High Molecular Weight Forms of Mammalian Respiratory Chain Complex II

Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CII_hmw) that can be resolved by clear native electrophoresis. CII_hmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 – over 1000 kDa) and cultured cells (400–670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CII_hmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CII_hmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase.     

Some properties of turnip yellow mosaic virus isolated from hybrid Brassica Perko PVH in Yugoslavia

Anhand mikroskopischer Untersuchungen und durch Mittelversuche an A. pisum wurden folgende Kenntnisse zur Endosymbiose gewonnen:

• In L₃‐Stadien von A. pisum sind zwischen 55 und 85 potentielle Bakteriocyten vorhanden, von dene ca. 60–80 % besiedelt sind.

• Eine Reduktion des besiedelten Anteils in der F₁‐Generation auf unter 50% läßt eine deutliche Depression in der F₂‐Generation erwarten.

• Das Kriterium Embryonenlänge ist großen Schwankungen unterworfen und eignet sich nur bedingt als Unterscheidungsmerkmal.

• Die von Fröhlich (1990) vorgeschlagene Methodik zum Symbiontizidscreening bei A. pisum mit dem Standard OTC 2000 ppm und der Auszählung der mit TTC angefärbten Bakteriocyten unter dem Mikroskop läßt eine praktikable Testung von Substanzen auf symbiontizide Wirkung bei A. pisum zu. Es wird jedoch als günstiger angesehen, nicht die Larven mit den Pflanzen zu behandeln, wie von Fröhlich (1990) vorgeschlagen, sondern erst nach dem Antrocknen des Spritzbelages Adulte zur Erzeugung von F₁‐Larven anzusetzen.

• Es konnte eindeutig nachgewiesen werden, daß die von den Prüfsubstanzen hervorgerufenen aphiziden Effekte, insbesondere durch Cycloheximid (100/500 ppm) sowie Neemkernextrakt (50%), nicht auf einem symbiontiziden Wirkungsmechanismus beruhen (Ausnahme Oxytetracyclin 2000 ppm als Standard).

     

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.     

Improving Education in Medical Statistics: Implementing a Blended Learning Model in the Existing Curriculum

Background

Although recent studies report on the benefits of blended learning in improving medical student education, there is still no empirical evidence on the relative effectiveness of blended over traditional learning approaches in medical statistics. We implemented blended along with on-site (i.e. face-to-face) learning to further assess the potential value of web-based learning in medical statistics.

Methods

This was a prospective study conducted with third year medical undergraduate students attending the Faculty of Medicine, University of Belgrade, who passed (440 of 545) the final exam of the obligatory introductory statistics course during 2013–14. Student statistics achievements were stratified based on the two methods of education delivery: blended learning and on-site learning. Blended learning included a combination of face-to-face and distance learning methodologies integrated into a single course.

Results

Mean exam scores for the blended learning student group were higher than for the on-site student group for both final statistics score (89.36±6.60 vs. 86.06±8.48; p = 0.001) and knowledge test score (7.88±1.30 vs. 7.51±1.36; p = 0.023) with a medium effect size. There were no differences in sex or study duration between the groups. Current grade point average (GPA) was higher in the blended group. In a multivariable regression model, current GPA and knowledge test scores were associated with the final statistics score after adjusting for study duration and learning modality (p<0.001).

Conclusion

This study provides empirical evidence to support educator decisions to implement different learning environments for teaching medical statistics to undergraduate medical students. Blended and on-site training formats led to similar knowledge acquisition; however, students with higher GPA preferred the technology assisted learning format. Implementation of blended learning approaches can be considered an attractive, cost-effective, and efficient alternative to traditional classroom training in medical statistics.     

Fluorescent substrates for ADAM15 useful for assaying and high throughput screening

A disintegrin and metalloproteinase 15 (ADAM15), also known as metargidin, plays important roles in regulating inflammation, wound healing, neovascularization, and is an attractive drug target. Fluorescence resonance energy transfer (FRET)-based peptide substrates were tested to identify candidate reagents for high throughput screening and detection of ADAM15 in biological samples. ADAM15 exhibits a unique and diverse activity profile compared to other metalloproteinases. Two FRET substrates, Dabcyl-Gly-Pro-Leu-Gly-Met-Arg-Gly-Lys(FAM)-NH2 (PEPDAB011) and Dabcyl-Ala-Pro-Arg-Trp-Ile-Gln-Asp-Lys(FAM)-NH2 (PEPDAB017), which also detect activities of several matrix metalloproteinases (MMPs −2, –9, and −13), were efficiently cleaved by ADAM15 with specificity constants of 5800 M⁻¹ s⁻¹ and 4300 M⁻¹ s⁻¹, respectively. Additionally, ADAM15 efficiently processed Dabcyl-Leu-Arg-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(FAM)-NH2 (PEPDAB022), which is based on a physiological CD23 cleavage site, with a specificity constant (k_cat/K_m) of 5200 M⁻¹ s⁻¹. PEPDAB022 was used to screen the ability of known metalloproteinase inhibitors including TAPI-2, marimastat, GI-254023, and the Tissue Inhibitor of Metalloproteinases(TIMPs) 1 and 3 to block ADAM15 activity. Even though ADAM15 exhibits similar substrate preferences to other metalloproteinases, many broad spectrum inhibitors failed to block ADAM15 activity at concentrations as high as 50 μM. Thus, a clear need exists to develop potent and selective ADAM15 inhibitors, and the FRET substrates described herein should aid future research efforts towards this aim.     

