Genetic and Molecular Basis of Encapsulation and Capsule Diversity in Kingella kingae

Kingella kingae is a bacterial pathogen that is increasingly recognized as an etiology of septic arthritis, osteomyelitis, bacteremia, and endocarditis in young children. The pathogenesis of K. kingae disease starts with bacterial adherence to the respiratory epithelium of the posterior pharynx. Previous work has identified type IV pili and a trimeric autotransporter protein called Knh (Kingella NhhA homolog) as critical factors for adherence to human epithelial cells. Additional studies established that the presence of a polysaccharide capsule interferes with Knh-mediated adherence. Given the inhibitory role of capsule during adherence we sought to uncover the genes involved in capsule expression to understand how capsule is elaborated on the cell surface. Additionally, this work aimed to further characterize capsule diversity among K. kingae clinical isolates and to investigate the relationship between capsule type and site of isolation.

We first set out to identify the carbohydrates present in the K. kingae capsule present in the prototype strain 269-492. Glycosyl composition and NMR analysis of surface extractable polysaccharides demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→.

To discern the two polysaccharides we disrupted the ctrA gene required for surface localization of the K. kingae polysaccharide capsule and observed a loss of GalNAc and Kdo but no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. These results established that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→.

Having established that K. kingae produces a capsule comprised of GalNAc and Kdo, we next set out to identify the genetic determinants of capsule through a transposon mutagenesis screen. In addition to the previously identified ctrABCD operon, lipA, lipB, and a putative glycosyltransferase termed csaA (capsule synthesis region A gene A) were found to be essential for the production of surface-localized capsule. The ctr operon, lipA, lipB, and csaA were found to be present at unlinked locations throughout the genome, which is atypical for gram-negative organisms that elaborate a capsule dependent on an ABC-type transporter for surface localization. Through examining capsule localization in the ctrA, lipA, lipB, and csaA mutant strains, we determined that the ctrABCD, lipA/lipB, and csaA gene products respectively function in capsule export, assembly, and synthesis, respectively. The GalNAc transferase and Kdo transferase domains found in CsaA further support its role in catalyzing the synthesis of the GalNAc-Kdo capsule in the K. kingae prototype strain.

To investigate the capsule diversity that exists in K. kingae we screened a panel of strains isolated from patients with invasive disease or healthy carriers for the csaA capsule synthesis locus. We discovered that Kingella kingae expresses one of 4 capsule synthesis loci (csa, csb, csc, or csd) associated with a capsule consisting of Kdo and GalNAc (type a), Kdo and GlcNAc (type b), Kdo and ribose (type c), and GlcNAc and galactose (type d), respectively. Cloning of the csa, csb, csc, or csd locus into the empty flanking gene region in a non-encapsulated mutant (creation of an isogenic capsule swap) was sufficient to produce either the type a, type b, or type c capsule, respectively, further supporting the role of these loci in expression of a specific polysaccharide linkage. Capsule type a and capsule type b accounted for 96% of invasive strains. Conversely, capsule type c and capsule type d were found disproportionately among carrier isolates, suggesting that capsule type is important in promoting invasion and dissemination.

In conclusion, we discovered that Kingella kingae expresses a polysaccharide capsule and an exopolysaccharide on its surface that require distinct genetic loci for surface localization. Further investigation into genetic determinants of encapsulation revealed the loci ctrABCD, lipA/lipB, and a putative glycosyltransferase are required for capsule expression, with the gene products having roles in capsule export, assembly, and synthesis, respectively. The putative glycosyltransferase CsaA was determined to be a bifunctional enzyme with both GalNAc-transferase and Kdo-transferase activity. Furthermore, we discovered a total of 4 capsule types expressed in clinical isolates of K. kingae, each with a distinct capsule synthesis locus. The variation in the proportion of capsule types found between invasive strains and carriage strains suggest that capsule type is important in promoting invasion and dissemination. Taken together, this work expands our knowledge of the capsule types expressed among K. kingae carrier and invasive isolates and provides insights into the common genetic determinants of capsule expression. These contributions may lead to selecting clinically relevant capsule types to develop into a capsule based vaccine to prevent K. kingae colonization.

