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Peptidoglycan—a mesh sac of glycans that are linked by peptides—is the main component of bacterial cell walls. Peptidoglycan provides structural strength, protects cells from osmotic pressure and contributes to shape. All bacterial glycans are repeating disaccharides of N-acetylglucosamine (GlcNAc) β-(1–4)-linked to N-acetylmuramic acid (MurNAc). Borrelia burgdorferi, the tick-borne Lyme disease pathogen, produces glycan chains in which MurNAc is occasionally replaced with an unknown sugar. Nuclear magnetic resonance, liquid chromatography–mass spectroscopy and genetic analyses show that B. burgdorferi produces glycans that contain GlcNAc–GlcNAc. This unusual disaccharide is chitobiose, a component of its chitinous tick vector. Mutant bacteria that are auxotrophic for chitobiose have altered morphology, reduced motility and cell envelope defects that probably result from producing peptidoglycan that is stiffer than that in wild-type bacteria. We propose that the peptidoglycan of B. burgdorferi probably evolved by adaptation to obligate parasitization of a tick vector, resulting in a biophysical cell-wall alteration to withstand the atypical torque associated with twisting motility., Tick chitobiose is co-opted to build the cell wall of Lyme disease pathogen Borrelia burgdorferi.

SummaryMembers of the Enterobacteriaceae family includingEscherichia coliare associated with persistent gut inflammation during disorders like inflammatory bowel disease. This is due to rapid microbial colonization during dysbiosis combined with pathogenic tendencies. We characterized the dysbiotic gut community, defined potential functional pathways, and investigated crosstalk between host gene expression and microbial detections in an intestinal inflammation murine model. Members of Enterobacteriaceae family and theEnterococcusgenus were highly detected in dysbiotic mice. These metagenome assembled genomes (MAGs) contained several virulence factors and metabolic pathways necessary to drive perpetual inflammation. Two Enterobacteriaceae MAGs with L-cysteine and L-taurine dioxygenases were strongly correlated with upregulation of the host gene CSAD, responsible for cysteine metabolism. Suggesting these bacteria compete with the host to utilize essential amino acids. We observed that bacterial isolates from dysbiotic mice displayed increased growth rates supplemented with L-cysteine, confirming that these microbes can utilize host nutrients to sustain inflammation.In BriefInflammatory bowel disease is associated with an increase in Enterobacteriaceae andEnterococcusspecies, however the mechanisms are unclear. Richieet al. show that these bacterial populations use sulfur metabolism and tolerate host-derived immune-response, to drive host inflammation and fuel growth in the dysbiotic colon. Cultured isolates from dysbiotic mice indicated faster growth supplemented with L-cysteine, showing these microbes can utilize these essential host nutrients.HighlightsMice receiving native microbial FMT showed lower colon inflammation scores, higher microbial diversity, detections and gene expression similar to control mice.Dysbiotic mice displayed increased colon inflammation, higher detection of potential pathogenic MAGs, and upregulation of cysteine dioxygenase and other inflammation response genesMAGs assigned toEnterococcusand Enterobacteriaceae species were more frequently detected in dysbiotic mice, while almost absent in mice receiving FMT or control mice, they also contain several virulence factors and antibiotic resistance genes.These MAGs also display potential functions of utilizing host products and nutrients including nitrate, cysteine, and taurine to further fuel their growth and metabolism, which results in persistent host intestinal inflammation.Isolates in the Enterobacteriaceae family from dysbiotic mice utilize L-cysteine for growth, whereas isolates from FMT and control mice show no significant difference, indicating these bacteria can utilize the host derived cysteine.

