Higher-level snake phylogeny inferred from mitochondrial DNA sequences of 12S rRNA and 16S rRNA genes

Portions of two mitochondrial genes (12S and 16S ribosomal RNA) were sequenced to determine the phylogenetic relationships among the major clades of snakes. Thirty-six species, representing nearly all extant families, were examined and compared with sequences of a tuatara and three families of lizards. Snakes were found to constitute a monophyletic group (confidence probability [CP] = 96%), with the scolecophidians (blind snakes) as the most basal lineages (CP = 99%). This finding supports the hypothesis that snakes underwent a subterranean period early in their evolution. Caenophidians (advanced snakes), excluding Acrochordus, were found to be monophyletic (CP = 99%). Among the caenophidians, viperids were monophyletic (CP = 98%) and formed the sister group to the elapids plus colubrids (CP = 94%). Within the viperids, two monophyletic groups were identified: true vipers (CP = 98%) and pit vipers plus Azemiops (CP = 99%). The elapids plus Atractaspis formed a monophyletic clade (CP = 99%). Within the paraphyletic Colubridae, the largely Holarctic Colubrinae was found to be a monophyletic assemblage (CP = 98%), and the Xenodontinae was found to be polyphyletic (CP = 91%). Monophyly of the henophidians (primitive snakes) was neither supported nor rejected because of the weak resolution of relationships among those taxa, except for the clustering of Calabaria with a uropeltid, Rhinophis (CP = 94%).      

Impaired mitochondrial Ca2+ homeostasis in respiratory chain-deficient cells but efficient compensation of energetic disadvantage by enhanced anaerobic glycolysis due to low ATP steady state levels

Energy-producing pathways, adenine nucleotide levels, oxidative stress response and Ca(2+) homeostasis were investigated in cybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A>G and 3302A>G in tRNA(Leu(UUR)), as well as Rho(0) cells and compared to their parental 143B osteosarcoma cell line. All cells suffering from a severe respiratory chain deficiency were able to proliferate as fast as controls. The major defect in oxidative phosphorylation was efficiently compensated by a rise in anaerobic glycolysis, so that the total ATP production rate was preserved. This enhancement of glycolysis was enabled by a considerable decrease of cellular total adenine nucleotide pools and a concomitant shift in the AMP+ADP/ATP ratios, while the energy charge potential was still in the normal range. Further important consequences were an increased production of superoxide which, however, was neither escorted by major changes in the antioxidative defence systems nor was it leading to substantial oxidative damage. Most interestingly, the lowered mitochondrial membrane potential led to a disturbed intramitochondrial calcium homeostasis, which most likely is a major pathomechanism in mitochondrial diseases.     

Clinical and diagnostic features of partially anomalous pulmonary venous connection in an adult female patient: a case report and review of the literature

A 40-year-old woman presented with dyspnoea, chest pain and fatigue. Her medical history was unremarkable. An early systolic ejection murmur was heard in the 3D left inter-costal space. Chest X-ray revealed normal cardiothoracic ratio with an anomalous vessel adjacent to the left pulmonary hilum. Echocardiography and exercise tolerance test were normal. Right heart catheterisation revealed normal pulmonary pressures with normal cardiac output. CT scan and MRI of the thorax were diagnostic for an aberrant pulmonary venous connection between the left lower lobe pulmonary vein and the left brachiocephalic vein without atrial septal defect. She was treated conservatively and remained well.     

Changes in the endurance shuttle walk test in COPD patients with chronic respiratory failure after pulmonary rehabilitation: the minimal important difference obtained with anchor- and distribution-based method

Background

Although the endurance shuttle walk test (ESWT) has proven to be responsive to change in exercise capacity after pulmonary rehabilitation (PR) for COPD, the minimally important difference (MID) has not yet been established. We aimed to establish the MID of the ESWT in patients with severe COPD and chronic hypercapnic respiratory failure following PR.

Methods

Data were derived from a randomized controlled trial, investigating the value of noninvasive positive pressure ventilation added to PR. Fifty-five patients with stable COPD, GOLD stage IV, with chronic respiratory failure were included (mean (SD) FEV₁ 31.1 (12.0) % pred, age 62 (9) y). MID estimates of the ESWT in seconds, percentage and meters change were calculated with anchor based and distribution based methods. Six minute walking distance (6MWD), peak work rate on bicycle ergometry (Wpeak) and Chronic Respiratory Questionnaire (CRQ) were used as anchors and Cohen’s effect size was used as distribution based method.

Results

The estimated MID of the ESWT with the different anchors ranged from 186–199 s, 76–82% and 154–164 m. Using the distribution based method the MID was 144 s, 61% and 137 m.

Conclusions

Estimates of the MID for the ESWT after PR showed only small differences using different anchors in patients with COPD and chronic respiratory failure. Therefore we recommend using a range of 186–199 s, 76–82% or 154–164 m as MID of the ESWT in COPD patients with chronic respiratory failure. Further research in larger populations should elucidate whether this cut-off value is also valid in other COPD populations and with other interventions.

Trial registration

ClinicalTrials.Gov (ID {"type":"clinical-trial","attrs":{"text":"NCT00135538","term_id":"NCT00135538"}}NCT00135538).

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0182-x) contains supplementary material, which is available to authorized users.     

