Letters to the metanarrative: Totalitarian “enjoyment” and self representation in postcommunist Serbia

Conclusion This outline of the instrumentality of the technology/technique of representation in postcommunist Serbia is but a brief attempt to address the modes of production of identity in postmodernism, its legitimation in/of dominant discourse, and their embedding in common sense language and practice. Postcommunist Serbia is obviously a special situation:²³ a particular case of the social appropriation and construction of enjoyment and desire, intimately related to the desires and various enjoyments of the members of the community, precarious in its collective affirmation. Based on the idea of contingent collectivity, faction-fiction, in its meta-critique seems to signal the emergence of a new régime in the relation between historical reality and its representation in historical narratives.This new régime, obviously, cannot be exhaustively (or even extensively) discussed in a brief paper like this one. Nevertheless, some (three) points of departure for the general discussion of postmodern modes of representation are still possible. One such point of departure is the contingence of representation, another the apparent paradigmatic similarity between trash talk-shows and Echoes and Reactions. And finally, much of iek's discussion does not seem to be restricted to the totalitarian metanarrative, and can be a significant contribution to the debate on power relations.Nikola Stojin is a Serbian Writer and Graduate Student in Comparative Literature at Rutgers University, New Brunswick, New Jersey.     

Plastid transformation in greening scales of the onion bulb (Allium cepa,Alliaceae)

The greening of the upper part of the outerAllium cepa L. bulb scales, in particular along the vascular regions, is limited to the hypodermal cells in which typical leucoplasts are transformed to normal and functional chloroplasts. This process is light dependent and cannot afterwards be reversed or modified by darkness. The changes in fine structure are described and briefly discussed.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday and 55 years after the publication of his Grundriß der Cytologie.     

Pressurized Pepsin Digestion in Proteomics: AN AUTOMATABLE ALTERNATIVE TO TRYPSIN FOR INTEGRATED TOP-DOWN BOTTOM-UP PROTEOMICS*