The use of guided waves to identify damage has become a popular method due to its robustness and fast execution, as well as the advantage of being able to inspect large areas and detect minor structural defects. When a travelling wave on a plate interacts with a defect, it generates a scattered field that will depend on the defects geometry. By analysing the scattered field, one can thus characterize the type and size of the plate damage. A Bayesian framework based on a guided waves interaction model for damage identification of infinite plate for the first time is presented here. A semi-analytical approach based on the lowest order plate theories is adopted to obtain the scattering features for damage geometries with circular symmetry, resulting in an efficient inversion procedure. Subsequently, ultrasound experiments are performed on a large aluminium plate with a circular indentation to generate wave reflection and transmission coefficients. With the aid of signal processing techniques, the effectiveness and efficiency of the proposed approach are verified. A full finite element model is used to test the damage identification scheme. Finally, the scattering coefficients are reconstructed, reliably matching the experimental results. The framework supports digital twin technology of structural health monitoring. ispartof: ULTRASONICS vol:125 ispartof: location:Netherlands status: published

Kinetic modeling has relied on using a tedious number of mathematical equations to describe molecular kinetics in interacting reactions. The long list of differential equations with associated abstract variables and parameters inevitably hinders readers’ easy understanding of the models. However, the mathematical equations describing the kinetics of biochemical reactions can be exactly mapped to the dynamics of voltages and currents in simple electronic circuits wherein voltages represent molecular concentrations and currents represent molecular fluxes. For example, we theoretically derive and experimentally verify accurate circuit models for Michaelis-Menten kinetics. Then, we show that such circuit models can be scaled via simple wiring among circuit motifs to represent more and arbitrarily complex reactions. Hence, we can directly map reaction networks to equivalent circuit schematics in a rapid, quantitatively accurate, and intuitive fashion without needing mathematical equations. We verify experimentally that these circuit models are quantitatively accurate. Examples include 1) different mechanisms of competitive, noncompetitive, uncompetitive, and mixed enzyme inhibition, important for understanding pharmacokinetics; 2) product-feedback inhibition, common in biochemistry; 3) reversible reactions; 4) multi-substrate enzymatic reactions, both important in many metabolic pathways; and 5) translation and transcription dynamics in a cell-free system, which brings insight into the functioning of all gene-protein networks. We envision that circuit modeling and simulation could become a powerful scientific communication language and tool for quantitative studies of kinetics in biology and related fields.

Here we demonstrate a switchable DNA electron-transfer catalyst, enabled by selective destabilization of secondary structure by the denaturant, perchlorate. The system is comprised of two strands, one of which can be selectively switched between a G-quadruplex and duplex or single-stranded conformations. In the G-quadruplex state, it binds hemin, enabling peroxidase activity. This switching ability arises from our finding that perchlorate, a chaotropic Hofmeister ion, selectively destabilizes duplex over G-quadruplex DNA. By varying perchlorate concentration, we show that the DNA structure can be switched between states that do and do not catalyze electron-transfer catalysis. State switching can be achieved in three ways: thermally, by dilution, or by concentration.

Peptidoglycan—a mesh sac of glycans that are linked by peptides—is the main component of bacterial cell walls. Peptidoglycan provides structural strength, protects cells from osmotic pressure and contributes to shape. All bacterial glycans are repeating disaccharides of N-acetylglucosamine (GlcNAc) β-(1–4)-linked to N-acetylmuramic acid (MurNAc). Borrelia burgdorferi, the tick-borne Lyme disease pathogen, produces glycan chains in which MurNAc is occasionally replaced with an unknown sugar. Nuclear magnetic resonance, liquid chromatography–mass spectroscopy and genetic analyses show that B. burgdorferi produces glycans that contain GlcNAc–GlcNAc. This unusual disaccharide is chitobiose, a component of its chitinous tick vector. Mutant bacteria that are auxotrophic for chitobiose have altered morphology, reduced motility and cell envelope defects that probably result from producing peptidoglycan that is stiffer than that in wild-type bacteria. We propose that the peptidoglycan of B. burgdorferi probably evolved by adaptation to obligate parasitization of a tick vector, resulting in a biophysical cell-wall alteration to withstand the atypical torque associated with twisting motility. Published version