UK consensus project on quality in palliative day services: developing a quality indicator set using the RAND/UCLA appropriateness method

Introduction
Evaluating quality of palliative day services is essential for assessing care across diverse settings, and for monitoring quality improvement approaches.

Aim
To develop a set of quality indicators for assessment of all aspects (structure, process and outcome) of care in palliative day services.

Methods
Using a modified version of the RAND/UCLA appropriateness method (Fitch et al., 2001), a multidisciplinary panel of 16 experts independently completed a survey rating the appropriateness of 182 potential quality indicators previously identified during a systematic evidence review. Panel members then attended a one day, face-to-face meeting where indicators were discussed and subsequently re-rated. Panel members were also asked to rate the feasibility and necessity of measuring each indicator.

Results
71 indicators classified as inappropriate during the survey were removed based on median appropriateness ratings and level of agreement. Following the panel discussions, a further 60 were removed based on appropriateness and feasibility ratings, level of agreement and assessment of necessity. Themes identified during the panel discussion and findings of the evidence review were used to translate the remaining 51 indicators into a final set of 27.

Conclusion
The final indicator set included information on rationale and supporting evidence, methods of assessment, risk adjustment, and recommended performance levels. Further implementation work will test the suitability of this ‘toolkit’ for measurement and benchmarking. The final indicator set provides the basis for standardised assessment of quality across services, including care delivered in community and primary care settings.

Reference

• Fitch K, Bernstein SJ, Aguilar MD, et al. The RAND/UCLA Appropriateness Method User’s Manual. Santa Monica, CA: RAND Corporation; 2001. http://www.rand.org/pubs/monograph_reports/MR1269

The genetic basis of psychological traits in infancy: Implications for understanding the causes of developmental psychopathology

This chapter reviews genetic studies that have aimed to identify genes influencing psychological traits in infancy (from birth to age 12 months), and considers how this research informs us about the causes of developmental psychopathology. Specifically, this chapter systematically reviews findings from studies that associated common genetic variants with individual variation in infants’ attention, temperament and behaviour, and attachment disorganisation. DRD4 and 5-HTTLPR genes were the most frequently studied candidate genes. Possibly the most coherent set of results relates to the L-DRD4 genotype, which is significantly associated with infant attention, temperament, and attachment style. Research in infant genetics has been strengthened by a careful focus on uniform age ranges within studies, by several longitudinal studies, and by exploration of gene-environment interactions between genes and maternal characteristics. However there is also considerable inconsistency in results in this field and possible reasons for this are discussed. The chapter outlines the main genetic methods that have been used and what new genetic approaches such as polygenic risk scoring could offer infant genetics. Recent findings suggest that some traits during infancy predict individual differences in developmental psychopathology in childhood. It is argued that infant genetic research has considerable potential for the identification of populations at risk for psychopathology in later life, and this remains an area open for future research.

Modelling of water removal during a paper vacuum dewatering process using a Level-Set method

Water removal in paper manufacturing is an energy-intensive process. The dewatering process generally consists of four stages of which the first three stages include mechanical water removal through gravity filtration, vacuum dewatering and wet pressing. In the fourth stage, water is removed thermally, which is the most expensive stage in terms of energy use. In order to analyse water removal during a vacuum dewatering process, a numerical model was created by using a Level-Set method. Various different 2D structures of the paper model were created in MATLAB code with randomly positioned circular fibres with identical orientation. The model considers the influence of the forming fabric which supports the paper sheet during the dewatering process, by using volume forces to represent flow resistance in the momentum equation. The models were used to estimate the dry content of the porous structure for various dwell times. The relation between dry content and dwell time was compared to laboratory data for paper sheets with basis weights of 20 and 50 g/m2 exposed to vacuum levels between 20 kPa and 60 kPa. The comparison showed reasonable results for dewatering and air flow rates. The random positioning of the fibres influences the dewatering rate slightly. In order to achieve more accurate comparisons, the random orientation of the fibres needs to be considered, as well as the deformation and displacement of the fibres during the dewatering process.

