3D Ultrastructural Organization of Whole Chlamydomonas reinhardtii Cells Studied by Nanoscale Soft X-Ray Tomography

The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.     

Dysferlin-Peptides Reallocate Mutated Dysferlin Thereby Restoring Function

Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca²⁺dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in DYSF lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients’ myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition.     

Binding of Lassa virus perturbs extracellular matrix-induced signal transduction via dystroglycan

The arenavirus Lassa virus (LASV) causes a severe haemorrhagic fever with high mortality in man. The cellular receptor for LASV is dystroglycan (DG). DG is a ubiquitous receptor for extracellular matrix (ECM) proteins, which cooperates with β1 integrins to control cell-matrix interactions. Here, we investigated whether LASV binding to DG triggers signal transduction, mimicking the natural ligands. Engagement of DG by LASV resulted in the recruitment of the adaptor protein Grb2 and the protein kinase MEK1 by the cytoplasmic domain of DG without activating the MEK/ERK pathway, indicating assembly of an inactive signalling complex. LASV binding to cells however affected the activation of the MEK/ERK pathway via α6β1 integrins. The virus-induced perturbation of α6β1 integrin signalling critically depended on high-affinity LASV binding to DG and DG's cytoplasmic domain, indicating that LASV-receptor binding perturbed signalling cross-talk between DG and β1 integrins.     

Sites of generation of reactive oxygen species in homogenates of brain tissue determined with the use of respiratory substrates and inhibitors

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of various neurological diseases and aging. But the exact sites of ROS generation in brain tissue remained so far elusive. Here, we provide direct experimental evidence that at least 50% of total ROS generation in succinate-oxidizing homogenates of brain tissue can be attributed to complex I of mitochondrial respiratory chain. Applying quantitative methods for ROS detection we observed in different preparations from human, rat and mouse brain (digitonin-permeabilized tissue homogenates and isolated mitochondria) a linear relationship between rate of oxygen consumption and ROS generation with succinate as mitochondrial substrate. This quantitative relationship indicates, that under the particular conditions of oxygen saturation about 1% of the corresponding respiratory chain electron flow is redirected to form superoxide. Since we observed in mouse and rat brain mitochondria a unique dependency of both forward and reverse electron flow-dependent mitochondrial H(2)O(2) production on NAD redox state, we substantiated previous evidence that the FMN moiety of complex I is the major donor of electrons for the single electron reduction of molecular oxygen.     

Do short courses in evidence based medicine improve knowledge and skills? Validation of Berlin questionnaire and before and after study of courses in evidence based medicine

ObjectiveTo develop and validate an instrument for measuring knowledge and skills in evidence based medicine and to investigate whether short courses in evidence based medicine lead to a meaningful increase in knowledge and skills.DesignDevelopment and validation of an assessment instrument and before and after study.SettingVarious postgraduate short courses in evidence based medicine in Germany.ParticipantsThe instrument was validated with experts in evidence based medicine, postgraduate doctors, and medical students. The effect of courses was assessed by postgraduate doctors from medical and surgical backgrounds.InterventionIntensive 3 day courses in evidence based medicine delivered through tutor facilitated small groups.ResultsThe questionnaire distinguished reliably between groups with different expertise in evidence based medicine. Experts attained a threefold higher average score than students. Postgraduates who had not attended a course performed better than students but significantly worse than experts. Knowledge and skills in evidence based medicine increased after the course by 57% (mean score before course 6.3 (SD 2.9) v 9.9 (SD 2.8), P<0.001). No difference was found among experts or students in absence of an intervention.ConclusionsThe instrument reliably assessed knowledge and skills in evidence based medicine. An intensive 3 day course in evidence based medicine led to a significant increase in knowledge and skills.

What is already known on this topic

Numerous observational studies have investigated the impact of teaching evidence based medicine to healthcare professionals, with conflicting resultsMost of the studies were of poor methodological quality

What this study adds

An instrument assessing basic knowledge and skills required for practising evidence based medicine was developed and validatedAn intensive 3 day course on evidence based medicine for doctors from various backgrounds and training level led to a clinically meaningful improvement of knowledge and skills     

Spectral properties of fluorescent flavoproteins of isolated rat liver mitochondria

The different flavoproteins contributing to flavin fluorescence of isolated rat liver mitochondria have distinct excitation and emission spectra. The NAD-linked flavin component was identified as alpha-lipoamide dehydrogenase, while the non-NAD-linked component was found to be electron transfer flavoprotein. The differences in excitation and emission properties of the mitochondrial flavoproteins permit selective recording of their redox state changes in isolated mitochondria.     

TbPDE1, a novel class I phosphodiesterase of Trypanosoma brucei.

Cyclic nucleotide specific phosphodiesterases (PDEs) are important components of all cAMP signalling networks. In humans, 11 different PDE families have been identified to date, all of which belong to the class I PDEs. Pharmacologically, they have become of great interest as targets for the development of drugs for a large variety of clinical conditions. PDEs in parasitic protozoa have not yet been extensively investigated, despite their potential as antiparasitic drug targets. The current study presents the identification and characterization of a novel class I PDE from the parasitic protozoon Trypanosoma brucei, the causative agent of human sleeping sickness. This enzyme, TbPDE1, is encoded by a single-copy gene located on chromosome 10, and it functionally complements PDE-deficient strains of Saccharomyces cerevisiae. Its C-terminal catalytic domain shares about 30% amino acid identity, including all functionally important residues, with the catalytic domains of human PDEs. A fragment of TbPDE1 containing the catalytic domain could be expressed in active form in Escherichia coli. The recombinant enzyme is specific for cAMP, but exhibits a remarkably high Km of > 600 microm for this substrate.