Boltzmann-exponential thermodynamic laws govern noisy molecular flux in chemical reactions as well as noisy subthreshold electron current flux in transistors. These common mathematical laws enable one to map and simulate arbitrary stochastic biochemical reaction networks in highly efficient cytomorphic systems built on subthreshold analog circuits. Such simulations can accurately model noisy, nonlinear, asynchronous, stiff, and non-modular feedback dynamics in interconnected networks in the physical circuits, automatically. The scaling in simulation time for stochastic networks with the number of reactions or molecules is constant in cytomorphic systems. In contrast, it grows rapidly in digital systems, which are not parallelizable. Therefore, cytomorphic systems enable large-scale supercomputing systems-biology simulations of arbitrary and highly computationally intensive biochemical reaction networks that can nevertheless be compiled to them via digitally programmable parameters and connectivity. We outline how cytomorphic systems can be utilized for rapid drug-cocktail formulation and discovery in future pandemics like COVID-19; can simulate networks important in cancer; and can help automate the design of synthetic biological circuits, e.g. a synthetic biological operational amplifier for robust and precise drug delivery. Thus, just as neuromorphic systems have enabled multiple applications in A.I., cytomorphic systems will enable multiple applications in biology and medicine.

Ions in the Hofmeister series exhibit varied effects on biopolymers. Those classed as kosmotropes generally stabilize secondary structure, and those classed as chaotropes generally destabilize secondary structure. Here, we report that several anionic chaotropes exhibit unique effects on one DNA secondary structure - a G quadruplex. These chaotropes exhibit the expected behaviour (destabilization of secondary structure) in two other structural contexts: a DNA duplex and i-Motifs. Uniquely among secondary structures, we observe that G quadruplexes are comparatively insensitive to the presence of anionic chaotropes, but not other denaturants. Further, the presence of equimolar NaCl provided greater mitigation of the destabilization caused by other non-anionic denaturants. These results are consistent with the presence of monovalent cations providing an especially pronounced stabilizing effect to G quadruplexes when studied in denaturing solution conditions.

The bacterial pathogen responsible for causing Lyme disease, Borrelia burgdorferi, is an atypical Gram-negative spirochete that is transmitted to humans via the bite of an infected Ixodes tick. In diderms, peptidoglycan (PG) is sandwiched between the inner and outer membrane of the cell envelope. In many other Gram-negative bacteria, PG is bound by protein(s), which provide both structural integrity and continuity between envelope layers. Here, we present evidence of a peptidoglycan-associated protein (PAP) in B. burgdorferi. Using an unbiased proteomics approach, we identified Neutrophil Attracting Protein A (NapA) as a PAP. Interestingly, NapA is a Dps homologue, which typically functions to bind and protect cellular DNA from damage during times of stress. While B. burgdorferi NapA is known to be involved in the oxidative stress response, it lacks the critical residues necessary for DNA binding. Biochemical and cellular studies demonstrate that NapA is localized to the B. burgdorferi periplasm and is indeed a PAP. Cryo-electron microscopy indicates that mutant bacteria, unable to produce NapA, have structural abnormalities. Defects in cell-wall integrity impact growth rate and cause the napA mutant to be more susceptible to osmotic and PG-specific stresses. NapA-linked PG is secreted in outer membrane vesicles and augments IL-17 production, relative to PG alone. Using microfluidics, we demonstrate that NapA acts as a molecular beacon—exacerbating the pathogenic properties of B. burgdorferi PG. These studies further our understanding of the B. burgdorferi cell envelope, provide critical information that underlies its pathogenesis, and highlight how a highly conserved bacterial protein can evolve mechanistically, while maintaining biological function., Author summary Diderms typically produce peptidoglycan-associated proteins (PAPs) to enhance structural integrity and continuity within the cell envelope. We have identified a PAP in B. burgdorferi with structural and sequence homology to the near ubiquitous bacterial protein Dps (DNA binding protein from starved bacteria). The B. burgdorferi Dps paralogue is incapable of binding DNA. Instead, it bolsters the protective properties of the PG layer, while playing an important role in host immune modulation. Collectively, our findings highlight the plasticity of bacterial proteins in that they may change how they perform a task despite maintaining the same basic biological function.

Lightsheet microscopy is a form of fluorescence microscopy that can be used to visualize specimen with high resolution, a large depth-of-field, and minimal photodamage and photobleaching as compared to traditional confocal microscopy. As this technology becomes much more readily available, it will be useful in revealing new findings in the cardiovascular development field that may be hidden or difficult to image. In this manuscript, we describe an approach for mounting and culturing postimplantation mouse embryos to visualize blood vessel development with a lightsheet microscope.

