Whole Blood Profiling of T-cell-Derived microRNA Allows the Development of Prognostic models in Inflammatory Bowel Disease.

BACKGROUND: MicroRNAs [miRNAs] are cell-specific small non-coding RNAs that can regulate gene expression and have been implicated in inflammatory bowel disease [IBD] pathogenesis. Here we define the cell-specific miRNA profiles and investigate its biomarker potential in IBD. METHODS: In a two-stage prospective multi-centre case control study, next generation sequencing was performed on a discovery cohort of immunomagnetically separated leukocytes from 32 patients (nine Crohn's disease [CD], 14 ulcerative colitis [UC], eight healthy controls) and differentially expressed signals were validated in whole blood in 294 patients [97 UC, 98 CD, 98 non-IBD, 1 IBDU] using quantitative PCR. Correlations were analysed with phenotype, including need for early treatment escalation as a marker of progressive disease using Cox proportional hazards. RESULTS: In stage 1, each leukocyte subset [CD4+ and CD8+ T-cells and CD14+ monocytes] was analysed in IBD and controls. Three specific miRNAs differentiated IBD from controls in CD4+ T-cells, including miR-1307-3p [p = 0.01], miR-3615 [p = 0.02] and miR-4792 [p = 0.01]. In the extension cohort, in stage 2, miR-1307-3p was able to predict disease progression in IBD (hazard ratio [HR] 1.98, interquartile range [IQR]: 1.20-3.27; logrank p = 1.80 × 10-3), in particular CD [HR 2.81; IQR: 1.11-3.53, p = 6.50 × 10-4]. Using blood-based multimarker miRNA models, the estimated chance of escalation in CD was 83% if two or more criteria were met and 90% for UC if three or more criteria are met. INTERPRETATION: We have identified and validated unique CD4+ T-cell miRNAs that are differentially regulated in IBD. These miRNAs may be able to predict treatment escalation and have the potential for clinical translation; further prospective evaluation is now indicated.

Causes of nasoenteral tube obstruction in tertiary hospital patients.

BACKGROUND/OBJECTIVES: Obstruction of the nasoenteral tube is one of the complications of enteral nutrition therapy, and its causes and frequency of occurrence are not well understood. To evaluate the causes of enteral nutrition feeding tube obstruction. To study the time elapsed between the beginning of the nutrition therapy and the obstruction of the tube. SUBJECTS/METHODS: This was a retrospective cohort study of 1170 patients aged 18 years or older who were hospitalized at Sírio-Libanês Hospital between January 2015 and October 2017, and who were undergoing enteral nutrition therapy delivered using an infusion pump through a nasogastric or nasoenteral tube. The study population included 683 (58%) men and 487 (42%) women. The median age was 79 years. Of these, 1084 patients received enteral nutrition and medication through the feeding tube, and 86 received medication alone. Variables investigated as causes of feeding tube obstruction were the administration of medication through the tube, type of diet, and use of symbiotics. RESULTS: Obstruction rates were 4% for up to 40 days of observation and 8% for the total observation time. The time for obstruction of 10% of the tubes in patients receiving rivaroxaban, linagliptin, metformin, and nystatin was 16, 19, 20, and 28 days, respectively. CONCLUSIONS: The main cause of nasoenteral tube obstruction (odds ratio) was the combination of metformin (2.0), nystatin (3.1), linagliptin (4.3), rivaroxaban (2.4), and a high-protein diet (1.9). Overall, proper tube care and strict compliance with tubal drug delivery guidelines can result in low tube obstruction rates.

Association Between Nighttime Discharge from the Intensive Care Unit and Hospital Mortality: A Multi-Center Retrospective Cohort Study.

