Analysis of stop-gain and frameshift variants in human innate immunity genes.

Loss-of-function variants in innate immunity genes are associated with Mendelian disorders in the form of primary immunodeficiencies. Recent resequencing projects report that stop-gains and frameshifts are collectively prevalent in humans and could be responsible for some of the inter-individual variability in innate immune response. Current computational approaches evaluating loss-of-function in genes carrying these variants rely on gene-level characteristics such as evolutionary conservation and functional redundancy across the genome. However, innate immunity genes represent a particular case because they are more likely to be under positive selection and duplicated. To create a ranking of severity that would be applicable to innate immunity genes we evaluated 17,764 stop-gain and 13,915 frameshift variants from the NHLBI Exome Sequencing Project and 1,000 Genomes Project. Sequence-based features such as loss of functional domains, isoform-specific truncation and nonsense-mediated decay were found to correlate with variant allele frequency and validated with gene expression data. We integrated these features in a Bayesian classification scheme and benchmarked its use in predicting pathogenic variants against Online Mendelian Inheritance in Man (OMIM) disease stop-gains and frameshifts. The classification scheme was applied in the assessment of 335 stop-gains and 236 frameshifts affecting 227 interferon-stimulated genes. The sequence-based score ranks variants in innate immunity genes according to their potential to cause disease, and complements existing gene-based pathogenicity scores. Specifically, the sequence-based score improves measurement of functional gene impairment, discriminates across different variants in a given gene and appears particularly useful for analysis of less conserved genes.

Influence of inter-electrode distance, contraction type, and muscle on the relationship between the sEMG power spectrum and contraction force.

INTRODUCTION: Spectral frequencies of the surface electromyogram (sEMG) increase with contraction force, but debate still exists on whether this increase is affected by various methodological and anatomical factors. This study aimed to investigate the influence of inter-electrode distance (IED) and contraction modality (step-wise vs. ramp) on the changes in spectral frequencies with increasing contraction strength for the vastus lateralis (VL) and vastus medialis (VM) muscles. METHODS: Twenty healthy male volunteers were assessed for isometric sEMG activity of the VM and VL, with the knee at 90° flexion. Subjects performed isometric ramp contractions in knee extension (6-s duration) with the force gradually increasing from 0 to 80 % MVC. Also, subjects performed 4-s step-wise isometric contractions at 10, 20, 30, 40, 50, 60, 70, and 80 % MVC. Interference sEMG signals were recorded simultaneously at different IEDs: 10, 20, 30, and 50 mm. The mean (F mean) and median (F median) frequencies and root mean square (RMS) of sEMG signals were calculated. RESULTS: For all IEDs, contraction modalities, and muscles tested, spectral frequencies increased significantly with increasing level of force up to 50-60 % MVC force. Spectral indexes increased systematically as IED was decreased. The sensitivity of spectral frequencies to changes in contraction force was independent of IED. The behaviour of spectral indexes with increasing contraction force was similar for step-wise and ramp contractions. CONCLUSIONS: In the VL and VM muscles, it is highly unlikely that a particular inter-electrode distance or contraction modality could have prevented the observation of the full extent of the increase in spectral frequencies with increasing force level.

Bone anabolic potential of soy isoflavones and plant extracts and genomic changes during differentiation of hPOB-tert osteoblast-like cells

Summary For the nutritional management of bone health and the prevention of osteoporosis it is important to identify nutrients that positively influence the bone remodeling process at the cellular level. Soy isoflavones show promising osteoprotective effects in animals and humans but their mechanism of action in bone cells is yet poorly understood. Firstly, soy tissue cultures were characterized as a new and optimized source of isoflavones. A large variability in the isoflavone content was observed and high-producing strains (46.3 mg/g dry wt isoflavones) were identified. In the Ishikawa cells bioassay, the estrogenicity of isoflavones was confirmed to be 1000 to 10000 less than 17Mestradiol and that of the malonyl forms was shown for the first time (EC50 of 350 nM and 1880 nM for malonylgenistin and malonyldaidzin, respectively). The estrogenic activity of soya tissue culture extracts correlated to their isoflavone content. Secondly, the effects of phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway, as key mediators of bone formation, were investigated. Dietary achievable concentrations of genistein and daidzein (10vM), and statins (4xM) but not 17M estradiol (10nM), induced BMP-2 gene expression (by up to 3-fold) and inhibited the cholesterol biosynthetic pathway (by up to 50%) in the human osteoblastic cell line hP0B¬tert. In addition, several plant extracts (Cyperus rotundus, Lindera benzoin and Cnidium monnieri) induced BMP-2 gene expression but this induction was not restricted to the inhibition of the cholesterol synthesis pathway neither to the estrogenicity. Finally, the gene expression profiles during hP0B-tert differentiation induced by vitamin D and dexamethasone were analyzed with the Affymetrix human GeneChip. 1665 different genes and 98 ESTs were significantly regulated. The expression profiles of bone-related genes was largely in agreement with previously documented patterns, supporting the physiological relevance of the genomic results and the hP0B-tert cell line as a valid model of human osteoblast differentiation. The expression of alternative differentiation markers during the osteogenic treatment of hP0B-tert cells indicated that the adipocyte and myoblast differentiation pathways were repressed, confirming that these culture conditions allowed only osteoblast differentiation. The gene ontology analysis identified further sub-groups of genes that may be involved in the bone formation process. Aims of the thesis In order to define new strategies for the nutritional management of bone health and for the prevention of osteoporosis the major goal of the present work was to investigate the potential of phytonutrients to positively modulate the bone formation process at the cellular level and, in particular: 1.To select and optimise alternative plant sources containing high levels of isoflavones with estrogenic activity (Chapter 3). 2.To compare the effects of statins and phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway and to select new plant extracts with a bone anabolic potential (Chapter 4). 3.To further characterize the new human periosteal cell line, hP0B-tert, as a bone- formation model, by elucidating its gene expression profile during differentiation induced by vitamin D and dexamethasone (Chapter 5).

Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy.

BACKGROUND: Second Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet's membrane, in the posterior cornea. METHODOLOGY/PRINCIPAL FINDINGS: We studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet's membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats. CONCLUSIONS/SIGNIFICANCE: Our study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.

Use of antibiotics in pediatric intensive care and potential savings.

OBJECTIVE: Minimizing unwarranted prescription of antibiotics remains an important objective. Because of the heterogeneity between units regarding patient mix and other characteristics, site-specific targets for reduction must be identified. Here we present a model to address the issue by means of an observational cohort study. SETTING: A tertiary, multidisciplinary, neonatal, and pediatric intensive care unit of a university teaching hospital. PATIENTS: All newborns and children present in the unit (n = 456) between September 1998 and March 1999. Reasons for admission included postoperative care after cardiac surgery, major neonatal or pediatric surgery, severe trauma, and medical conditions requiring critical care. METHODS: Daily recording of antibiotics given and of indications for initiation. After discontinuation, each treatment episode was assessed as to the presence or absence of infection. RESULTS: Of the 456 patients 258 (56.6%) received systemic antibiotics, amounting to 1815 exposure days (54.6%) during 3322 hospitalization days. Of these, 512 (28%) were prescribed as prophylaxis and 1303 for suspected infection. Treatment for suspected ventilator-associated pneumonia accounted for 616 (47%) of 1303 treatment days and suspected sepsis for 255 days (20%). Patients were classified as having no infection or viral infection during 552 (40%) treatment days. The average weekly exposure rate in the unit varied considerably during the 29-week study period (range: 40-77/100 hospitalization days). Patient characteristics did not explain this variation. CONCLUSION: In this unit the largest reduction in antibiotic treatment would result from measures assisting suspected ventilator-associated pneumonia to be ruled out and from curtailing extended prophylaxis.

Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure.

Loss of T-tubules (TT), sarcolemmal invaginations of cardiomyocytes (CMs), was recently identified as a general heart failure (HF) hallmark. However, whether TT per se or the overall sarcolemma is altered during HF process is still unknown. In this study, we directly examined sarcolemmal surface topography and physical properties using Atomic Force Microscopy (AFM) in living CMs from healthy and failing mice hearts. We confirmed the presence of highly organized crests and hollows along myofilaments in isolated healthy CMs. Sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows and related to the presence of few packed subsarcolemmal mitochondria (SSM) as evidenced by electron microscopy. Three days after myocardial infarction (MI), CMs already exhibit an overall sarcolemma disorganization with general loss of crests topography thus becoming smooth and correlating with a decreased elasticity while interfibrillar mitochondria (IFM), myofilaments alignment and TT network were unaltered. End-stage post-ischemic condition (15days post-MI) exacerbates overall sarcolemma disorganization with, in addition to general loss of crest/hollow periodicity, a significant increase of cell surface stiffness. Strikingly, electron microscopy revealed the total depletion of SSM while some IFM heaps could be visualized beneath the membrane. Accordingly, mitochondrial Ca(2+) studies showed a heterogeneous pattern between SSM and IFM in healthy CMs which disappeared in HF. In vitro, formamide-induced sarcolemmal stress on healthy CMs phenocopied post-ischemic kinetics abnormalities and revealed initial SSM death and crest/hollow disorganization followed by IFM later disarray which moved toward the cell surface and structured heaps correlating with TT loss. This study demonstrates that the loss of crest/hollow organization of CM surface in HF occurs early and precedes disruption of the TT network. It also highlights a general stiffness increased of the CM surface most likely related to atypical IFM heaps while SSM died during HF process. Overall, these results indicate that initial sarcolemmal stress leading to SSM death could underlie subsequent TT disarray and HF setting.

