Engineering repeat proteins of the immune system

Limitations associated with immunoglobulins have motivated the search for novel binding scaffolds. Repeat proteins have emerged as one promising class of scaffolds, but often are limited to binding protein and peptide targets. An exception is the repeat proteins of the immune system, which have in recent years served as an inspiration for binding scaffolds which can bind glycans and other classes of biomolecule. Like other repeat proteins, these proteins can be very stable and have a monomeric mode of binding, with elongated and highly variable binding surfaces. The ability to target glycans and glycoproteins fill an important gap in current tools for research and biomedical applications.     

Three parallel linkage groups of human acidic keratin genes.

Two regions of human genomic DNA, each containing several keratin genes, were isolated and partially sequenced. The keratin genes are inactive, having suffered deleterious mutations. Both regions contain at least four keratin genes arranged in a head-to-tail orientation including a pseudogene for keratin K#16. Within each segment there are two keratin genes in close linkage with only 1.5 kb of DNA between them. Sequence comparison of the two regions showed 98.9% identity in both the coding and the intronic segments of the pseudogenes. The pseudogenes show 94% identity to their functional counterparts. Southern hybridization analysis showed that the segments are paralogous, not allelic. The regions are products of two independent, recent duplication events. The first occurred approximately 24 million years ago, after the separation of primates from the rhesus/baboon line. The second is specific for the human lineage, having occurred approximately 3.8 million years ago. Analysis of the genomic DNAs of primates showed the presence of only one of the regions in the DNAs of gibbon and gorilla, while rhesus monkey and baboon were missing both copies. We conclude that the human keratin genes are still actively evolving, with new duplications having occurred as recently as after the separation of human and gorilla ancestors.     

Optimized Null Model for Protein Structure Networks

Much attention has recently been given to the statistical significance of topological features observed in biological networks. Here, we consider residue interaction graphs (RIGs) as network representations of protein structures with residues as nodes and inter-residue interactions as edges. Degree-preserving randomized models have been widely used for this purpose in biomolecular networks. However, such a single summary statistic of a network may not be detailed enough to capture the complex topological characteristics of protein structures and their network counterparts. Here, we investigate a variety of topological properties of RIGs to find a well fitting network null model for them. The RIGs are derived from a structurally diverse protein data set at various distance cut-offs and for different groups of interacting atoms. We compare the network structure of RIGs to several random graph models. We show that 3-dimensional geometric random graphs, that model spatial relationships between objects, provide the best fit to RIGs. We investigate the relationship between the strength of the fit and various protein structural features. We show that the fit depends on protein size, structural class, and thermostability, but not on quaternary structure. We apply our model to the identification of significantly over-represented structural building blocks, i.e., network motifs, in protein structure networks. As expected, choosing geometric graphs as a null model results in the most specific identification of motifs. Our geometric random graph model may facilitate further graph-based studies of protein conformation space and have important implications for protein structure comparison and prediction. The choice of a well-fitting null model is crucial for finding structural motifs that play an important role in protein folding, stability and function. To our knowledge, this is the first study that addresses the challenge of finding an optimized null model for RIGs, by comparing various RIG definitions against a series of network models.     

Key for European species of the Cheilosia proxima group (Diptera,Syrphidae) with a description of a new species

A new hoverfly species, Cheilosia barbafacies Vujić & Radenković sp. n. (Diptera, Syrphidae), is described and distinguished from the closely related species Cheilosia pascuorum Becker, 1894, based on material collected from the mountains of the Balkan Peninsula. Diagnostic characteristics and an identification key for the members of the proxima group of Cheilosia s. str., including the new taxon, are provided.     

Silibinin inhibits hepatitis C virus entry into hepatocytes by hindering clathrin‐dependent trafficking

Hepatitis C virus (HCV) is a global health concern infecting 170 million people worldwide. Previous studies indicate that the extract from milk thistle known as silymarin and its main component silibinin inhibit HCV infection. Here we investigated the mechanism of anti‐HCV action ofsilymarin‐derived compounds at the molecular level. By using live‐cell confocal imaging, single particle tracking, transmission electron microscopy and biochemical approaches on HCV‐infected human hepatoma cells and primary hepatocytes, we show that silibinin potently inhibits HCV infection and hinders HCV entry by slowing down trafficking through clathrin‐coated pits and vesicles. Detailed analyses revealed that silibinin altered the formation of both clathrin‐coated pits and vesicles in cells and caused abnormal uptake and trafficking of transferrin, a well‐known cargo of the clathrin endocytic pathway. Silibinin also inhibited infection by other viruses that enter cells by clathrin‐mediated endocytosis including reovirus, vesicular stomatitis and influenza viruses. Our study demonstrates that silibinin inhibits HCV early steps of infection by affecting endosomal trafficking of virions. It provides new insights into the molecular mechanisms of action of silibinin against HCV entry and also suggests that silibinin is a potential broad‐spectrum antiviral therapy.     

