Human-mouse alignments with BLASTZ

The Mouse Genome Analysis Consortium aligned the human and mouse genome sequences for a variety of purposes, using alignment programs that suited the various needs. For investigating issues regarding genome evolution, a particularly sensitive method was needed to permit alignment of a large proportion of the neutrally evolving regions. We selected a program called BLASTZ, an independent implementation of the Gapped BLAST algorithm specifically designed for aligning two long genomic sequences. BLASTZ was subsequently modified, both to attain efficiency adequate for aligning entire mammalian genomes and to increase its sensitivity. This work describes BLASTZ, its modifications, the hardware environment on which we run it, and several empirical studies to validate its results.     

Tissue and serum levofloxacin concentrations in diabetic foot infection patients

OBJECTIVES: Levofloxacin has a high bioavailability and a broad antibacterial spectrum which covers the common pathogens found in acute and chronic diabetic foot infections. The purpose of our study was to determine the serum and tissue concentrations of levofloxacin when administered orally in patients with infected diabetic foot ulcers and to compare these values with microbiological findings. PATIENTS AND METHODS: Ten outpatients with diabetes and ulcerations of the lower extremity were included. All patients received oral levofloxacin therapy at a dose of 500 mg once daily. Wound tissue and serum samples were collected and levofloxacin concentrations determined by HPLC with fluorescence detection. Additionally, microbiological cultures were performed from swabs and debrided wound tissue, both before and after treatment. MICs of levofloxacin for all bacterial isolates were determined using the Etest. RESULTS: Following oral treatment with levofloxacin for an average of 10 +/- 3.8 days, all patients received debridement at the affected limbs. The levofloxacin concentrations in necrotic wound tissue were between 2.33-23.23 mg/kg and between 0.12-6.41 mg/L in serum. Tissue-to-serum ratios of levofloxacin concentrations for each patient were >1.0. The MIC values for all 17 initially isolated bacteria were < or = 2 mg/L. In half of our patients, fluoroquinolones were one of the few oral monotherapy options where the spectrum covered all initially isolated pathogens. CONCLUSION: Our data showed good tissue penetration of levofloxacin in diabetic foot ulcers. In combination with adequate surgical debridement, levofloxacin seems well suited to the treatment of skin structure infections of diabetics caused by susceptible organisms.     

