KrakenUniq: confident and fast metagenomics classification using unique k-mer counts.

False-positive identifications are a significant problem in metagenomics classification. We present KrakenUniq, a novel metagenomics classifier that combines the fast k-mer-based classification of Kraken with an efficient algorithm for assessing the coverage of unique k-mers found in each species in a dataset. On various test datasets, KrakenUniq gives better recall and precision than other methods and effectively classifies and distinguishes pathogens with low abundance from false positives in infectious disease samples. By using the probabilistic cardinality estimator HyperLogLog, KrakenUniq runs as fast as Kraken and requires little additional memory. KrakenUniq is freely available at https://github.com/fbreitwieser/krakenuniq .

The perils of gene patents.

I argue here that gene patents, and patented genetic tests based on them, are a very bad idea. First, I discuss whether genes can reasonably be the subject of patents in the first place; I maintain that the answer is no. Second, I explain how gene patents interfere with scientific progress, slowing down the development of new cures and treatments for genetic diseases.

[Renaissance of surgery for coronary artery disease]. / Renaissance der Bypasschirurgie.

Coronary bypass surgery is now a standard therapy for the treatment of coronary heart disease. Developed in the 1960s, the coronary artery bypass operation was the most performed operation at all in the 1980s. There was and still is rapidly advancing. The recovery of coronary surgery was held back in the 1970s with the introduction of heart catheterisation. To date, the number of interventional revascularizations has increased, while the number of bypass operations has reached a plateau. 30 resp. 40 years after the introduction of the two techniques it is still controversial which is the method of choice. According to the latest guidelines there is strong evidence that coronary artery surgery for patients with complex coronary artery disease is the gold standard: A Renaissance of bypass surgery.

Sternal plate closure: indications, surgical procedure and follow-up.

OBJECTIVES: Titanium plate osteosynthesis (Synthes) is an alternative option for sternal closure. The indications and time point of application are still debated. This study investigated the application and feasibility of this technique after median sternotomy. METHODS: Forty-one patients (29 M/12F, mean age 63 ± 17 years) received the plate system for complicated sternal conditions. Indications, intraoperative course and postoperative follow-up were assessed. RESULTS: Sternal deformity was present in 5 % (2/41), sternal fractures in 17 % (7/41), bone defect in 12 % (5/41), wire loosening in 39 % (16/41) and pseudoarthrosis in 27 % (11/41). 54 % (22/41) of patients showed concomitant sternal infection. Two intraoperative complications were noted: mammary artery injury (1 patient), pleural injury (1 patient). At discharge the patients reported no pain (90 %, 37/41) or only occasional discomfort (10 %, 4/41). Postoperative complications were subcutaneous hematoma in 12 % (5/41), seroma in 12 % (5/41) and sternal reinfection in 7 % (3/41). 12 % (5/41) showed occasional discomfort and 7 % (3/41) had persistent pain leading to plate removal. CONCLUSION: The Titanium Sternal Fixation System is comfortable and easy to use. It can be used to treat a wide spectrum of indications, especially for pseudoarthrosis, an entity which has not yet received sufficient attention.

"When Aneurysm Ain't Aneurysm": sinus of Valsalva aneurysm mimicked by healed abscess cavity under the aortic valve.

In a 70-year-old patient with severe aortic valve stenosis, preoperative standard imaging (transthoracic echocardiography and angiography) detected an unclear subannular cavity structure. Initially interpreted as an aneurysm of Valsalva, the structure was identified intraoperatively as a huge chronic abscess cavity and exclusion was carried out by pericardial patch plasty. This case draws attention to the importance of a differential diagnosis of an abscess due to infective endocarditis in cases of unclear subannular structures rashly diagnosed as aneurysm of Valsalva.

Analysis of Carica papaya Telomeres and Telomere-Associated Proteins: Insights into the Evolution of Telomere Maintenance in Brassicales.