RATIONALE. Risky choice can be measured using the risky decision task (RDT). In the RDT, animals choose between a large, risky option that is paired with probabilistic foot shock and a small, safe option that is never paired with shock. To date, studies examining the neurochemical basis of decision making in the RDT have focused primarily on the dopaminergic system but have not focused on the glutamatergic system, which has been implicated in risky decision making. OBJECTIVES. Because glutamate is known to play a critical role in decision making, we wanted to determine the contribution of the glutamatergic system to performance in the RDT. METHODS. In the experiment, 32 rats (16 male; 16 female) were tested in the RDT. The probability of receiving a foot shock increased across the session (ascending schedule) for half of the rats but decreased across the session (descending schedule) for half of the rats. Following training, rats received injections of the N-methyl-D-aspartate (NMDA) receptor competitive antagonist CGS 19755 (0, 1.0, 2.5, 5.0 mg/kg; s.c.) and the GluN2B-selective antagonist Ro 63–1908 (0, 0.1, 0.3, 1.0 mg/kg; s.c.). RESULTS. CGS 19755 (2.5 and 5.0 mg/kg) increased risky choice in males and females trained on the ascending schedule. Ro 63–1908 (1.0 mg/kg) decreased risky choice, but only in male rats trained on the ascending schedule. CONCLUSIONS. Although NMDA receptor antagonists differentially alter risky choice in the RDT, the current results show that NMDA receptors are an important mediator of decision making involving probabilistic delivery of positive punishment.

Probability discounting is often measured with independent schedules. Independent schedules have several limitations, such as confounding preference for one alternative with frequency of reward presentation and generating ceiling/floor effects at certain probabilities. To address this potential caveat, a controlled reinforcer frequency schedule can be used, in which the manipulandum that leads to reinforcement is pseudo-randomly determined before each trial. This schedule ensures subjects receive equal presentations of the small and large magnitude reinforcers across each block of trials. A total of 24 pair-housed and 11 individually housed female Sprague Dawley rats were tested in a controlled reinforcer frequency procedure. For half of the rats, the odds against (OA) receiving the large magnitude reinforcer increased across the session (ascending schedule); the OA decreased across the session for half of the rats (descending schedule). Following training, rats received treatments of amphetamine (AMPH; 0, 0.25, 0.5, 1.0 mg/kg; s.c.). For pair-housed rats, AMPH (0.5 mg/kg) increased risky choice, regardless of probability presentation order, whereas a higher dose of AMPH (1.0 mg/kg) decreased discriminability of reinforcer magnitude for rats trained on the descending schedule only. For individually housed rats, probability presentation order modulated the effects of AMPH on probability discounting, as AMPH (0.25 and 0.5 mg/kg) increased risky choice in rats trained on the ascending schedule but not on the descending schedule. These results show that pair-housing animals, but not using a controlled reinforcer frequency procedure, attenuates the modulatory effects of probability presentation order on drug effects on risky choice.

Structural biology education commonly employs molecular visualization software, such as PyMol, RasMol, and VMD, to allow students to appreciate structure-function relationships in biomolecules. In on-ground, classroom-based education, these programs are commonly used on University-owned devices with software preinstalled. Remote education typically involves the use of student-owned devices, which complicates the use of such software, owing to the fact that (a) student devices have differing configurations (e.g., Windows vs MacOS) and processing power, and (b) not all student devices are suitable for use with such software. Smartphones are near-ubiquitous devices, with smartphone ownership exceeding personal computer ownership, according to a recent survey. Here, we show the use of a smartphone-based augmented reality app, Augment, in a structural biology classroom exercise, which students installed independently without IT support. Post-lab attitudinal survey results indicate positive student experiences with this app. Based on our experiences, we suggest that smartphone-based molecular visualization software, such as that used in this exercise, is a powerful educational tool that is particularly well-suited for use in remote education.