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.In-depth characterization and quantitation of protein isoforms, including post-translationally modified proteins, are challenging goals of contemporary proteomics. Traditionally, top-down (1, 2) and bottom-up (3, 4) proteomics have been two distinct analytical paths for liquid-based proteomics analysis. Top-down proteomics is the mass spectrometry (MS)-based characterization of intact proteins, whereas bottom-up proteomics requires a chemical or enzymatic proteolytic digestion of all proteins into peptides prior to MS analysis. Both strategies have their own strengths and challenges and can be thought of as complementary rather than competing analytical techniques.In a top-down proteomics approach, proteins are usually separated by one- or two-dimensional liquid chromatography (LC) and identified using high performance MS (5, 6). This approach is very attractive because it allows the identification of protein isoforms arising from various amino acid modifications, genetic variants (e.g. single nucleotide polymorphisms), mRNA splice variants, and multisite modifications (7) (e.g. specific histone modifications) as well as characterization of proteolytic processing events. However, there are several challenges that have limited the broad application of the approach. Typically, intact proteins are less soluble than their peptide complement, which effectively results in greater losses during various stages of sample handling (i.e. limited sensitivity). Similarly, proteins above ∼40–50 kDa in size are more difficult to ionize, detect, and dissociate in most high throughput MS work flows. Additionally, major challenges associated with MS data interpretation and sensitivity, especially for higher molecular mass proteins (>100 kDa) and highly hydrophobic proteins (e.g. integral membrane proteins), remain largely unsolved, thus limiting the applicability of top-down proteomics on a large scale.Bottom-up proteomics approaches have broad application because peptides are easier to separate and analyze via LC coupled with tandem mass spectrometry (MS/MS), offering a basis for more comprehensive protein identification. As this method relies on protein digestion (which produces multiple peptides for each protein), the sample complexity can become exceedingly large, requiring several dimensions of chromatographic separations (e.g. strong cation exchange and/or high pH reversed phase) prior to the final LC separation (typically reversed phase (RP)¹ C₁₈), which is oftentimes directly coupled with the mass spectrometer (3, 8). In general, the bottom-up analysis rarely achieves 100% sequence coverage of the original proteins, which can result in an incorrect/incomplete assessment of protein isoforms and combinatorial PTMs. Additionally, the digested peptides are not detected with uniform efficiency, which challenges and distorts protein quantification efforts.Because the data obtained from top-down and bottom-up work flows are complementary, several attempts have been made to integrate the two strategies (9, 10). Typically, these efforts have utilized extensive fractionation of the intact protein separation followed by bottom-up analysis of the collected fractions. Results so far have encouraged us to consider on-line digestion methods for integrating top-down and bottom-up proteomics in a higher throughput fashion. Such an on-line digestion approach would not only benefit in terms of higher sample throughput and improved overall sensitivity but would also allow a better correlation between the observed intact protein and its peptide digestion products, greatly aiding data analysis and protein characterization efforts.So far, however, none of the on-line integrated methods have proven robust enough for routine high throughput analyses. One of the reasons for this limited success relates to the choice of the proteolytic enzyme used for the bottom-up segment. Trypsin is by far the most widely used enzyme for proteome analyses because it is affordable (relative to other proteases), it has been well characterized for proteome research, and it offers a nice array of detectable peptides due to a fairly even distribution of lysines and arginines across most proteins. However, protein/peptide RPLC separations (optimal at low pH) are fundamentally incompatible with on-line trypsin digestion (optimal at pH ∼ 8) (11, 12). Therefore, on-line coupling of trypsin digestion and RPLC separations is fraught with technological challenges, and proposed solutions (12) have not proven to be robust enough for integration into demanding high throughput platforms.Our approach to this challenge was to investigate alternative proteases that may be more compatible with automated on-line digestion, peptide separation, and MS detection. Pepsin, which is acid-compatible (i.e. it acts in the stomach to initially aid in the digestion of food) (13), is a particularly promising candidate. This protease has previously been successfully used for the targeted analyses of protein complexes, hydrogen/deuterium exchange experiments (14, 15), and characterization of biopharmaceuticals (16, 17). Generally, pepsin preferentially cleaves the peptide bond located on the N-terminal side of hydrophobic amino acids, such as leucine and phenylalanine, although with less specificity than the preferential cleavage observed for trypsin at arginine and lysine. The compatibility of pepsin with typical LC-MS operation makes it an ideal choice for the development of novel approaches combining protein digestion, protein/peptide separation, and MS-based protein/peptide identification.To develop an automated system capable of simultaneously capturing top-down and bottom-up data, enzyme kinetics of the chosen protease must be extremely fast (because one cannot wait hours as is typical when performing off-line proteolysis). Another requirement is the use of immobilized enzyme or a low enough concentration of the enzyme such that autolysis products do not obscure the detection of substrate peptides. The latter was a concern when using pepsin because prior hydrogen/deuterium exchange experiments used enzyme:substrate ratios up to 1:2 (18, 19). To test whether or not such a large concentration of pepsin was necessary, we performed pepsin digestion at ratios of 1:20. Many alternative energy inputs into the system were considered for speeding up the digestion. For instance, it has been shown that an input of ultrasonic energy could accelerate the reaction rate of a typical trypsin digestion while using small amounts of a protease (20). Because ultrasonic energy results in an increase of temperature and microenvironments of high pressure, it has been hypothesized that the higher temperature was the component responsible for the enhanced enzyme activity (21). López-Ferrer et al. (22, 23), however, have demonstrated that application of higher pressure with incorporation of a Barocycler alone can make trypsin display faster enzyme kinetics. This phenomenon can easily be integrated with an LC separation (which already operates at elevated pressure) to enable an automatable ultrarapid on-line digestion LC-MS proteomics platform. Herein, we refer to this platform as the fast on-line digestion system (FOLDS) (23). Although FOLDS has been described before using trypsin, here the system is characterized with pepsin, and the results obtained are compared with results attainable with trypsin. Like trypsin, pepsin produced efficient protein digestion in just a few minutes when placed under pressure. Because of the natural maximal activity of pepsin at low pH, the FOLDS can be incorporated with a RePlay (Advion Biosciences, Ithaca, NY) system, and this powerful combination is what ultimately makes the integration of top-down and bottom-up proteomics analyses possible. The integrated analysis begins with a chromatographic separation of intact proteins. The separated proteins are then split into two streams. One stream proceeds directly to the mass spectrometer for MS and/or tandem MS analysis. The second stream is split into a long capillary where the chromatographic separation of the proteins is maintained, but their arrival to the mass spectrometer for detection is delayed. This is in essence the concept of RePlay (24, 25). Herein, we have taken the RePlay a step further by implementing our FOLDS technology into the second split delayed stream of proteins. While these delayed proteins travel down the long and narrow capillary, we exposed them to pepsin where, in combination with the pressure, the proteins are quickly and reproducibly digested. These peptide fragments are subsequently subjected to MS and/or tandem MS analysis. The FOLDS RePlay system allows the rapid and robust incorporation of the integrated top-down bottom-up proteomics work flow with the ability to not only identify proteins but also to sequence multisite/combinatorial PTMs because all detected peptides (from the FOLDS analysis) are confined to the original chromatographic peak of the protein they were derived from. The analysis of protein mixtures using this integrated strategy reduces the total amount of samples required to obtain both the top-down and bottom-up data, increases throughput, and improves protein sequence coverage.     