BACKGROUND: We aimed to determine the impact of nighttime discharge from the intensive care unit (ICU) to the ward on hospital mortality and readmission rates in consecutive critically ill patients admitted to five Canadian ICUs. We hypothesized that hospital mortality and readmission rates would be higher for patients discharged after hours compared with discharge during the day. METHODS: A multi-center retrospective cohort study was carried out at five hospitals in Edmonton, Canada, between July 2002 and December 2009. Nighttime discharge was defined as discharge from the ICU occurring between 07:00 pm and 07:59 am. Logistic regression analysis was used to explore the associations between nighttime discharge and outcomes. RESULTS: Of 19,622 patients discharged alive from the ICU, 3,505 (17.9%) discharges occurred during nighttime. Nighttime discharge occurred more commonly among medical than surgical patients (19.9% vs. 13.8%, P < 0.001) and among those with more comorbid conditions, compared with daytime discharged patients. Crude hospital mortality (11.8% versus 8.8%, P < 0.001) was greater for nighttime discharged as compared to daytime discharged patients. In a multivariable analysis, after adjustment for comorbidities, diagnosis and source of admission, nighttime discharge remains associated with higher mortality (odds ratio [OR] 1.29; 95% CI, 1.14 to 1.46, P < 0.001). This finding was robust in two sensitivity analyses examining discharges occurring between 00:00 am and 04:59 am (OR 1.28; 1.12-1.47; P < 0.001) and for those who died within 48 h of ICU discharge without readmission (OR 1.24; 1.07-1.42, P = 0.002). There was no difference in ICU readmission for nighttime compared with daytime discharges (7.4% vs. 6.9 %, p = 0.26). However, rates were higher for nighttime discharges in community compared with tertiary hospitals (7.7% vs. 5.7%, P = 0.023). CONCLUSIONS: In a large integrated health region, 1 in 5 ICU patients are discharged at nighttime, a factor with increasing occurrence during our study and shown to be independently associated with higher hospital mortality.

Reproduction and dispersal in an ant-associated root aphid community.

Clonal organisms with occasional sex are important for our general understanding of the costs and benefits that maintain sexual reproduction. Cyclically parthenogenetic aphids are highly variable in their frequency of sexual reproduction. However, studies have mostly focused on free-living aphids above ground, whereas dispersal constraints and dependence on ant-tending may differentially affect the costs and benefits of sex in subterranean aphids. Here, we studied reproductive mode and dispersal in a community of root aphids that are obligately associated with the ant Lasius flavus. We assessed the genetic population structure of four species (Geoica utricularia, Tetraneura ulmi, Forda marginata and Forda formicaria) in a Dutch population and found that all species reproduce predominantly if not exclusively asexually, so that populations consist of multiple clonal lineages. We show that population viscosity is high and winged aphids rare, consistent with infrequent horizontal transmission between ant host colonies. The absence of the primary host shrub (Pistacia) may explain the absence of sex in three of the studied species, but elm trees (Ulmus) that are primary hosts of the fourth species (T. ulmi) occurred within a few km of the study population. We discuss the extent to which obligate ant-tending and absence of primary hosts may have affected selection for permanent parthenogenesis, and we highlight the need for further study of these aphids in Southern Europe where primary hosts may occur close to L. flavus populations, so that all four root aphid species would have realistic opportunities for completing their sexual life cycle.

Association between implementation of an intensivist-led medical emergency team and mortality.

PURPOSE: To evaluate the impact of implementation of a dedicated intensivist-led medical emergency team (IL-MET) on mortality in patients admitted to the intensive care unit (ICU). METHODS: All adult ward admissions to the ICU between July 2002 and December 2009 were reviewed (n=1920) after excluding readmissions and admissions for <24 h. IL-MET hours were defined as 8:00-15:59 (Monday to Friday). The following periods were analysed: period 1: 1 July 2002-31 August 2004 (control); period 2: 1 September 2004-11 February 2007 (partial MET without dedicated intensivist); and period 3: 12 February 2007-31 December 2009 (hospital-wide IL-MET). RESULTS: During all three periods, there were no significant differences in length of stay or mortality (IL-MET vs non-IL-MET hours, p>0.1 for all). On multivariate analysis, Acute Physiology and Chronic Health Evaluation (APACHE) II score and age were independently associated with mortality in all three periods (p<0.05 for all). During period 3, there was a non-significant trend towards decreased mortality if admitted during IL-MET hours (OR 0.73, 95% CI 0.51 to 1.03, p=0.08). During period 3, there was a non-significant trend towards decreased mortality if admitted during IL-MET hours (OR 0.73, 95% CI 0.51 to 1.03, p=0.08). However, this result likely reflects the observed increase in mortality during non-IL MET hours rather than improved mortality during IL-MET hours. CONCLUSION In a single centre experience, implementation of an IL-MET did not reduce the rate of in-hospital death or lengths of stay.