Development of the embryonic heart involves an intricate network of biochemical and genetic cues to ensure its proper growth and morphogenesis. However, studies from avian and teleost models reveal that biomechanical force, namely hemodynamic loading (blood pressure and shear stress), plays a significant role in regulating heart development. To study how hemodynamic loading impacts development of the mammalian embryonic heart, we utilized mouse embryo culture and manipulation techniques and performed optical projection tomography imaging followed by morphometric analysis to determine how reduced-loading affects heart volume, myocardial thickness, trabeculation and looping. Our results reveal that hemodynamic loading can regulate these features at different thresholds. Intermediate levels of hemodynamic loading are sufficient to promote proper myocardial growth and heart size, but insufficient to promote looping and trabeculation. Whereas, low levels of hemodynamic loading fails to promote proper growth of the myocardium and heart size. These results reveal that the regulation of heart development by biomechanical force is conserved across many vertebrate classes, and this study begins to elucidate how these specific forces regulate development of the mammalian heart.

We are nearing the end of a remarkable period that began in the 1960s in which semiconductor manufacturers succeeded in shrinking die and feature sizes logarithmically, thus growing transistor counts exponentially with time. As we reach the theoretical physical limits of classical MOSFET semiconductors, DNA is a highly attractive candidate for future miniaturization of microprocessors. Here we show a foundational electronic device - a transistor - can be constructed from DNA. The nanodevice is comprised of two strands, one of which can be selectively switched between a G-quadruplex and duplex or single-stranded conformations. This switching ability arises from our discovery that perchlorate, a chaotropic Hofmeister ion, selectively destabilizes duplex over G-quadruplex DNA. By varying perchlorate concentration, we show that the device can be operated as a switch or signal amplifier. State switching can be achieved in three ways: thermally, by dilution, or by concentration. In each case, when operated in the presence of the cofactor hemin, the device catalyzes electron transfer in only the G-quadruplex state.

Thermal denaturation is a common technique in the biophysical study of nucleic acids. These experiments are typically performed by monitoring the increase in absorbance (hyperchromism) of a sample at 260nm with temperature (MergnyLacroix, 2003; PuglisiTinoco, 1989). This wavelength is chosen as nucleic acids of mixed sequence typically exhibit their maximum absorbance here. Exceptions exist, however, some noncanonical nucleic acid structures exhibit differing spectral changes with temperature, resulting in other wavelengths being convenient reporters of secondary structure. In the case of nucleic acids that bind visible light-absorbing ligands, such as fluorogenic aptamers, another wavelength can be a convenient reporter of secondary structure stability and RNA-ligand recognition. As it can be difficult, if not impossible, to know which wavelength to employ a priori, we have developed a system for obtaining the full UV-visible spectrum of a sample at each wavelength, allowing for the subsequent extraction of the absorbance-temperature profile at the desired wavelength. Here, we describe the apparatus and software used to do so. We also describe another technique for the use of a qPCR instrument for measuring secondary structure stability of fluorescent nucleic acid-ligand complexes.

Thermal denaturation is a common technique in the biophysical study of nucleic acids. These experiments are typically performed by monitoring the increase in absorbance (hyperchromism) of a sample at 260nm with temperature (Mergny & Lacroix, 2003; Puglisi & Tinoco, 1989). This wavelength is chosen as nucleic acids of mixed sequence typically exhibit their maximum absorbance here. Exceptions exist, however, some noncanonical nucleic acid structures exhibit differing spectral changes with temperature, resulting in other wavelengths being convenient reporters of secondary structure. In the case of nucleic acids that bind visible light-absorbing ligands, such as fluorogenic aptamers, another wavelength can be a convenient reporter of secondary structure stability and RNA-ligand recognition. As it can be difficult, if not impossible, to know which wavelength to employ a priori, we have developed a system for obtaining the full UV-visible spectrum of a sample at each wavelength, allowing for the subsequent extraction of the absorbance-temperature profile at the desired wavelength. Here, we describe the apparatus and software used to do so. We also describe another technique for the use of a qPCR instrument for measuring secondary structure stability of fluorescent nucleic acid-ligand complexes.