Telomeres are terminal regions of linear eukaryotic chromosomes that are critical for genome stability and continued cell proliferation. The draft assembly of the papaya genome provides an opportunity to analyze and compare the evolution of telomeric DNA sequence composition and telomere maintenance machinery in this and other organisms of the Brassicales Order, which includes Arabidopsis. Here we investigate telomere size and sequence variation at papaya chromosome ends. As with most other plant species, papaya telomeres consist of TTTAGGG repeats. However, in contrast to members of the closely related Brassicaceae family, telomeres in papaya are ~10-fold longer. Sequence analysis reveals that many centromereproximal telomere repeats in papaya harbor nucleotide substitutions and insertions of Gs and Ts. In contrast, we found very few N-to-C substitutions, and even fewer instances of nucleotide deletion, suggesting that a six-nucleotide telomere repeat is not well tolerated. The papaya genome encodes single-copy sequence homologues of several genes involved in telomere maintenance and chromosome end protection, including the Telomerase Reverse Transcriptase (TERT) and Protection Of Telomeres (POT1). Notably, unlike Arabidopsis, which encodes six Telomere Repeat binding Factor-like (TRFL) proteins that bind double-stranded telomere DNA, papaya appears to encode only two such proteins. Thus, the more streamlined genome of papaya will provide an excellent resource for comparative and functional analysis of telomeres in plants.

Efficient implementation of a generalized pair hidden Markov model for comparative gene finding.

MOTIVATION: The increased availability of genome sequences of closely related organisms has generated much interest in utilizing homology to improve the accuracy of gene prediction programs. Generalized pair hidden Markov models (GPHMMs) have been proposed as one means to address this need. However, all GPHMM implementations currently available are either closed-source or the details of their operation are not fully described in the literature, leaving a significant hurdle for others wishing to advance the state of the art in GPHMM design. RESULTS: We have developed an open-source GPHMM gene finder, TWAIN, which performs very well on two related Aspergillus species, A.fumigatus and A.nidulans, finding 89% of the exons and predicting 74% of the gene models exactly correctly in a test set of 147 conserved gene pairs. We describe the implementation of this GPHMM and we explicitly address the assumptions and limitations of the system. We suggest possible ways of relaxing those assumptions to improve the utility of the system without sacrificing efficiency beyond what is practical. AVAILABILITY: Available at http://www.tigr.org/software/pirate/twain/twain.html under the open-source Artistic License.

Influence of peroxisome proliferator-activated receptor gamma activation by its endogenous ligand 15-deoxy Delta12,14 prostaglandin J2 on nitric oxide production in term placental tissues from diabetic women.

Diabetes induces alterations which condition placental remodelling. The levels of nitric oxide (NO) (a modulator of placental invasiveness, differentiation and proliferation) were higher in term placental explants from diabetic patients when compared to controls. Peroxisome proliferator-activated receptor gamma (PPARgamma) activation by its endogenous ligand 15-deoxy Delta(12,14)prostaglandin J(2) (15dPGJ(2)), is a differentiating factor of adipocytes and other cell types, such as trophoblasts. 15dPGJ(2) is also able to down-regulate NO production in different cell types. Our study evaluated the levels of 15dPGJ(2) and PPARgamma and the influence of PPARgamma activation by 15dPGJ(2) on the production of NO, in term placental tissues from control, pre-gestational and gestational diabetic patients. Our results showed that 15dPGJ(2) was present in human term placenta, and that its levels were diminished in gestational (P<0.05) and pre-gestational (P<0.002) diabetic women when compared to controls. Exogenous 15dPGJ(2) addition (2 x 10(-6) mol/l) down-regulated NO production in placenta from control (P<0.001) and pre-gestational diabetic (P<0.01) patients, but failed to do so in gestational diabetic women, whose placental PPARgamma expression was diminished in comparison to controls (P<0.001). As the exogenous activation of PPARgamma prevented NO overproduction in placenta from pre-gestational diabetic women, it may have the potential to improve fetal outcome in this pathology.

TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders.

UNLABELLED: We describe two new Generalized Hidden Markov Model implementations for ab initio eukaryotic gene prediction. The C/C++ source code for both is available as open source and is highly reusable due to their modular and extensible architectures. Unlike most of the currently available gene-finders, the programs are re-trainable by the end user. They are also re-configurable and include several types of probabilistic submodels which can be independently combined, such as Maximal Dependence Decomposition trees and interpolated Markov models. Both programs have been used at TIGR for the annotation of the Aspergillus fumigatus and Toxoplasma gondii genomes. AVAILABILITY: Source code and documentation are available under the open source Artistic License from http://www.tigr.org/software/pirate

Left ventricular assist device as bridge to heart transplantation--lessons learned with the MicroMed DeBakey axial blood flow pump.