Comparative genomics of two ‘Candidatus Accumulibacter' clades performing biological phosphorus removal

Members of the genus Candidatus Accumulibacter are important in many wastewater treatment systems performing enhanced biological phosphorus removal (EBPR). The Accumulibacter lineage can be subdivided phylogenetically into multiple clades, and previous work showed that these clades are ecologically distinct. The complete genome of Candidatus Accumulibacter phosphatis strain UW-1, a member of Clade IIA, was previously sequenced. Here, we report a draft genome sequence of Candidatus Accumulibacter spp. strain UW-2, a member of Clade IA, assembled following shotgun metagenomic sequencing of laboratory-scale bioreactor sludge. We estimate the genome to be 80–90% complete. Although the two clades share 16S rRNA sequence identity of >98.0%, we observed a remarkable lack of synteny between the two genomes. We identified 2317 genes shared between the two genomes, with an average nucleotide identity (ANI) of 78.3%, and accounting for 49% of genes in the UW-1 genome. Unlike UW-1, the UW-2 genome seemed to lack genes for nitrogen fixation and carbon fixation. Despite these differences, metabolic genes essential for denitrification and EBPR, including carbon storage polymer and polyphosphate metabolism, were conserved in both genomes. The ANI from genes associated with EBPR was statistically higher than that from genes not associated with EBPR, indicating a high selective pressure in EBPR systems. Further, we identified genomic islands of foreign origins including a near-complete lysogenic phage in the Clade IA genome. Interestingly, Clade IA appeared to be more phage susceptible based on it containing only a single Clustered Regularly Interspaced Short Palindromic Repeats locus as compared with the two found in Clade IIA. Overall, the comparative analysis provided a genetic basis to understand physiological differences and ecological niches of Accumulibacter populations, and highlights the importance of diversity in maintaining system functional resilience.     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.     

alpha2-Adrenoceptors control the release of noradrenaline but not neuropeptide Y from perivascular nerve terminals

Neuropeptide Y (NPY) and noradrenaline (NA) are co-transmitters at many sympathetic synapses, but it is not yet clear if their release is independently regulated. To address this question, we quantified the electrically evoked release of these co-transmitters from perivascular nerve terminals to the mesenteric circulation in control and drug-treated rats. 6-Hydroxydopamine reduced the tissue content and the electrically evoked release of ir-NPY and NA as well as the rise in perfusion pressure. A 0.001 mg/kg reserpine reduced the content of ir-NPY and NA, but did not modify their release nor altered the rise in perfusion pressure elicited by the electrical stimuli. However, 0.1mg/kg reserpine reduced both the content and release of NA but decreased only the content but not the release of ir-NPY; the rise in perfusion pressure was halved. Clonidine did not affect the release of ir-NPY while it lowered the outflow of NA, not altering the rise in perfusion pressure elicited by the electrical stimuli. Yohimbine, did not modify the release of ir-NPY but increased the NA outflow, it antagonized the clonidine effect. Therefore, presynaptic alpha2-adrenoceptors modulate the release of NA but not NPY, implying separate regulatory mechanisms.     

30-Day Survival Probabilities as a Quality Indicator for Norwegian Hospitals: Data Management and Analysis

Background

The Norwegian Knowledge Centre for the Health Services (NOKC) reports 30-day survival as a quality indicator for Norwegian hospitals. The indicators have been published annually since 2011 on the website of the Norwegian Directorate of Health (www.helsenorge.no), as part of the Norwegian Quality Indicator System authorized by the Ministry of Health. Openness regarding calculation of quality indicators is important, as it provides the opportunity to critically review and discuss the method. The purpose of this article is to describe the data collection, data pre-processing, and data analyses, as carried out by NOKC, for the calculation of 30-day risk-adjusted survival probability as a quality indicator.

Methods and Findings

Three diagnosis-specific 30-day survival indicators (first time acute myocardial infarction (AMI), stroke and hip fracture) are estimated based on all-cause deaths, occurring in-hospital or out-of-hospital, within 30 days counting from the first day of hospitalization. Furthermore, a hospital-wide (i.e. overall) 30-day survival indicator is calculated. Patient administrative data from all Norwegian hospitals and information from the Norwegian Population Register are retrieved annually, and linked to datasets for previous years. The outcome (alive/death within 30 days) is attributed to every hospital by the fraction of time spent in each hospital. A logistic regression followed by a hierarchical Bayesian analysis is used for the estimation of risk-adjusted survival probabilities. A multiple testing procedure with a false discovery rate of 5% is used to identify hospitals, hospital trusts and regional health authorities with significantly higher/lower survival than the reference. In addition, estimated risk-adjusted survival probabilities are published per hospital, hospital trust and regional health authority. The variation in risk-adjusted survival probabilities across hospitals for AMI shows a decreasing trend over time: estimated survival probabilities for AMI in 2011 varied from 80.6% (in the hospital with lowest estimated survival) to 91.7% (in the hospital with highest estimated survival), whereas it ranged from 83.8% to 91.2% in 2013.

Conclusions

Since 2011, several hospitals and hospital trusts have initiated quality improvement projects, and some of the hospitals have improved the survival over these years. Public reporting of survival/mortality indicators are increasingly being used as quality measures of health care systems. Openness regarding the methods used to calculate the indicators are important, as it provides the opportunity of critically reviewing and discussing the methods in the literature. In this way, the methods employed for establishing the indicators may be improved.