Blood vessel maturation, which is characterized by the investment of vascular smooth muscle cells (vSMCs) around developing blood vessels, begins when vessels remodel into a hierarchy of proximal arteries and proximal veins that branch into smaller distal capillaries. The ultimate result of maturation is formation of the tunica media-the middlemost layer of a vessel that is composed of vSMCs and acts to control vessel integrity and vascular tone. Though many studies have implicated the role of various signaling molecules in regulating maturation, no studies have determined a role for hemodynamic force in the regulation of maturation in the mouse. In the current study, we provide evidence that a hemodynamic force-dependent mechanism occurs in the mouse because reduced blood flow mouse embryos exhibited a diminished or absent coverage of vSMCs around vessels, and in normal-flow embryos, extent of coverage correlated to the amount of blood flow that vessels were exposed to. We also determine that the cellular mechanism of force-induced maturation was not by promoting vSMC differentiation/proliferation, but instead involved the recruitment of vSMCs away from neighboring low-flow distal capillaries towards high-flow vessels. Finally, we hypothesize that hemodynamic force may regulate expression of specific signaling molecules to control vSMC recruitment to high-flow vessels, as reduction of flow results in the misexpression of Semaphorin 3A, 3F, 3G, and the Notch target gene Hey1, all of which are implicated in controlling vessel maturation. This study reveals another role for hemodynamic force in regulating blood vessel development of the mouse, and opens up a new model to begin elucidating mechanotransduction pathways regulating vascular maturation.

Many traits of cancer progression (e.g., development of metastases or resistance to therapy) are facilitated by tumour evolution: Darwinian selection of subclones with distinct genotypes or phenotypes that enable such progression. Characterising these subclones provide an opportunity to develop drugs to better target their specific properties but requires the accurate identification of somatic mutations shared across multiple spatiotemporal tumours from the same patient. Current best practices for calling somatic mutations are optimised for single samples, and risk being too conservative to identify shared mutations with low prevalence in some samples. We reasoned that datasets from multiple matched tumours can be used for mutual validation and thus propose an adapted two‐stage approach: (1) low‐stringency mutation calling to identify mutations shared across samples irrespective of the weight of evidence in a single sample; (2) high‐stringency mutation calling to further characterise mutations present in a single sample. We applied our approach to three‐independent cohorts of paired primary and recurrent glioblastoma tumours, two of which have previously been analysed using existing approaches, and found that it significantly increased the amount of biologically relevant shared somatic mutations identified. We also found that duplicate removal was detrimental when identifying shared somatic mutations. Our approach is also applicable when multiple datasets e.g. DNA and RNA are available for the same tumour.

Gap junctions, transmembrane protein channels that directly connect the cytoplasm of neighboring cells and enable the exchange of molecules between cells, are a promising new frontier for therapeutic delivery. Specifically, cell-derived lipid vesicles that contain functional gap junction channels, termed Connectosomes, have recently been demonstrated to substantially increase the effectiveness of small molecule chemotherapeutics. However, because gap junctions are present in nearly all tissues, Connectosomes have no intrinsic ability to target specific cell types, which potentially limits their therapeutic effectiveness. To address this challenge, here we display targeting ligands consisting of single-domain antibodies on the surfaces of Connectosomes. We demonstrate that these targeted Connectosomes selectively interact with cells that express a model receptor, promoting the selective delivery of the chemotherapeutic doxorubicin to this target cell population. More generally, our approach has the potential to boost cytoplasmic delivery of diverse therapeutic molecules to specific cell populations while protecting off-target cells, a critical step toward realizing the therapeutic potential of gap junctions.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. The resonant frequency of a diamond shaped monopole antennas is analysed by using a finite difference time domain method. The diamond shaped monopole antennas was fabricated and tested experimentally on RT Duroid material and then the results were directly compared with simple monopole antennas., Tthe work shows that the diamond shaped monopole antennas has smaller dimensions, higher bandwidth and gain at millimeteric wave frequency . Experimentally measured diamond shaped monopole antennas has good agreement with simulated results.