Measuring, Modeling, and Forecasting Volatility and Correlations from High-Frequency Data

This dissertation contains four essays that all share a common purpose: developing new methodologies to exploit the potential of high-frequency data for the measurement, modeling and forecasting of financial assets volatility and correlations. The first two chapters provide useful tools for univariate applications while the last two chapters develop multivariate methodologies. In chapter 1, we introduce a new class of univariate volatility models named FloGARCH models. FloGARCH models provide a parsimonious joint model for low frequency returns and realized measures, and are sufficiently flexible to capture long memory as well as asymmetries related to leverage effects. We analyze the performances of the models in a realistic numerical study and on the basis of a data set composed of 65 equities. Using more than 10 years of high-frequency transactions, we document significant statistical gains related to the FloGARCH models in terms of in-sample fit, out-of-sample fit and forecasting accuracy compared to classical and Realized GARCH models. In chapter 2, using 12 years of high-frequency transactions for 55 U.S. stocks, we argue that combining low-frequency exogenous economic indicators with high-frequency financial data improves the ability of conditionally heteroskedastic models to forecast the volatility of returns, their full multi-step ahead conditional distribution and the multi-period Value-at-Risk. Using a refined version of the Realized LGARCH model allowing for time-varying intercept and implemented with realized kernels, we document that nominal corporate profits and term spreads have strong long-run predictive ability and generate accurate risk measures forecasts over long-horizon. The results are based on several loss functions and tests, including the Model Confidence Set. Chapter 3 is a joint work with David Veredas. We study the class of disentangled realized estimators for the integrated covariance matrix of Brownian semimartingales with finite activity jumps. These estimators separate correlations and volatilities. We analyze different combinations of quantile- and median-based realized volatilities, and four estimators of realized correlations with three synchronization schemes. Their finite sample properties are studied under four data generating processes, in presence, or not, of microstructure noise, and under synchronous and asynchronous trading. The main finding is that the pre-averaged version of disentangled estimators based on Gaussian ranks (for the correlations) and median deviations (for the volatilities) provide a precise, computationally efficient, and easy alternative to measure integrated covariances on the basis of noisy and asynchronous prices. Along these lines, a minimum variance portfolio application shows the superiority of this disentangled realized estimator in terms of numerous performance metrics. Chapter 4 is co-authored with Niels S. Hansen, Asger Lunde and Kasper V. Olesen, all affiliated with CREATES at Aarhus University. We propose to use the Realized Beta GARCH model to exploit the potential of high-frequency data in commodity markets. The model produces high quality forecasts of pairwise correlations between commodities which can be used to construct a composite covariance matrix. We evaluate the quality of this matrix in a portfolio context and compare it to models used in the industry. We demonstrate significant economic gains in a realistic setting including short selling constraints and transaction costs.

Enhancing SPH using moving least-squares and radial basis functions

In this paper we consider two sources of enhancement for the meshfree Lagrangian particle method smoothed particle hydrodynamics (SPH) by improving the accuracy of the particle approximation. Namely, we will consider shape functions constructed using: moving least-squares approximation (MLS); radial basis functions (RBF). Using MLS approximation is appealing because polynomial consistency of the particle approximation can be enforced. RBFs further appeal as they allow one to dispense with the smoothing-length - the parameter in the SPH method which governs the number of particles within the support of the shape function. Currently, only ad hoc methods for choosing the smoothing-length exist. We ensure that any enhancement retains the conservative and meshfree nature of SPH. In doing so, we derive a new set of variationally-consistent hydrodynamic equations. Finally, we demonstrate the performance of the new equations on the Sod shock tube problem.

First insight into personality traits in Northern pike (Esox lucius) larvae: a basis for behavioural studies of early life stages