What underlies localization and urbanization economies? Evidence from the location of new firms

The objective of this paper is to analyze why firms in some industries locate in specialized economic environments (localization economies) while those in other industries prefer large city locations (urbanization economies). To this end, we examine the location decisions of new manufacturing firms in Spain at the city level and for narrowly defined industries (three-digit level). First, we estimate firm location models to obtain estimates that reflect the importance of localization and urbanization economies in each industry. In a second step, we regress these estimates on industry characteristics that are related to the potential importance of three agglomeration theories, namely, labor market pooling, input sharing and knowledge spillovers. Localization effects are low and urbanization effects are high in knowledge-intensive industries, suggesting that firms (partly) locate in large cities to reap the benefits of inter-industry knowledge spillovers. We also find that localization effects are high in industries that employ workers whose skills are more industry-specific, suggesting that industries (partly) locate in specialized economic environments to share a common pool of specialized workers.

Predation Potential of the Water Bugs Sphaerodema rusticum on the Sewage Snails Physa acuta

The sewage snail Physa acuta is a serious threat to certain economic plants and to the purification plant of sewage works by rendering the biofilters ineffective. Various attempts are being made to control it. The efficacy of the predacious water bugs Sphaerodema rusticum was judged experimentally, in the laboratory in the potential control of P. acuta. It is revealed that, when supplied separately, the first, second and third instar and the adult S. rusticum did not attack P. acuta belonging to 3.1-8 mm, 5.1-8 mm, 7.1-8 mm and <= 3 mm size classes respectively. In the remaining trials predation rate varied from zero to eight (average 2.3) individuals per predator per day. In experiments with P. acuta belonging to all the size classes supplied together, none, except the first instar S. rusticum, attacked the prey individuals belonging to the lowest (<= 3 mm) size class. The first and second instar S. rusticum, in both trials did not attack P. acuta larger than 4 mm and 5 mm in shell length respectively. The water bugs belonging to the third, fourth, fifth instar and adult stages though preyed upon P. acuta with 3.1-8 mm shell length. The average rate of predation by a single S. rusticum varied from 0.14-3.08 individuals per day depending upon the size of P. acuta and the stage of S. rusticum. A single S. rusticum, irrespective of instar and adult stages, destroyed on average 4.16 P. acuta daily irrespective of sizes. It is estimated that one S. rusticum could destroy 1,360 P. acuta in its life time. The results clearly indicate that the water bug S. rusticum may be used to control the snails P. acuta.

Search for Borrelia sp. in ticks collected from potential reservoirs in an urban forest reserve in the State of Mato Grosso do Sul, Brazil: a short report

A total of 128 ticks of the genus Amblyomma were recovered from 5 marsupials (Didelphis albiventris) - with 4 recaptures - and 17 rodents (16 Bolomys lasiurus and 1 Rattus norvegicus) captured in an urban forest reserve in Campo Grande, State of Mato Grosso do Sul, Brazil. Of the ticks collected, 95 (78.9%) were in larval form and 22 (21.1%) were nymphs; the only adult (0.8%) was identified as A. cajennense. Viewed under dark-field microscopy in the fourth month after seeding, 9 cultures prepared from spleens and livers of the rodents, blood of the marsupials, and macerates of Amblyomma sp. nymphs revealed spiral-shaped, spirochete-like structures resembling those of Borrelia sp. Some of them showed little motility, while others were non-motile. No such structures could be found either in positive Giemsa-stained culture smears or under electron microscopy. No PCR amplification of DNA from those cultures could be obtained by employing Leptospira sp., B. burgdorferi, and Borrelia sp. primers. These aspects suggest that the spirochete-like structures found in this study do not fit into the genera Borrelia or Leptospira, requiring instead to be isolated for proper identification.

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I.

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.

Towards a Standardised Framework for Inter-Country Adoption - Report of the Implementation Group

The report contains Background to the commissioning of the Report â?~â?~Towards a Standardised Framework for Intercountry Adoption Assessment Proceduresâ?Tâ?T; the Government decision arising; and the principal findings and recommendations of the Report (Chapter 1); Detailed information on progress made in relation to the recommendations contained in the Report (Chapters 2-5); Statistical data in relation to intercountry adoption services at June, 2000 (Chapter 6); A summary of key findings of the Implementation Group (Chapter 7); and The Implementation Groupâ?Ts recommendations regarding the future of intercountry adoption services (Chapter 8). Download the Report here

Towards a Standardised Framework for Inter-Country Adoption - Assessment Procedures

In the first part the background on inter-country adoption and assessment of prospective adopters are considered in general terms in Chapters Two and Three respectively. The second chapter places the objectives of the research into context. Due to the declining numbers of children being put forward for domestic adoption, there is increasing recourse to inter-country adoption. An overview of the situation is outlined. The legislation section outlines both Irish legislation and the pivotal international agreement relevant to inter-country adoption, namely the Hague Convention. The implications for inter-country adoption processes are described Download the Report here

A potential snail host of schistosomiasis in Bolivia: Biomphalaria amazonica paraense, 1966

Biomphalaria amazonica Paraense, 1996 was collected from a permanent pond in the outskirts of the Bolivian city of Santa Cruz. Identification of the collected specimens was made by comparison with the original description of the species and with topotypic material in the collection of Instituto Oswaldo Cruz. Phylogenetic analysis confirmed that these Bolivian specimens belong to B. amazonica.