OBJECTIVE: The MicroMed DeBakey left ventricular assist device (LVAD) axial blood flow pump was used as bridge to heart transplantation (HTx) in patients with terminal heart failure. The aim was to evaluate this novel mechanical circulatory support system in regard to overall outcome. METHODS: Prospective study in 15 HTx candidates (mean age 40+/-7 years) with terminal heart failure and maximal medical treatment due to ischemic cardiomyopathy (CMP, n=5), dilated CMP (n=3), restrictive CMP (n=2), unclassified CMP (n=1), metabolic CMP (n=1), valvular CMP (n=1) and congenital CMP (n=2). All patients were implanted with a MicroMed DeBakey LVAD. A rescue procedure was necessary in eight critical patients, while seven underwent elective LVAD implantation. Procedures were performed via median sternotomy, in normotherm femoro-femoral CPB (mean duration 59+/-1 min). Oral Marcoumar (INR 2.0-3.0) and Aspirin (100 mg daily) were started as soon as possible. Patients were discharged into a specialized rehabilitation clinic from which it was possible to release them home after a few weeks. RESULTS: Successful implantation and discharge from ICU (mean stay 10+/-7 days) was possible in 11 patients. Seven were transplanted (mean support 50.7 days) and one is awaiting HTx (support >310 days) in the comfort of his home (NYHA I). Survival was 100% among the transplanted patients. Of the seven elective implants, five, and of the eight rescue procedures three patients underwent successful HTx. Four patients died early, while three patients died late on pump support due to intracranial hemorrhage (n=2, 73 and 76 days) and chest infection (n=1, 124 days). All survivors were discharged from hospital, with significant decrease in NYHA class (mean 3.8-2.4 (n=11)). Treadmill testing showed increased exercise tolerance, from 35 to 71W (n=4). Plasma BNP values (mean 950-162 ng/l (n=4)) and pulmonary resistance (mean 316-194.5 dyne s/cm(5) (n=3)) decreased significantly during LVAD support. CONCLUSIONS: The MicroMed DeBakey LVAD is simple to implant; outpatient treatment is safe and efficient. Patients' condition and pulmonary resistances normalize within 6 weeks, making previously considered inoperable patients amenable for HTx. HTx can be performed in low-risk situation, allowing better donor-recipient matching and improving overall outcome.

Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae.

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains.

Virulence and immunity are poorly understood in Mycobacterium tuberculosis. We sequenced the complete genome of the M. tuberculosis clinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample of M. tuberculosis clinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms among M. tuberculosis strains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.

A probabilistic method for identifying start codons in bacterial genomes.

As the pace of genome sequencing has accelerated, the need for highly accurate gene prediction systems has grown. Computational systems for identifying genes in prokaryotic genomes have sensitivities of 98-99% or higher (Delcher et al., Nucleic Acids Res., 27, 4636-4641, 1999). These accuracy figures are calculated by comparing the locations of verified stop codons to the predictions. Determining the accuracy of start codon prediction is more problematic, however, due to the relatively small number of start sites that have been confirmed by independent, non-computational methods. Nonetheless, the accuracy of gene finders at predicting the exact gene boundaries at both the 5' and 3' ends of genes is of critical importance for microbial genome annotation, especially in light of the important signaling information that is sometimes found on the 5' end of a protein coding region. In this paper we propose a probabilistic method to improve the accuracy of gene identification systems at finding precise translation start sites. The new system, RBSfinder, is tested on a validated set of genes from Escherichia coli, for which it improves the accuracy of start site locations predicted by computational gene finding systems from the range 67-77% to 90% correct.

Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence.