Mosaic retroposon insertion patterns in placental mammals

One and a half centuries after Charles Darwin and Alfred Russel Wallace outlined our current understanding of evolution, a new scientific era is dawning that enables direct observations of genetic variation. However, pure sequence-based molecular attempts to resolve the basal origin of placental mammals have so far resulted only in apparently conflicting hypotheses. By contrast, in the mammalian genomes where they were highly active, the insertion of retroelements and their comparative insertion patterns constitute a neutral, virtually homoplasy-free archive of evolutionary histories. The “presence” of a retroelement at an orthologous genomic position in two species indicates their common ancestry in contrast to its “absence” in more distant species. To resolve the placental origin controversy we extracted ∼2 million potentially phylogenetically informative, retroposon-containing loci from representatives of the major placental mammalian lineages and found highly significant evidence challenging all current single hypotheses of their basal origin. The Exafroplacentalia hypothesis (Afrotheria as the sister group to all remaining placentals) is significantly supported by five retroposon insertions, the Epitheria hypothesis (Xenarthra as the sister group to all remaining placentals) by nine insertion patterns, and the Atlantogenata hypothesis (a monophyletic clade comprising Xenarthra and Afrotheria as the sister group to Boreotheria comprising all remaining placentals) by eight insertion patterns. These findings provide significant support for a “soft” polytomy of the major mammalian clades. Ancestral successive hybridization events and/or incomplete lineage sorting associated with short speciation intervals are viable explanations for the mosaic retroposon insertion patterns of recent placental mammals and for the futile search for a clear root dichotomy.Genomic variation is based on genetic convertibility and is amplified by gene flow and sexual recombination. While only a small fraction of genetic variation is directly exposed to the natural selection driving the evolution of species, the majority of changes are neutral (Kimura 1968). In the molecular Darwinian and post-genomic age when such variations are now directly observable in prodigious numbers, one still needs only compare the volumes of controversy in scientific reports to dispel the notion that all the mysteries of evolutionary history are now an open book. The basal node of the placental mammals is a prime example of such a controversy. After continuous morphological inconsistencies and a lack of resolution due to narrow temporal speciation events at the deeper divergences of the placental mammalian tree, the hope was that large-scale DNA sequencing and an increasing number of sampled species might resolve the higher-level mammalian relationships by reducing systematic biases. But the extraction and analysis of molecular traces of evolutionary history has been no less sensitive to misinterpretation than was the understanding of dusty specimens 150 years ago. The reliability of sequence data in genome-scale phylogenetic approaches is subject to unequal evolutionary rates among lineages, regional- or lineage-specific compositional biases, shifts in site-specific evolutionary rates over time and sequence regions (e.g., Nishihara et al. 2007), variable accuracies of multisequence alignments and analytical tools, and last but not least, the general liability of such data to homoplasy.In a seminal work, Murphy et al. (2001a) investigated ∼10 kb of sequence information to substantiate the previously proposed (Waddell et al. 1999) major mammalian groups Afrotheria, Xenarthra, Laurasiatheria, and Euarchontoglires. Moderate support was found for Afrotheria as the basal split of placentals (Exafroplacentalia; Fig. 1A). A subsequent combination and expansion of previously published data (Madsen et al. 2001; Murphy et al. 2001a) found significant support for a basal split between Afrotheria and other placentals (Murphy et al. 2001b). Nikolaev et al. (2007) also found support for the Afrotheria as the first placental split, drawing on ∼200 kb of protein-coding sequences from the 1% of the human genome studied in the ENCODE pilot project (The ENCODE Project Consortium 2007). However, other studies of large-scale sequences (e.g., Hallström et al. 2007) and previous studies of smaller data sets could not significantly confirm an Afrotherian root (e.g., Madsen et al. 2001; Waddell et al. 2001; Delsuc et al. 2002; Amrine-Madsen et al. 2003). A sister-group relationship of Afrotheria and Xenarthra (Atlantogenata) also received significant support in analyses of sequences from ∼1700 conserved genome loci (Wildman et al. 2007), 2840 protein-coding genes (Hallström et al. 2007), and mitochondrial genomes (Kjer and Honeycutt 2007); and this was recently confirmed by large-scale analyses of 2.8 Mbp of protein-coding data (Hallström and Janke 2008) and large-scale genomic sequences (Prasad et al. 2008). Thus, despite many different molecular studies, the basal origin of placental mammals remains controversial and unresolved. Unfortunately, pure sequence data cannot resolve these apparent contradictions.Open in a separate windowFigure 1.Different hypotheses of the placental origin. (A) The Exafroplacentalia hypothesis proposes a mammalian clade merging Boreotheria (Supraprimates plus Laurasiathera) and Xenarthra, with Afrotheria as the sister group. (B) The Epitheria hypothesis merges Boreotheria and Afrotheria, with Xenarthra as the sister group. (C) The Atlantogenata hypothesis proposes Xenarthra and Afrotheria in one clade.By contrast, the presence/absence patterns of inserted retroelements constitute a virtually homoplasy-free marker system with theoretically infinite character states (Steel and Penny 2000). Specific genomic insertions of such elements in the ancestor of two species reliably document their common ancestry. The very few known examples of discordance in retroelement presence/absence data can be explained by deletion via illegitimate recombination between perfect direct repeats flanking each insertion (van de Lagemaat et al. 2005), exact parallel insertions (Cantrell et al. 2001), or lineage sorting, a phenomenon related to incomplete allele fixation and frequent intervals of speciation events (Shedlock et al. 2004; Ray et al. 2006).Analyses of a smaller number of retroelements as phylogenetic markers in mammals validated the four superordinal mammalian clades (Nishihara et al. 2005; Kriegs et al. 2006; Möller-Krull et al. 2007). However, in support of Shoshani and McKenna (1998), but in contradiction to most other molecular investigations, we found the first evidence, two L1MB5 retroelements, that placed Xenarthra (Epitheria hypothesis) (Fig. 1B) instead of Afrotheria at the base of the placental tree (Kriegs et al. 2006). Meanwhile, Murphy et al. (2007) presented two L1MB5 retroelements and four additional indels that favored a sister group relationship of Afrotheria and Xenarthra (Atlantogenata) (Fig. 1C) at the base of the placental tree. Thus, to date, even retroposon insertion patterns have not satisfactorily resolved the basal split of placental mammals.With the intention of resolving the conflicting topologies of placental lineages, we aimed to examine a much larger sample of retroposed elements and in more species. For this study we took a slightly different tack and aligned whole genomes of three representative placental species, including additional species for informative loci to represent a maximum divergence within each of the groups, and scanned them for diagnostic retroelement insertions. In an analysis of ∼2 million potential phylogenetically informative loci, we found multiple retroposon insertion patterns significantly supporting all three placental speciation hypotheses, indicating a complex ancestral speciation scenario including successive early divergences in close temporal proximity and hybridization and/or lineage sorting events as viable sources for an effective “soft” polytomy. This very rare example of multiple retroposon incongruence goes a long way toward explaining the decades-long stream of apparently conflicting evidence for one or the other placental evolutionary history based on multiple marker systems including both morphological and molecular sequence evidence.     