Host acceptance and sex allocation of Nasonia wasps in response to conspecifics and heterospecifics.

Species recognition is an important aspect of an organism's biology. Here, we consider how parasitoid wasps vary their reproductive decisions when their offspring face intra- and interspecific competition for resources and mates. We use host acceptance and sex ratio behaviour to test whether female Nasonia vitripennis and Nasonia longicornis discriminate between conspecifics and heterospecifics when ovipositing. We tested pairs of conspecific or heterospecific females ovipositing either simultaneously or sequentially on a single host, using strains varying in their recent history of sympatry. Both N. vitripennis and N. longicornis rejected parasitized hosts more often than unparasitized hosts, although females were more likely to superparasitize their own species in the sequential treatment. However, sex ratio behaviour did not vary, suggesting similar responses towards conspecifics and heterospecifics. This contrasts with theory predicting that heterospecifics should not influence sex ratios as their offspring do not influence local mate competition, where conspecifics would. These non-adaptive sex ratios reinforce the lack of adaptive kin discrimination in N. vitripennis and suggest a behavioural constraint. Discrimination between closely related species is therefore context dependent in Nasonia. We suggest that isolating mechanisms associated with the speciation process have influenced behaviour to a greater extent than selection on sex ratios.

Genetic diversity amongst isolates of Neospora caninum, and the development of a multiplex assay for the detection of distinct strains.

Infection with Neospora caninum is regarded as a significant cause of abortion in cattle. Despite the economic impact of this infection, relatively little is known about the biology of this parasite. In this study, mini and microsatellite DNAs were detected in the genome of N. caninum and eight loci were identified that each contained repetitive DNA which was polymorphic among different isolates of this parasite. A multiplex PCR assay was developed for the detection of genetic variation within N. caninum based on length polymorphism associated with three different repetitive markers. The utility of the multiplex PCR was demonstrated in that it was able to distinguish amongst strains of N. caninum used as either vaccine or challenge strains in animal vaccination experiments and that it could genotype N. caninum associated with naturally acquired infections of animals. The multiplex PCR is simple, rapid, informative and sensitive and should provide a valuable tool for further studies on the epidemiology of N. caninum in different host species.

The genome of the simian and human malaria parasite Plasmodium knowlesi.

Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the 'kra' monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated, and it has a close phylogenetic relationship to Plasmodium vivax, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or 'hypnozoite' in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome and other sequenced Plasmodium genomes. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.

Secondary DNA structure analysis of the coding strand switch regions of five Leishmania major Friedlin chromosomes.

As part of the EULEISH international genome project, a region of 74,674 nucleotides from chromosome 21 of Leishmania major Friedlin was subcloned and sequenced; and 31 new coding sequences were predicted. Of particular interest was a unique coding strand switching region covering 1.6 kb of DNA; and this was subjected to further investigation. Bioinformatic analysis of this region revealed an unusually high AT composition, a lack of putative hairpins and a strong curvature of the DNA in agreement with the structural characteristics of similar regions of other Leishmania chromosomes. These observations and a comparison with the secondary DNA structure of four other Leishmania chromosomes and chromosomes of different organisms could suggest a functional role of this region in transcription and mitotic division.

Identification and cloning of Lmairk, a member of the Aurora/Ipl1p protein kinase family, from the human protozoan parasite Leishmania.