The genome of the halophilic archaeon Halobacterium sp. NRC-1 and predicted proteome have been analyzed by computational methods and reveal characteristics relevant to life in an extreme environment distinguished by hypersalinity and high solar radiation: (1) The proteome is highly acidic, with a median pI of 4.9 and mostly lacking basic proteins. This characteristic correlates with high surface negative charge, determined through homology modeling, as the major adaptive mechanism of halophilic proteins to function in nearly saturating salinity. (2) Codon usage displays the expected GC bias in the wobble position and is consistent with a highly acidic proteome. (3) Distinct genomic domains of NRC-1 with bacterial character are apparent by whole proteome BLAST analysis, including two gene clusters coding for a bacterial-type aerobic respiratory chain. This result indicates that the capacity of halophiles for aerobic respiration may have been acquired through lateral gene transfer. (4) Two regions of the large chromosome were found with relatively lower GC composition and overrepresentation of IS elements, similar to the minichromosomes. These IS-element-rich regions of the genome may serve to exchange DNA between the three replicons and promote genome evolution. (5) GC-skew analysis showed evidence for the existence of two replication origins in the large chromosome. This finding and the occurrence of multiple chromosomes indicate a dynamic genome organization with eukaryotic character.

A clustering method for repeat analysis in DNA sequences.

BACKGROUND: A computational system for analysis of the repetitive structure of genomic sequences is described. The method uses suffix trees to organize and search the input sequences; this data structure has been used previously for efficient computation of exact and degenerate repeats. RESULTS: The resulting software tool collects all repeat classes and outputs summary statistics as well as a file containing multiple sequences (multi fasta), that can be used as the target of searches. Its use is demonstrated here on several complete microbial genomes, the entire Arabidopsis thaliana genome, and a large collection of rice bacterial artificial chromosome end sequences. CONCLUSIONS: We propose a new clustering method for analysis of the repeat data captured in suffix trees. This method has been incorporated into a system that can find repeats in individual genome sequences or sets of sequences, and that can organize those repeats into classes. It quickly and accurately creates repeat databases from small and large genomes. The associated software (RepeatFinder), should prove helpful in the analysis of repeat structure for both complete and partial genome sequences.

Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.

Microbial genes in the human genome: lateral transfer or gene loss?

The human genome was analyzed for evidence that genes had been laterally transferred into the genome from prokaryotic organisms. Protein sequence comparisons of the proteomes of human, fruit fly, nematode worm, yeast, mustard weed, eukaryotic parasites, and all completed prokaryote genomes were performed, and all genes shared between human and each of the other groups of organisms were collected. About 40 genes were found to be exclusively shared by humans and bacteria and are candidate examples of horizontal transfer from bacteria to vertebrates. Gene loss combined with sample size effects and evolutionary rate variation provide an alternative, more biologically plausible explanation.

Structural characterization of erythroid and megakaryocytic differentiation in Friend erythroleukemia cells.

OBJECTIVE: The aim of this study was to examine the structural characterization of erythroid and megakaryocytic cell differentiation in Friend erythroleukemic cells using spectral imaging and electron microscopy. MATERIALS AND METHODS: Two variants of Friend erythroleukemia cells were treated with hexamethylene bisacetamide (HMBA) to induce differentiation: 1) MEL, which exhibit the normal phenotype and are susceptible to differentiation; and 2) the resistant R1 cells. The cells were analyzed by spectral imaging along with transmission and scanning electron microscopy. The expression of cell cycle regulatory proteins was analyzed by Western blotting. RESULTS: Spectral imaging of HMBA-treated MEL and R1 cells stained by May-Grünwald-Giemsa and subjected to spectral similarity mapping revealed five morphologic cell types: proerythroblast-like cells, normoblast-like cells, reticulocyte-like cells, megakaryocytes, and apoptotic cells. In MEL cells, both megakaryocytic differentiation characterized by nuclear lobes and erythroid differentiation characterized by accumulation of hemoglobin were detected; R1 cells were not committed to terminal differentiation. HMBA-induced cell cycle arrest at G(1) affected the expression of regulatory proteins in a similar manner in both types of cells. Expression of cyclin-dependent kinase 4 decreased and expression of p21(WAF1) increased. The level of the underphosphorylated form of phosphorylated retinoblastoma protein increased, inducing a decrease in the level of c-myc. In addition, we detected a decrease in the expression of the anti-apoptotic regulator, Bcl-2, and an increased expression of the pro-apoptotic regulator, Bax. CONCLUSIONS: Spectral imaging provides new insight for the morphologic characterization of erythroid and megakaryocytic cell differentiation as well as apoptosis. Image analysis was well correlated to cell cycle arrest and the expression of regulatory proteins.