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.     

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas.In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters^{1, 2}.Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals^{3, 4}. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks^{3, 5}.Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice^{5, 6}.     

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.     

Fluctuations of Serum Neuron Specific Enolase and Protein S-100B Concentrations in Relation to the Use of Shunt during Carotid Endarterectomy

Objective

To evaluate the changes in serum neuron specific enolase and protein S-100B, after carotid endarterectomy performed using the conventional technique with routine shunting and patch closure, or eversion technique without the use of shunt.

Materials and Methods

Prospective non-randomized study included 43 patients with severe (>80%) carotid stenosis undergoing carotid endarterectomy in regional anesthesia. Patients were divided into two groups: conventional endarterectomy with routine use of shunt and Dacron patch (csCEA group) and eversion endarterectomy without the use of shunt (eCEA group). Protein S-100B and NSE concentrations were measured from peripheral blood before carotid clamping, after declamping and 24 hours after surgery.

Results

Neurologic examination and brain CT findings on the first postoperative day did not differ from preoperative controls in any patients. In csCEA group, NSE concentrations decreased after declamping (P<0.01), and 24 hours after surgery (P<0.01), while in the eCEA group NSE values slightly increased (P=ns), accounting for a significant difference between groups on the first postoperative day (P=0.006). In both groups S-100B concentrations significantly increased after declamping (P<0.05), returning to near pre-clamp values 24 hours after surgery (P=ns). Sub-group analysis revealed significant decline of serum NSE concentrations in asymptomatic patients shunted during surgery after declamping (P<0.05) and 24 hours after surgery (P<0.01), while no significant changes were noted in non-shunted patients (P=ns). Decrease of NSE serum levels was also found in symptomatic patients operated with the use of shunt on the first postoperative day (P<0.05). Significant increase in NSE serum levels was recorded in non-shunted symptomatic patients 24 hours after surgery (P<0.05).

Conclusion

Variations of NSE concentrations seemed to be influenced by cerebral perfusion alterations, while protein S-100B values were unaffected by shunting strategy. Routine shunting during surgery for symptomatic carotid stenosis may have the potential to prevent postoperative increase of serum NSE levels, a potential marker of brain injury.     

Inhibition of Rho‐kinase protects cerebral barrier from ischaemia‐evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood–brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho‐kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho‐kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil‐ versus vehicle‐treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post‐ischaemia or 4 h post‐ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress‐ and tight junction‐related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen–glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin‐5. Cotreatment of cells with Y‐27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho‐kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions.

     

A 3-year longitudinal study of quality-of-life outcomes of elderly patients with implant- and tooth-supported fixed partial dentures in posterior dental regions

doi: 10.1111/j.1741‐2358.2011.00592.x
A 3‐year longitudinal study of quality‐of‐life outcomes of elderly patients with implant‐ and tooth‐supported fixed partial dentures in posterior dental regions Background: Clinical studies have mainly been focused on oral health‐related quality‐of‐life (OHRQoL) outcomes of removable dentures. Objective: To evaluate therapy of elderly patients with implant‐supported fixed partial dentures (IFPD) and tooth‐supported fixed partial dentures (FPD) in the posterior dental regions. Patients and methods: The OHIP49 was used to measure OHRQoL in 64 patients with IFPD and 38 patients with FPD, before, 3 weeks and 3 years after rehabilitation. A control group (CG) consisted of 62 individuals. Results: The Oral Health Impact Profile questionnaire (OHIP) follow‐up scores of the patients with FPD and the patients with IFPD were significantly smaller in comparison with the baseline scores (p < 0.01). The OHIP scores were further reduced at the 3‐year follow‐up. The patients with IFPD had significantly higher scores than the patients with FPD and the CG at the baseline and at the follow‐ups. In the patients with FPD, both age groups (≤60 and >60) showed equal improvement of the OHRQoL. In the IFPD group, patients older than 60 years showed better improvement (p < 0.05). There were no significant differences dependent on gender and antagonistic teeth (p > 0.05). Conclusion: The FPD and the IFPD treatment showed significant improvement of OHRQoL. The FPD treatment improved OHRQoL equally in both age groups, while the IFPD treatment improved OHRQoL better in older patients.