Lmairk, a gene encoding a member of the Aurora/Ipl1p family of protein kinases (AIRK), was cloned from the protozoan parasite Leishmania major. Aurora kinases are key enzymes involved in the regulation of normal chromosome segregation during mitosis and cytokenesis of eukaryotic cells. This single-copy gene located on L. major chromosome 28 encodes a 301 amino acid polypeptide. All 11 conserved eukaryotic protein kinase catalytic subdomains are present and the proposed AIRK signature sequence was identified in the activation loop between subdomains VII and VIII. Lmairk is expressed, as an approximately 2.4 kb message, in at least three different species of Leishmania. This report represents the first identification of an AIRK from the trypanosomatid family of early divergent eukaryotes.

Parasite genome initiatives.

During 1993-1994, scientists from developing and developed countries planned and initiated a number of parasite genome projects and several consortiums for the mapping and sequencing of these medium-sized genomes were established, often based on already ongoing scientific collaborations. Financial and other support came from WHO/TDR, Wellcome Trust and other funding agencies. Thus, the genomes of Plasmodium falciparum, Schistosoma mansoni, Trypanosoma cruzi, Leishmania major, Trypanosoma brucei, Brugia malayi and other pathogenic nematodes are now under study. From an initial phase of network formation, mapping efforts and resource building (EST, GSS, phage, cosmid, BAC and YAC library constructions), sequencing was initiated in gene discovery projects but soon also on a small chromosome, and now on a fully fledged genome scale. Proteomics, functional analysis, genetic manipulation and microarray analysis are ongoing to different degrees in the respective genome initiatives, and as the funding for the whole genome sequencing becomes secured, most of the participating laboratories, apart from larger sequencing centres, become oriented to post-genomics. Bioinformatics networks are being expanded, including in developing countries, for data mining, annotation and in-depth analysis.

Purification, characterization and crystallization of thermostable anthranilate phosphoribosyltransferase from Sulfolobus solfataricus.

Anthranilate phosphoribosyltransferase (TrpD; EC 2.4.2.18) from the hyperthermophilic archaeon Sulfolobus solfataricus (ssTrpD) was expressed in Escherichia coli, purified and crystallized. Analytical gel permeation chromatography revealed a homodimeric composition of the enzyme. The steady-state kinetic characteristics suggest tight binding of the substrate anthranilic acid and efficient catalysis at the physiological growth temperature of S. solfataricus. Crystals of ssTrpD diffract to better than 2.6 A resolution and preliminary X-ray characterization was carried out. The crystals are suitable for structure determination.

A re-annotation of the Saccharomyces cerevisiae genome.

Discrepancies in gene and orphan number indicated by previous analyses suggest that S. cerevisiae would benefit from a consistent re-annotation. In this analysis three new genes are identified and 46 alterations to gene coordinates are described. 370 ORFs are defined as totally spurious ORFs which should be disregarded. At least a further 193 genes could be described as very hypothetical, based on a number of criteria. It was found that disparate genes with sequence overlaps over ten amino acids (especially at the N-terminus) are rare in both S. cerevisiae and Sz. pombe. A new S. cerevisiae gene number estimate with an upper limit of 5804 is proposed, but after the removal of very hypothetical genes and pseudogenes this is reduced to 5570. Although this is likely to be closer to the true upper limit, it is still predicted to be an overestimate of gene number. A complete list of revised gene coordinates is available from the Sanger Centre (S. cerevisiae reannotation: ftp://ftp/pub/yeast/SCreannotation).

The Leishmania genome project: new insights into gene organization and function.