Personality traits have been studied for some decades in fish species. Yet, most often, studies focused on juveniles or adults. Thus, very few studies tried to demonstrate that traits could also be found in fish larvae. In this study, we aimed at identifying personality traits in Northern pike (Exos lucius) larvae. Twenty first-feeding larvae aged 21 days post hatch (16.1 +/− 0.4 mm in total length, mean +/− SD) were used to establish personality traits with two tests: a maze and a novel object. These tests are generally used for evaluating the activity and exploration of specimens as well as their activity and boldness, respectively. The same Northern pike twenty larvae were challenged in the two tests. Their performances were measured by their activity, their exploratory behaviour and the time spent in the different arms of the maze or near the novel object. Then, we used principal component analysis (PCA) and a hierarchical ascendant classification (HAC) for analysis of each data set separately. Finally, we used PCA reduction for the maze test data to analyse the relationship between a synthetic behavioural index (PCA1) and morphometric variables. Within each test, larvae could be divided in two sub groups, which exhibited different behavioural traits, qualified as bold (n = 7 for the maze test and n = 13 for the novel object test) or shy (n = 9 for the maze test and n = 11 for the novel object test). Nevertheless, in both tests, there was a continuum of boldness/shyness. Besides, some larvae were classified differently between the two tests but 40 % of the larvae showed cross context consistency and could be qualified as bold and/or proactive individuals. This study showed that it is possible to identify personality traits of very young fish larvae of a freshwater fish species.

Triangular mesh parameterization with trimmed surfaces

Given a 2manifold triangular mesh \(M \subset {\mathbb {R}}^3\), with border, a parameterization of \(M\) is a FACE or trimmed surface \(F=\{S,L_0,\ldots, L_m\}\) -- \(F\) is a connected subset or region of a parametric surface \(S\), bounded by a set of LOOPs \(L_0,\ldots ,L_m\) such that each \(L_i \subset S\) is a closed 1manifold having no intersection with the other \(L_j\) LOOPs -- The parametric surface \(S\) is a statistical fit of the mesh \(M\) -- \(L_0\) is the outermost LOOP bounding \(F\) and \(L_i\) is the LOOP of the ith hole in \(F\) (if any) -- The problem of parameterizing triangular meshes is relevant for reverse engineering, tool path planning, feature detection, redesign, etc -- Stateofart mesh procedures parameterize a rectangular mesh \(M\) -- To improve such procedures, we report here the implementation of an algorithm which parameterizes meshes \(M\) presenting holes and concavities -- We synthesize a parametric surface \(S \subset {\mathbb {R}}^3\) which approximates a superset of the mesh \(M\) -- Then, we compute a set of LOOPs trimming \(S\), and therefore completing the FACE \(F=\ {S,L_0,\ldots ,L_m\}\) -- Our algorithm gives satisfactory results for \(M\) having low Gaussian curvature (i.e., \(M\) being quasi-developable or developable) -- This assumption is a reasonable one, since \(M\) is the product of manifold segmentation preprocessing -- Our algorithm computes: (1) a manifold learning mapping \(\phi : M \rightarrow U \subset {\mathbb {R}}^2\), (2) an inverse mapping \(S: W \subset {\mathbb {R}}^2 \rightarrow {\mathbb {R}}^3\), with \ (W\) being a rectangular grid containing and surpassing \(U\) -- To compute \(\phi\) we test IsoMap, Laplacian Eigenmaps and Hessian local linear embedding (best results with HLLE) -- For the back mapping (NURBS) \(S\) the crucial step is to find a control polyhedron \(P\), which is an extrapolation of \(M\) -- We calculate \(P\) by extrapolating radial basis functions that interpolate points inside \(\phi (M)\) -- We successfully test our implementation with several datasets presenting concavities, holes, and are extremely nondevelopable -- Ongoing work is being devoted to manifold segmentation which facilitates mesh parameterization

Vegetation series as a basis for habitats and species conservation: methodological aspects

Vegetation series, defined as the sequence of stages in a sucession, and know as sigmetum (synassociation), describes the set of plant communities or stages that can be found in similar tesselar spaces as a result of the sucession process. This establishes the concept of vegetation series; a climatophilous series is one that depends on the climate, whereas an edaphoxerophilous series depends on the dryness of the soil, and is found on crests, spurs, ledges and limestone and siliceous rock fields. Edaphohygrophilous series are located in valleys, dry water courses and river terraces, and depend on the water present in the soil, which may become temporarily flooded and thus condition the temporihygrophilous series; they represent the transition between the clearly edaphohygrophilous and climatophilous series. The vegetation permaseries represents the perennial communities of permatesselae or similar permatesselar complexes, as occurs in polar territories, hyperdesert, high-mountain peaks, and non-stratified communities lacking in serial communities. The edaphoxerophilous series may include -in addition to the series head- permaseries (permanent communities) and other habitats, such as annual and crevice habitats. A territory behaves undergoes soil-loss phenomena it may become an edaphoseries, if the loss of the soil factor produces a situation of rocky crest. Thus the edaphoseries may act as dynamic transitional stage between the climatophilous series and the permaseries.