Microvascular reconstruction of the upper extremity with the rectus abdominis muscle

Large soft-tissue defects of the upper extremity are difficult to reconstruct. Defects in 21 patients (15–75 years old) were treated by free tissue transfer of the rectus abdominis muscle. The defects were the result of trauma or resection of tumor and measured more than 15 × 15 cm. The muscle was transferred on the inferior epigastric pedicle and covered with a skin graft within 48 hours. All transfers were successful, and early soft-tissue healing occurred. This procedure offers the advantages of easy positioning, large donor vessels, and a highly vascular soft tissue reconstruction. The long-term functional and cosmetic results have been excellent. © 1994 Wiley-Liss, Inc.     

Access and closure of the left ventricular apex: state of play

Calcific aortic stenosis is the most frequent manifestation of valvular heart disease. The preferred treatment for patients of all age groups is surgical aortic valve replacement. Recently, transcatheter aortic valve implantation (TAVI) has become the standard of care for patients that are deemed to be at high risk for open heart surgery. The most common access route for TAVI is the retrograde transfemoral (TF) approach, followed by the antegrade transapical (TA) approach. Both access routes have distinct indications. While the TF route is least invasive and the access of choice at most centers, the apical route is used complementary in patients with poor femoral access. In addition, the TA approach holds various benefits such as a short distance from the operator to the annulus facilitating exact positioning of the valve and the possibility to accommodate larger sheaths. Furthermore, the TA approach not only provides direct access to the aortic valve but also the mitral valve allowing for a wide range of interventions. Various apical closure devices are currently being developed under the premise of increasing overall safety of the TA-TAVI approach by further standardizing the procedure, alleviating left ventricular access and minimizing the risk of complications, such as apical bleeding. The aim of this article is to give an overview of current devices for apical closure. The ideal apical closure device should be easy to put in place, leave a minimum of foreign material, provide complete hemostasis and have a minimal risk of displacement. So far the range of commercially available devices in Europe is very limited with only one CE-certified device on the market and one device that is expected to receive CE-certification soon. Off-the-shelf closure devices could help flatten the initial operator learning curve and facilitate a safe apical access, ultimately leading to an entirely percutaneous TA-TAVI approach.     

Morphometric analysis of postoperative corneal neovascularization after high-risk keratoplasty: herpetic versus non-herpetic disease

Background

Postoperative complications after high-risk corneal grafting are decisively associated with corneal neovascularization (CNV). This study aimed to identify the incidence, extent, speed, localization, and influence of surgery-related factors on CNV after high-risk penetrating keratoplasty (PK) and to evaluate the effect of removing the angiogenic stimulus, i.e., residual components of herpes simplex virus (HSV) on postkeratoplasty CNV in patients with herpetic stromal keratitis (HSK).

Methods

All primary high-risk PK performed for HSK and non-herpetic keratitis (controls) between 1 January 1998 and 31 December 2003 at our department with available standardized corneal photographs taken preoperatively as well as 6?weeks, 3, 6 and 12?months postoperatively were evaluated (n _herpes?=?19, n _controls?=?5 patients). Maximal extension of CNV, limbus suture distance (LSD), limbus graft distance (LGD) and graft size in digitalized pictures were measured in each of the 16 sectors of the cornea at every visit.

Results

One hundred percent of the prevascularized corneas (n?=?24) showed further CNV outgrowth within 1?year after keratoplasty, while 58?% of these patients featured high-grade CNV reaching the host–graft junction or invading the donor tissue. Overall, CNV outgrowth was fastest during the first 6?weeks after PK, with a mean speed of 48?μm/week. Mean CNV growth speed within 6?months post-PK was significantly lower in the herpes group (13?μm/week) than in the non-herpes group (25?μm/week, p?=?0.017). Corneal location around the 12 o'clock position showed the most intense vessel outgrowth, which proved to be an independent risk factor for high-grade CNV (p?=?0.025). Inverse correlation was evident between CNV growth speed and LSD (p?=?0.032).

Conclusions

Additional intense CNV outgrowth is a common phenomenon after high-risk keratoplasty, strongly marked in the early postoperative period. The removal of residual HSV components representing a potential angiogenic stimulus leads to a reduction in corneal angiogenesis not in the short term, but in the long term after PK in patients with HSK. In addition to preferable atraumatic operation techniques, modern antiviral prophylaxis and anti-angiogenic therapy should be applied early, possibly even prior to transplantation. Short LSD seems to be an intraoperative adjustable risk factor for CNV in high-risk setting. Attention should also be paid to the superior site around the 12 o'clock position.