The 5.5-year results of MegaOATS – autologous transfer of the posterior femoral condyle: a case-series study

Introduction

Large osteochondral defects of the weight-bearing zones of femoral condyles in young and active patients were treated by autologous transfer of the posterior femoral condyle (large osteochondral autogenous transplantation system (MegaOATS)). The technique presented is a sound and feasible salvage procedure to address large osteochondral defects in weight-bearing zones.

Methods

Thirty-six patients between July 1996 and December 2000 were included. Thirty-three patients (10 females, 23 males) were evaluated by the Lysholm score and X-ray scans. A random sample of 16 individuals underwent magnetic resonance imaging analysis. The average age at the date of surgery was 34.3 (15 to 59) years, and the mean follow up was 66.4 (46 to 98) months. The mean defect size was 6.2 (2 to 10.5) cm², in 27 patients affecting the medial femoral condyle and in six patients affecting the lateral femoral condyle. Trauma or osteochondrosis dissecans were pathogenetic in 82%.

Results

The Lysholm score in all 33 individuals showed a highly significant increase from a preoperative median 49.0 points to a median 86.0 points (P ≤ 0.001). Twenty-seven patients returned to recreational sports. X-ray scans showed a rounding of the osteotomy edge in 24 patients, interpreted as a partial remodelling of the posterior femoral condyle. Preoperative osteoarthritis in 17 individuals was related to significant lower Lysholm scores (P = 0.014), but progression in 17 patients did not significantly influence the score results (P = 0.143). All 16 magnetic resonance imaging examinations showed vital and congruent grafts.

Conclusion

Patients significantly improve in the Lysholm score, in daily-life activity levels and in return to recreational sports. Thirty-one out of 33 patients were comfortable with the results and would undergo the procedure again. The MegaOATS technique is therefore recommended as a salvage procedure for young individuals with large osteochondral defects in the weight-bearing zone of the femoral condyle.     

LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ?L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.     

Systematic Identification of Rhythmic Genes Reveals camk1gb as a New Element in the Circadian Clockwork

A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA–seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, camk1gb, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of camk1gb disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that camk1gb plays a role in linking the pineal master clock with the periphery.     

Inter-chromosomal Contact Properties in Live-Cell Imaging and in Hi-C

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Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b₅₆₁ proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b₅₆₁-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b₅₆₁ paralogs from Arabidopsis thaliana (Acytb₅₆₁-A, Acytb₅₆₁-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb₅₆₁-A resembles the best characterised member of the CYBASC family, the cytochrome b₅₆₁ from adrenomedullary chromaffin vesicles, and that Acytb₅₆₁-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (E_M) values were found to be fully consistent with ascorbate oxidation activities and Fe^3 +-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b₅₆₁ from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E_M values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E_M values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe^3 +-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E_M values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b₅₆₁ paralogs exist as homodimers.     

Mikroskopische Studien zur Innervation des Magen-Darmkanales V.

Zusammenfassung Die interstitielle Zelle läßt sich vielleicht als die kleinste Form einer vegetativen Ganglienzelle betrachten.Im Auerbachschen Plexus des menschlichen Colons kommen Zellen vom Typus 1 und 2 nach Dogiel und viele kleine und mittelgroße, der Form nach sehr mannigfache Gnanglienzellan vor.Der Auerbachsche Plexus zeigt eine Gliederung in ein Primär-, Sekundär- und Tertiärgeflecht. Der mit dem Auerbachschen Plexus kontinuierlich zusammenhängende Plexus muscularis profundus besitzt in verhältnismäßig spärlicher, aber gleichmäßiger Verteilung Ganglienzellen.Die großen Ganglienzellen des Meissnerschen Plexus gehören vorwiegend dem Typus 2 nach Dogiel an; daneben gibt es noch eine Fülle kleiner, teils multipolarer, teils der Form nach schwer bestimmbarer Ganglienzellen.Die an die Muscularis mucosae grenzenden Maschen des Meissnerschen Plexus sind von außerordentlicher Feinheit und enthalten auch interstitielle Zellen.Der Meissnersche Plexus geht mit feinsten, netzartigen Faserzügen ohne scharfe Grenze in den in der Schleimhaut ausgebreiteten Plexus mucosus über. Letzterer enthält zwar in seinem an die Submucosa grenzenden Gebiet noch vereinzelte kleine multipolare Ganglienzellen, weist jedoch in seinen übrigen, dem Epithel genäherten Lagen nur noch interstitielle Zellen auf.Der Plexus mucosus besitzt die Form des Terminalretikulums, den Charakter einer netzartigen Endformation des vegetativen Nervensystems, das hier afferente, efferente und (Sekretorische Nervenelemente in einer gemeinsamen plasmodialen Leitbahn beherbergt.In der Schleimhaut des Processus vermiformis entwickelt der dort ausgebreitete Plexus mucosus eine außerordentliche Zartheit und Reichhaltigkeit seiner nervösen Elemente.In einem Falle von rein neurogener Appandizitis kommen im Plexus mucosus des menschlichen Processus vermiformis bei sonst intakter Schleimhaut neuromatöse Gewebsneubildungen vor, die als das Resultat eines im Terminalretikulum zutage tretenden Wucherungsprozesses gedeutet werden können.In einem Falle von Megacolon werden schwere pathologische Veränderungen, vor allem an den Zellen und Fasern des Auerbachschen Plexus und des Plexus muscularis profundus beschrieben.     

Interpretability of bi-level variable selection methods

Variable selection is usually performed to increase interpretability, as sparser models are easier to understand than full models. However, a focus on sparsity is not always suitable, for example, when features are related due to contextual similarities or high correlations. Here, it may be more appropriate to identify groups and their predictive members, a task that can be accomplished with bi-level selection procedures. To investigate whether such techniques lead to increased interpretability, group exponential LASSO (GEL), sparse group LASSO (SGL), composite minimax concave penalty (cMCP), and least absolute shrinkage, and selection operator (LASSO) as reference methods were used to select predictors in time-to-event, regression, and classification tasks in bootstrap samples from a cohort of 1001 patients. Different groupings based on prior knowledge, correlation structure, and random assignment were compared in terms of selection relevance, group consistency, and collinearity tolerance. The results show that bi-level selection methods are superior to LASSO in all criteria. The cMCP demonstrated superiority in selection relevance, while SGL was convincing in group consistency. An all-round capacity was achieved by GEL: the approach jointly selected correlated and content-related predictors while maintaining high selection relevance. This method seems recommendable when variables are grouped, and interpretation is of primary interest.     

NUMB does not impair growth and differentiation status of experimental gliomas

The cell fate determinant NUMB orchestrates asymmetric cell division in flies and mammals and has lately been suggested to have a tumor suppressor function in breast and lung cancer. Here, we studied NUMB in the context of malignant gliomas. We used ectopic expression of NUMB in order to inhibit proliferation and induce differentiation in glioma cells by alteration of Notch, Hedgehog and p53 signaling. We found that NUMB is consistently expressed in glioma biopsies with predominance of NUMB2/4 isoforms as determined by isoform-specific real-time PCR and Western blotting. Upon lentiviral overexpression, in vitro proliferation rate and the grade of differentiation as assessed by morphology and expression of neural and glial markers remained unchanged. Orthotopic xenografts of NUMB-transduced human U87 glioma cells could be established in nude rats without impairing engraftment or causing significant changes in morphology based on magnetic resonance imaging (MRI). The previously reported alteration of Hedgehog and p53 signaling by NUMB could not be recapitulated in glioma cells. We thus show that in experimental gliomas, NUMB overexpression most likely does not exert a tumor suppressor function such as seen in epithelial cancers.