The sequencing of Leishmania major Friedlin chromosome 1 (Chr1), Chr3, and Chr4 has been completed. and several other chromosomes are well underway. The complete genome sequence should be available by 2003. Over 1,000 full-length new genes have been identified, with the majority (approximately 75%) having unknown function. Many of these may be Leishmania (or kinetoplastid) specific. Most interestingly, the genes are organized into large (> 100-500 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a "divergent" manner, i.e., the mRNAs for the two sets of genes are both transcribed towards the telomeres. Nuclear run-on analysis suggests that transcription is initiated in both directions within the "divergent" region. Chr3 and Chr4 contain two "convergent" clusters, with a single "divergent" gene at one telomere of Chr3. Sequence analysis of several genes from the LD1 region of Chr35 indicates a high degree of sequence conservation between L. major and L. donovani/L. infantum within protein-coding open reading frames (ORFs), with a lower degree of conservation within the non-coding regions. Immunization of mice with recombinant antigen from two of these genes, BTI (formerly ORFG) and ORFF, results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.

Genomic organization and gene function in Leishmania.

Sequencing of the Leishmania major Friedlin genome is well underway with chromosome 1 (Chr1) and Chr3 having been completely sequenced, and Chr4 virtually complete. Sequencing of several other chromosomes is in progress and the complete genome sequence may be available as soon as 2003. A large proportion ( approximately 70%) of the newly identified genes remains unclassified, with many of these being potentially Leishmania- (or kinetoplastid-) specific. Most interestingly, the genes are organized into large (>100-300 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a 'divergent' manner, i. e. the mRNAs for the two sets of genes are both transcribed towards the telomeres. Chr3 contains two 'convergent' clusters, with a single 'divergent' gene at one telomere, with the two large clusters separated by a tRNA gene. We have characterized several genes from the LD1 (Leishmania DNA 1) region of Chr35. BT1 (formerly ORFG) encodes a biopterin transporter and ORFF encodes a nuclear protein of unknown function. Immunization of mice with recombinant antigens from these genes results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.

Reports from the cutting edge of parasitic genome analysis.

This new feature in Parasitology Today will host reports from the laboratories involved in genomics of parasites, be that sequencing, mapping or 'functional genomics' - the mining and analysis of the sequence datasets, and the development of postgenomics tools to examine gene expression, response to drugs and population variability. It will publicize new technology to wider audiences, let communities of researchers know about novel resources (particularly those available through the World Wide Web) and highlight significant advances in the understanding of parasitic genomes through functional genomics.

The crystal structure of anthranilate synthase from Sulfolobus solfataricus: functional implications.

Anthranilate synthase catalyzes the synthesis of anthranilate from chorismate and glutamine and is feedback-inhibited by tryptophan. The enzyme of the hyperthermophile Sulfolobus solfataricus has been crystallized in the absence of physiological ligands, and its three-dimensional structure has been determined at 2.5-A resolution with x-ray crystallography. It is a heterotetramer of anthranilate synthase (TrpE) and glutamine amidotransferase (TrpG) subunits, in which two TrpG:TrpE protomers associate mainly via the TrpG subunits. The small TrpG subunit (195 residues) has the known "triad" glutamine amidotransferase fold. The large TrpE subunit (421 residues) has a novel fold. It displays a cleft between two domains, the tips of which contact the TrpG subunit across its active site. Clusters of catalytically essential residues are located inside the cleft, spatially separated from clustered residues involved in feedback inhibition. The structure suggests a model in which chorismate binding triggers a relative movement of the two domain tips of the TrpE subunit, activating the TrpG subunit and creating a channel for passage of ammonia toward the active site of the TrpE subunit. Tryptophan presumably blocks this rearrangement, thus stabilizing the inactive states of both subunits. The structure of the TrpE subunit is a likely prototype for the related enzymes 4-amino 4-deoxychorismate synthase and isochorismate synthase.

The Leishmania genome comes of Age.

The Leishmania Genome Network (LGN) was born in Rio de Janeiro, Brazil in 1994. In the short period that has elapsed since then, the LGN has focused solely on the acquisition of the resources, and hence data, that have enabled a rational approach to genomic sequencing of the reference strain, Leishmania major Friedlin. This has now been achieved. In this review, Alasdair Ivens and Jennie Blackwell, secretary and chairman of the LGN, respectively, re-examine the approaches that were adopted, comment on some of the interesting data that have been obtained and introduce some genome-wide approaches that will facilitate functional studies of the parasite.