Data-stream driven Fuzzy-granular approaches for system maintenance

Intelligent systems are currently inherent to the society, supporting a synergistic human-machine collaboration. Beyond economical and climate factors, energy consumption is strongly affected by the performance of computing systems. The quality of software functioning may invalidate any improvement attempt. In addition, data-driven machine learning algorithms are the basis for human-centered applications, being their interpretability one of the most important features of computational systems. Software maintenance is a critical discipline to support automatic and life-long system operation. As most software registers its inner events by means of logs, log analysis is an approach to keep system operation. Logs are characterized as Big data assembled in large-flow streams, being unstructured, heterogeneous, imprecise, and uncertain. This thesis addresses fuzzy and neuro-granular methods to provide maintenance solutions applied to anomaly detection (AD) and log parsing (LP), dealing with data uncertainty, identifying ideal time periods for detailed software analyses. LP provides deeper semantics interpretation of the anomalous occurrences. The solutions evolve over time and are general-purpose, being highly applicable, scalable, and maintainable. Granular classification models, namely, Fuzzy set-Based evolving Model (FBeM), evolving Granular Neural Network (eGNN), and evolving Gaussian Fuzzy Classifier (eGFC), are compared considering the AD problem. The evolving Log Parsing (eLP) method is proposed to approach the automatic parsing applied to system logs. All the methods perform recursive mechanisms to create, update, merge, and delete information granules according with the data behavior. For the first time in the evolving intelligent systems literature, the proposed method, eLP, is able to process streams of words and sentences. Essentially, regarding to AD accuracy, FBeM achieved (85.64+-3.69)%; eGNN reached (96.17+-0.78)%; eGFC obtained (92.48+-1.21)%; and eLP reached (96.05+-1.04)%. Besides being competitive, eLP particularly generates a log grammar, and presents a higher level of model interpretability.

Changes In Motor Behavior During Pregnancy In Rats: The Basis For A Possible Animal Model Of Restless Legs Syndrome.

Pregnant women have a 2-3 fold higher probability of developing restless legs syndrome (RLS - sleep-related movement disorders) than general population. This study aims to evaluate the behavior and locomotion of rats during pregnancy in order to verify if part of these animals exhibit some RLS-like features. We used 14 female 80-day-old Wistar rats that weighed between 200 and 250 g. The rats were distributed into control (CTRL) and pregnant (PN) groups. After a baseline evaluation of their behavior and locomotor activity in an open-field environment, the PN group was inducted into pregnancy, and their behavior and locomotor activity were evaluated on days 3, 10 and 19 of pregnancy and in the post-lactation period in parallel with the CTRL group. The serum iron and transferrin levels in the CTRL and PN groups were analyzed in blood collected after euthanasia by decapitation. There were no significant differences in the total ambulation, grooming events, fecal boli or urine pools between the CTRL and PN groups. However, the PN group exhibited fewer rearing events, increased grooming time and reduced immobilization time than the CTRL group (ANOVA, p<0.05). These results suggest that pregnant rats show behavioral and locomotor alterations similar to those observed in animal models of RLS, demonstrating to be a possible animal model of this sleep disorder.

Kinase Inhibitor Profile For Human Nek1, Nek6, And Nek7 And Analysis Of The Structural Basis For Inhibitor Specificity.