The distal hinge of the reactive site loop and its proximity: a target to modulate plasminogen activator inhibitor-1 activity

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulatory role in various physiological processes (e.g. fibrinolysis and pericellular proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclonal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-reactivities of these monoclonals with PAI-1 from different species and sequence alignments between these PAI-1s, combined with the three-dimensional structure, revealed several charged residues as possible candidates to contribute to the respective epitopes. The production, characterization, and subsequent evaluation of a variety of alanine mutants using surface plasmon resonance revealed that the residues His(185), Arg(186), and Arg(187) formed the major sites of interaction for MA-44E4. In contrast, the epitopes of MA-42A2F6 and MA-56A7C10 were found to be conformational. The epitope of MA-42A2F6 comprises residues Lys(243) and Glu(350), whereas the epitope of MA-56A7C10 comprises residues Glu(242), Lys(243), Glu(244), Glu(350), Asp(355), and Arg(356). The participation of Glu(350), Asp(355), and Arg(356) provides a molecular explanation for the differential exposure of this epitope in the different conformations of PAI-1 and for the effect of these antibodies on the kinetics of the formation of the initial PAI-1-proteinase complexes. The localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucidates two previously unidentified molecular mechanisms to modulate PAI-1 activity and opens new perspectives for the rational development of PAI-1 neutralizing compounds.     

Cytomegalovirus infection aggravates atherogenesis in apoE knockout mice by both local and systemic immune activation

Since the 1970s, cytomegalovirus (CMV) infection has been associated with atherosclerotic disease. However, the exact contribution of the virus remains uncertain. In this article we describe both a direct and indirect immune-mediated effect of the virus on the disease process. Eight-week-old apolipoprotein E (apoE) knockout mice were infected with mouse CMV (MCMV) or mock injected, and they were sacrificed at 2 and 20 weeks post-injection (p.i.) to study atherosclerosis, vascular wall IFNgamma and TNFalpha expression and MCMV spread. To study plasma IFNgamma and TNFalpha levels, blood was collected at 1, 2, 4 and 6 days p.i. in addition to days of sacrifice. Plasma cytokine levels were increased after MCMV infection at early time points and decreased to mock levels at 2 and 20 weeks p.i. At 2 weeks p.i., more aortic arch samples showed local cytokine expression after MCMV infection. The number of early atherosclerotic lesions and the percentage of mice containing early lesions were increased at 2 weeks p.i., while at 20 weeks p.i., the MCMV-induced effect on atherogenesis was seen on the late lesions. In conclusion, MCMV infection induces a systemic immune response reflecting an indirect effect of MCMV infection on atherosclerosis in addition to a local aortic immune response reflecting a direct effect of the virus on the atherosclerotic process.     

Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR

Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the bacteriophage M13 is hindered by a specific protein aggregation effect. Conditions are described for which NMR spectra of the protein can best be recorded. The aromatic part of the spectrum has been reinvestigated by means of two-dimensional total correlation spectroscopy. Sequence-specific assignments were obtained for all of the aromatic amino acid residues with the help of a series of single-site mutant proteins. The solution properties of the mutants of the aromatic amino acid residues have been fully investigated. It has been shown that, for these proteins, either none or only local changes occur compared to the wild-type molecule. Spin-labeled oligonucleotide-binding studies of wild-type and mutant gene V proteins indicate that tyrosine 26 and phenylalanine 73 are the only aromatic residues involved in binding to short stretches of single-stranded DNA. The degree of aggregation of wild-type gene V protein is dependent on both the total protein and salt concentration. The data obtained suggest the occurrence of specific protein-protein interactions between dimeric gene V protein molecules in which the tyrosine residue at position 41 is involved. This hypothesis is further strengthened by the observation that the solubility of tyrosine 41 mutants of gene V protein is significantly higher than that of the wild-type protein. The discovery of the so-called 'solubility' mutants of M13 gene V protein has finally made it possible to study the solution structure of gene V protein and its interaction with single-stranded DNA by means of two-dimensional NMR.     

Genetic variation determines mast cell functions in experimental asthma

Mast cell-deficient mice are a key for investigating the function of mast cells in health and disease. Allergic airway disease induced as a Th2-type immune response in mice is employed as a model to unravel the mechanisms underlying inception and progression of human allergic asthma. Previous work done in mast cell-deficient mouse strains that otherwise typically mount Th1-dominated immune responses revealed contradictory results as to whether mast cells contribute to the development of airway hyperresponsiveness and airway inflammation. However, a major contribution of mast cells was shown using adjuvant-free protocols to achieve sensitization. The identification of a traceable genetic polymorphism closely linked to the Kit(W-sh) allele allowed us to generate congenic mast cell-deficient mice on a Th2-prone BALB/c background, termed C.B6-Kit(W-sh). In accordance with the expectations, C.B6-Kit(W-sh) mice do not develop IgE- and mast cell-dependent passive cutaneous anaphylaxis. Yet, unexpectedly, C.B6-Kit(W-sh) mice develop full-blown airway inflammation, airway hyperresponsiveness, and mucus production despite the absence of mast cells. Thus, our findings demonstrate a major influence of genetic background on the contribution of mast cells in an important disease model and introduce a novel strain of mast cell-deficient mice.