Peak-load pricing (PLP), a two-tariffs charging scheme commonly used in public transport and utilities, is tested on the European Air Traffic Management (ATM) system as a means for reducing capacity-demand imbalances. In particular, a centralised approach to PLP (CPLP) where a Central Planner (CP) sets en-route charges on the network is presented. CPLP consists of two phases: in the first, congested airspace sectors and their peak and off-peak hours are identified; in the second, CP assesses and sets en-route charges in order to reduce overall shift on the network. Such charges should guarantee that Air Navigation Service Providers (ANSPs) are able to recover their operational costs while inducing the Airspace Users (AUs) to route their flights in a way that respects airspace capacity. The interaction between CP and AUs is modelled as a Stackelberg game and formulated by means of bilevel linear programming. Two heuristic approaches, based on Coordinate-wise Descent and Genetic Algorithms are implemented to solve the CPLP model on a data set obtained from historical data for an entire day of traffic on European airspace. Results show that significant improvements in traffic distribution in terms of shift and sector load can be achieved through this simple en-route charges modulation scheme.

We describe a multi-component boundary element method for predicting wave energy distributions in a complex built-up system with material properties changing discontinuously at boundaries between sub-components. We point out that depending on the boundary conditions and the number of interfaces between sub-components, it may be advantageous to use a normal derivative method to set up the integral kernels. We describe how the resulting hypersingular integral kernels can be regularised. The method can be used to minimise the number of weakly singular integrals thus leading to BEM formulations which are easier to handle.

Contains fulltext : 69886.pdf (Publisher’s version ) (Open Access) PURPOSE: The goal of this study was to identify mutations in X-chromosomal genes associated with retinitis pigmentosa (RP) in patients from Germany, The Netherlands, Denmark, and Switzerland. METHODS: In addition to all coding exons of RP2, exons 1 through 15, 9a, ORF15, 15a and 15b of RPGR were screened for mutations. PCR products were amplified from genomic DNA extracted from blood samples and analyzed by direct sequencing. In one family with apparently dominant inheritance of RP, linkage analysis identified an interval on the X chromosome containing RPGR, and mutation screening revealed a pathogenic variant in this gene. Patients of this family were examined clinically and by X-inactivation studies. RESULTS: This study included 141 RP families with possible X-chromosomal inheritance. In total, we identified 46 families with pathogenic sequence alterations in RPGR and RP2, of which 17 mutations have not been described previously. Two of the novel mutations represent the most 3'-terminal pathogenic sequence variants in RPGR and RP2 reported to date. In exon ORF15 of RPGR, we found eight novel and 14 known mutations. All lead to a disruption of open reading frame. Of the families with suggested X-chromosomal inheritance, 35% showed mutations in ORF15. In addition, we found five novel mutations in other exons of RPGR and four in RP2. Deletions in ORF15 of RPGR were identified in three families in which female carriers showed variable manifestation of the phenotype. Furthermore, an ORF15 mutation was found in an RP patient who additionally carries a 6.4 kbp deletion downstream of the coding region of exon ORF15. We did not identify mutations in 39 sporadic male cases from Switzerland. CONCLUSIONS: RPGR mutations were confirmed to be the most frequent cause of RP in families with an X-chromosomal inheritance pattern. We propose a screening strategy to provide molecular diagnostics in these families.