Human Neks are a conserved protein kinase family related to cell cycle progression and cell division and are considered potential drug targets for the treatment of cancer and other pathologies. We screened the activation loop mutant kinases hNek1 and hNek2, wild-type hNek7, and five hNek6 variants in different activation/phosphorylation statesand compared them against 85 compounds using thermal shift denaturation. We identified three compounds with significant Tm shifts: JNK Inhibitor II for hNek1(Δ262-1258)-(T162A), Isogranulatimide for hNek6(S206A), andGSK-3 Inhibitor XIII for hNek7wt. Each one of these compounds was also validated by reducing the kinases activity by at least 25%. The binding sites for these compounds were identified by in silico docking at the ATP-binding site of the respective hNeks. Potential inhibitors were first screened by thermal shift assays, had their efficiency tested by a kinase assay, and were finally analyzed by molecular docking. Our findings corroborate the idea of ATP-competitive inhibition for hNek1 and hNek6 and suggest a novel non-competitive inhibition for hNek7 in regard to GSK-3 Inhibitor XIII. Our results demonstrate that our approach is useful for finding promising general and specific hNekscandidate inhibitors, which may also function as scaffolds to design more potent and selective inhibitors.

Non-gaussian Spatial Correlations Dramatically Weaken Localization.

We perform variational studies of the interaction-localization problem to describe the interaction-induced renormalizations of the effective (screened) random potential seen by quasiparticles. Here we present results of careful finite-size scaling studies for the conductance of disordered Hubbard chains at half-filling and zero temperature. While our results indicate that quasiparticle wave functions remain exponentially localized even in the presence of moderate to strong repulsive interactions, we show that interactions produce a strong decrease of the characteristic conductance scale g^{*} signaling the crossover to strong localization. This effect, which cannot be captured by a simple renormalization of the disorder strength, instead reflects a peculiar non-Gaussian form of the spatial correlations of the screened disordered potential, a hitherto neglected mechanism to dramatically reduce the impact of Anderson localization (interference) effects.

Microsatellite Markers For Urochloa Humidicola (poaceae) And Their Transferability To Other Urochloa Species.

Urochloa humidicola is a warm-season grass commonly used as forage in the tropics and is recognized for its tolerance to seasonal flooding. This grass is an important forage species for the Cerrado and Amazon regions of Brazil. U. humidicola is a polyploid species with variable ploidy (6X-9X) and facultative apomixis with high phenotypic plasticity. However, this apomixis and ploidy, as well as the limited knowledge of the genetic basis of the germplasm collection, have constrained genetic breeding activities, yet microsatellite markers may enable a better understanding of the species' genetic composition. This study aimed to develop and characterize new polymorphic microsatellite molecular markers in U. humidicola and to evaluate their transferability to other Urochloa species. A set of microsatellite markers for U. humidicola was identified from two new enriched genomic DNA libraries: the first library was constructed from a single sexual genotype and the second from a pool of eight apomictic genotypes selected on the basis of previous results. Of the 114 loci developed, 72 primer pairs presented a good amplification product, and 64 were polymorphic among the 34 genotypes tested. The number of bands per simple sequence repeat (SSR) locus ranged from 1 to 29, with a mean of 9.6 bands per locus. The mean polymorphism information content (PIC) of all loci was 0.77, and the mean discrimination power (DP) was 0.87. STRUCTURE analysis revealed differences among U. humidicola accessions, hybrids, and other Urochloa accessions. The transferability of these microsatellites was evaluated in four species of the genus, U. brizantha, U. decumbens, U. ruziziensis, and U. dictyoneura, and the percentage of transferability ranged from 58.33% to 69.44% depending on the species. This work reports new polymorphic microsatellite markers for U. humidicola that can be used for breeding programs of this and other Urochloa species, including genetic linkage mapping, quantitative trait loci identification, and marker-assisted selection.

Análise bayesiana do modelo de herança monogênica no melhoramento vegetal: um exemplo com abobrinha

A common breeding strategy is to carry out basic studies to investigate the hypothesis of a single gene controlling the trait (major gene) with or without polygenes of minor effect. In this study we used Bayesian inference to fit genetic additive-dominance models of inheritance to plant breeding experiments with multiple generations. Normal densities with different means, according to the major gene genotype, were considered in a linear model in which the design matrix of the genetic effects had unknown coefficients (which were estimated in individual basis). An actual data set from an inheritance study of partenocarpy in zucchini (Cucurbita pepo L.) was used for illustration. Model fitting included posterior probabilities for all individual genotypes. Analysis agrees with results in the literature but this approach was far more efficient than previous alternatives assuming that design matrix was known for the generations. Partenocarpy in zucchini is controlled by a major gene with important additive effect and partial dominance.