with clinical covariates. Causal modeling provided insight into specific pQTL-disease relationships for airflow obstruction and emphysema. In conclusion, given the frequency of highly significant local pQTLs, the large amount of variance potentially explained by pQTL, and the differences observed between pQTLs and eQTLs SNPs, we recommend that protein biomarker-disease association studies take into account the potential effect of common local SNPs and that pQTLs be integrated along with eQTLs to uncover disease mechanisms. Large-scale blood biomarker studies would also benefit from close attention to the ABO blood group

Kepler-18b, c, and d: a system of three planets confirmed by transit timing variations, light curve validation, Warm-Spitzer photometry, and radial velocity measurements

We report the detection of three transiting planets around a Sun-like star, which we designate Kepler-18. The transit signals were detected in photometric data from the Kepler satellite, and were confirmed to arise from planets using a combination of large transit-timing variations (TTVs), radial velocity variations, Warm-Spitzer observations, and statistical analysis of false-positive probabilities. The Kepler-18 star has a mass of 0.97 M ⊙, a radius of 1.1 R ⊙, an effective temperature of 5345K, and an iron abundance of [Fe/H] = +0.19. The planets have orbital periods of approximately 3.5, 7.6, and 14.9 days. The innermost planet 'b' is a 'super-Earth' with a mass of 6.9 ± 3.4 M ⊕, a radius of 2.00 ± 0.10 R ⊕, an' a mean density of 4.9 ± 2.4gcm3. The two outer planets 'c' and 'd' are both low-density Neptune-mass planets. Kepler-18c has a mass of 17.3 ± 1.9 M ⊕, a radius of 5.49 ± 0.26 R ⊕, and a mean density of 0.59 0.07gcm 3, while Kepler-18d has a mass of 16.4 ± 1.4 M ⊕, a radius of 6.98 ± 0.33 R ⊕ and a mean density of 0.27 ± 0.03gcm3. Kepler-18c and Kepler-18d have orbital periods near a 2:1 mean-motion resonance, leading to large and readily detected TTVs