Antioxidant and anti‐inflammatory effects of the monocarbonyl curcumin analogs B2BRBC and C66 in monocrotaline‐induced right ventricular hypertrophy

For 22 days after monocrotaline injection two groups of rats received either of the monocarbonyl curcumin analogs (2E,6E)‐2,6‐bis(2‐bromobenzylidene)cycloxehanone (B2BrBC) and (2E,6E)‐2,6‐bis([2‐tri?uoromethyl]benzylidene)cyclohexanone (C66), and their right ventricle parameters were compared to those from the control and the monocrotaline injected animals. B2BrBC and C66 treatments did not prevent the monocrotaline‐induced right ventricular hypertrophy but attenuated the changes in antioxidant enzyme activities and reduced inflammation. The level of thiol‐based nonenzymatic antioxidants did not change in the function of monocrotaline or curcumin analogs treatment. However, due to its stronger antioxidant properties, only B2BrBC treatment was effective in the reduction of monocrotaline‐associated lipid peroxidation. The obtained results suggest that increasing the levels of antioxidant enzymes may not be sufficient to reduce oxidative stress and chronic inflammation optimally and our current study supports the potential of compounds with more than one beneficial biological activity as a promising treatment against the progression of cardiac hypertrophy.     

Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry

We investigated the combination of weak anion exchange (WAX) fractionation and on-line reversed-phase liquid chromatography (RPLC) separation using a 12 T FTICR mass spectrometer for the detection of intact proteins from a Shewanella oneidensis MR-1 cell lysate. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification state. A total of 715 intact proteins were detected, and the combined results from the WAX fractions and the unfractionated cell lysate were aligned using LC-MS features to facilitate protein abundance measurements. Protein identifications and post-translational modifications were assigned for approximately 10% of the detected proteins by comparing intact protein mass measurements to proteins identified in peptide MS/MS analysis of an aliquot of the same fraction. Intact proteins were also detected for S. oneidensis lysates obtained from cells grown on 13C-, 15N-depleted media under aerobic and sub-oxic conditions. The strategy can be readily applied for measuring differential protein abundances and provides a platform for high-throughput selection of biologically relevant targets for further characterization.     

Pharmacogenetic testing, informed consent and the problem of secondary information

Numerous benefits for patients have been predicted if prescribing decisions were routinely accompanied by pharmacogenetic testing. So far, little attention has been paid to the possibility that the routine application of this new technology could result in considerable harm to patients. This article emphasises that pharmacogenetic testing shares both the opportunities and the pitfalls with 'conventional' disease-genetic testing. It demonstrates that performing pharmacogenetic tests as well as interpreting the results are extraordinarily complex issues requiring a high level of expertise. It further argues that pharmacogenetic testing can have a huge impact on clinical decisions and may influence the therapeutic strategy as well as the clinical monitoring of a patient. This view challenges the predominant paradigm that pharmacogenetic testing will predict patients' responses to medicines, but that it will not provide any other significant disease-specific predictive information about the patient or family members. The article also questions published proposals to reduce the consent procedure for pharmacogenetic testing to a simple statement that the physician wishes to test a sample of the patient's DNA to see if a drug will be safe or whether it will work, and presents an alternative model that is better suited to protect patient's interests and to obtain meaningful informed consent. The paper concludes by outlining conditions for the application of pharmacogenetic testing in clinical practice in a way that can make full use of its potential benefits while minimising possible harm to patients and their families.     

Effect of antioxidant vitamins on radiation-induced apoptosis in cells of a human lymphoblastic cell line

Modulating the amount of radiation-induced apoptosis by administering antioxidant vitamins offers a possible way to influence radiation-induced side effects in normal tissues. Therefore, we investigated the effect of beta-carotene, vitamin C and alpha-tocopherol on radiation-induced apoptosis in cells in culture. Human T-lymphoblastic MOLT-3 cells were irradiated with a dose of 3 Gy 1 h after or immediately prior to the addition of vitamins in three concentrations (0.01 microM, 1 microM and 100 microM). Eight hours later, apoptosis was scored morphologically by staining the nuclear DNA with Hoechst 33342. When given prior to irradiation, beta-carotene and vitamin E reduced the amount of radiation-induced apoptosis significantly at concentrations of 0.01 microM and 1 microM. In contrast, vitamin C did not show any protective effect when given at these two concentrations and caused a slight but significant radiosensitization at 100 microM. At 0.01 microM, all combinations of two vitamins showed a protective effect. This was also observed for the combination of all three vitamins at concentrations of 0.01 and 1 microM. When given immediately after irradiation, each of the three vitamins showed a protective effect at 0.01 microM. In addition, the combination of alpha-tocopherol and vitamin C reduced radiation-induced apoptosis slightly when given at 1 microM. In all other cases, no statistically significant modulation of radiation-induced apoptosis was observed. In our experimental system, the protective effect of beta-carotene and vitamin E was dependent on concentration and occurred only in the micromolar and sub-micromolar concentration range, while vitamin C alone, but not in combinations, had a sensitizing effect, thus arguing for a careful consideration of vitamin concentrations in clinical settings.