El crecimiento de los niños representa un indicador muy sensible para valorar su estado de salud. Cualquier desviación de los parámetros normales constituye una alarma sobre la posible existencia de algún trastorno, ya sea congénito o adquirido. El conocer con precisión cual es la edad ósea de los niños durante el periodo de crecimiento constituye un dato útil en múltiples situaciones. Desde el punto de vista clínico es una herramienta diagnóstica con posibilidad de monitorizar los efectos del tratamiento, en caso de desviaciones de la normalidad. Es por ello que se debe de profundizar en el conocimiento de los métodos usados y establecer revisiones periódicas que certifiquen su validez. En la actualidad, los métodos radiológicos más frecuentemente empleados en la medida o cálculo de la edad ósea de los niños de nuestro entorno fueron diseñados a mediados del siglo XX basándose en las radiografías de niños de otros países. Sería, pues, interesante conocer si los cambios sociales, económicos, nutricionales y sanitarios han influido en el crecimiento y desarrollo de los niños españoles de una forma significativa, restando validez a los métodos tradicionales de determinación de la edad ósea. Este estudio partió de una hipótesis principal que afirma que el entorno del niño tiene una influencia significativa en la concordancia de la edad ósea con la edad cronológica. Siendo nuestro objetivo estudiar la posible relación entre la alimentación y el desarrollo óseo. Por tanto, el diseño que de una forma más rápida podía testar esta afirmación era indudablemente un estudio transversal o de prevalencia, que determinase el porcentaje de niños que presentaban una edad cronológica concordante con la edad ósea. El trabajo es realizado en el Centro de Rehabilitación y Traumatología de Granada, perteneciente al Hospital Universitario Virgen de las Nieves. Para ello aprovechamos la consulta al Servicio de Urgencias de niños y adolescentes, por traumatismos banales. El tamaño de muestra se ha adaptado a las hipótesis principales del trabajo, el total de los pacientes incluidos fue de 100, por lo que se supera con creces los casos necesarios para testar la validez del estudio en particular. Para determinar la concordancia con la edad ósea con las distintas variables se ha realizado un estudio analítico en el que se incluye, análisis univariante, bivariante y multivariante, con los distintos test que caracterizan a cada uno de ellos. Hemos encontrado que los niños con edad ósea atrasada toman menos pan que los niños con edad ósea adelantada. Sería preciso determinar si la edad ósea y la masa ósea tienen valores inversamente relacionados para saber si estos datos pueden ser concordantes. Sólo se ha podido determinar que actualmente la maduración ósea precoz se suele asociar con el crecimiento rápido. Es probable que los niños que toman más pan tengan una edad ósea adelantada, un crecimiento más rápido y la masa ósea disminuida. En este trabajo, los resultados han de considerarse preliminares a causa del tamaño de la muestra. En el futuro, nuestra investigación sobre la influencia de la nutrición deberá incluir un estudio longitudinal. Child growth constitutes a very sensitive indicator for the assessment of their health state. Any deviation from standard parameters could entail the appearance of any congenital or acquired disorder. Identifying with precision children's bone age during their growth period can be very useful in different situations. From a clinical point of view, it is a tool for diagnosis, which can offer the possibility of monitoring treatment effects in the case of standard deviations. This is why we need to study in depth current methods and carry out regular controls that insure their reliability. Currently, the most frequent radiological methods in our country for the determination of children's bone age were designed in the middle of the 20th century on the basis of X-rays performed on children from other countries. Thus, it would be interesting to find out whether social, economic, nutritional and health changes in Spain have influenced child growth and development in a significant way. Hence it would invalidate traditional methods for the determination of bone age. This study came from a main hypothesis: the child's environment significantly influences the concordance of bone age and chronological age. Our aim was to study a possible relationship between nutrition and bone development. Thus, the fastest way of testing this hypothesis was to implement a cross-sectional prevalence study in order to determine the percentage of children showing a chronological age concording with bone age. The study was entirely carried out in the Traumatology Hospital of Granada, which belongs to the University Hospital Virgen de las Nieves. We used the information provided by the Emergency Department about slight traumatisms. The cross-section sample was adapted to our main hypothesis. The overall number of patients was 100, which exceeds the necessary cases to test the reliability of this particular study. On the other hand, in order to determine the concordance of bone age and its different variables, an analitical study was also carried out, which includes: an univariant analysis, a bivariate analysis and a multivariate analysis, with the various tests they usually include. We found out that children showing a delayed bone age eat less bread than those showing an advanced bone age. It would be necessary to determine whether bone age and bone mass have inversely related values before considering it as concordant information. We could only determine that, currently, advanced bone maturation is usually associated with rapid growth. It is possible that children who eat more bread actually show an advanced bone age, a more rapid growth and a reduced bone mass. However, in this study, the outcomes must be considered just as preliminar, due to the size of the cross-section sample. Our research on the influence of nutrition should also include a further longitudinal study.

Objectives of this paper are: briefly examine solutions applied in other network industries and based on that, set the policy and pricing context for development of market-based mechanisms for strategic air traffic re-distribution to avoid congestion, which is a main goal of SATURN project. Further, focus on current and possible future ATM pricing policy goals, by summarising current practice in Europe and introducing two possible future scenarios developed within the project. The implementation plan is outlined, discussing both the modelling challenges and the parallel consultation and validation processes. We conclude with a short look ahead.