Oxidised LDL-lipids increase beta amyloid production by SH-SY5Y cells through glutathione depletion and lipid raft formation

Elevated total cholesterol in midlife has been associated with increased risk of dementia in later life. We have previously shown that low-density lipoprotein (LDL) is more oxidized in the plasma of dementia patients, although total cholesterol levels are not different from those of age-matched controls. β-Amyloid (Aβ) peptide, which accumulates in Alzheimer disease (AD), arises from the initial cleavage of amyloid precursor protein by β-secretase-1 (BACE1). BACE1 activity is regulated by membrane lipids and raft formation. Given the evidence for altered lipid metabolism in AD, we have investigated a mechanism for enhanced Aβ production by SH-SY5Y neuronal-like cells exposed to oxidized LDL (oxLDL). The viability of SH-SY5Y cells exposed to 4 μg oxLDL and 25 μM 27-hydroxycholesterol (27OH-C) was decreased significantly. Lipids, but not proteins, extracted from oxLDL were more cytotoxic than oxLDL. In parallel, the ratio of reduced glutathione (GSH) to oxidized glutathione was decreased at sublethal concentrations of lipids extracted from native and oxLDL. GSH loss was associated with an increase in acid sphingomyelinase (ASMase) activity and lipid raft formation, which could be inhibited by the ASMase inhibitor desipramine. 27OH-C and total lipids from LDL and oxLDL independently increased Aβ production by SH-SY5Y cells, and Aβ accumulation could be inhibited by desipramine and by N-acetylcysteine. These data suggest a mechanism whereby oxLDL lipids and 27OH-C can drive Aβ production by GSH depletion, ASMase-driven membrane remodeling, and BACE1 activation in neuronal cells. © 2014 The Authors.

Dehdyroepiandrosterone sulfate directly activates protein kinase C-β to increase human neutrophil superoxide generation

Dehydroepiandrosterone sulfate (DHEAS) is the most abundant steroid in the human circulation and is secreted by the adrenals in an age-dependent fashion, with maximum levels during the third decade and very low levels in old age. DHEAS is considered an inactive metabolite, whereas cleavage of the sulfate group generates dehydroepiandrosterone (DHEA), a crucial sex steroid precursor. However, here we show that DHEAS, but not DHEA, increases superoxide generation in primed human neutrophils in a dose-dependent fashion, thereby impacting on a key bactericidal mechanism. This effect was not prevented by coincubation with androgen and estrogen receptor antagonists but was reversed by the protein kinase C inhibitor Bisindolylmaleimide 1. Moreover, we found that neutrophils are unique among leukocytes in expressing an organic anion-transporting polypeptide D, able to mediate active DHEAS influx transport whereas they did not express steroid sulfatase that activates DHEAS to DHEA. A specific receptor for DHEAS has not yet been identified, but we show that DHEAS directly activated recombinant protein kinase C-ß (PKC-ß) in a cell-free assay. Enhanced PKC-ß activation by DHEAS resulted in increased phosphorylation of p47phox, a crucial component of the active reduced nicotinamide adenine dinucleotide phosphate complex responsible for neutrophil superoxide generation. Our results demonstrate that PKC-ß acts as an intracellular receptor for DHEAS in human neutrophils, a signaling mechanism entirely distinct from the role of DHEA as sex steroid precursor and with important implications for immunesenescence, which includes reduced neutrophil superoxide generation in response to pathogens.

The implementation of a design of experiments strategy to increase recombinant protein yields in yeast (review)

Biological processes are subject to the influence of numerous factors and their interactions, which may be non-linear in nature. In a recombinant protein production experiment, understanding the relative importance of these factors, and their influence on the yield and quality of the recombinant protein being produced, is an essential part of its optimisation. In many cases, implementing a design of experiments (DoE) approach has delivered this understanding. This chapter aims to provide the reader with useful pointers in applying a DoE strategy to improve the yields of recombinant yeast cultures.

Towards e+Learning or How to Increase the Learning into e-Learning

It is discussed some changes in the traditional e-learning notion on the point of view of R. Koper’s question 'where is the learning in e-learning?’. We put a focus on the conception of learning as a management process and present the project Bulgarian Educational Site (BEST) – a possible answer to Koper’s question. The BEST is a virtual learning environment, based on the following principles: learning is a goal-directed and didactics-managed process; learners may define their own learning objectives, monitor and regulate the learning process; collaborative e-learning is more effective; etc. The BEST is based on two famous e-learning systems (Moodle, LAMS) and Plovdiv e-University (versions 1.0 and 2.0). The paper brings up a mater about the new ‘electronic’ pedagogy and proposes an approach for pedagogical modeling and interpretation of e-learning applied in the BEST.

Is it safe to increase diabetic retinopathy screening intervals in patients with no/background diabetic retinopathy?

Patients who present with background DR should continue to be screened annually as a high prportion of these patients develop sight threatening DR (12%). A low prportion of patients with no DR at baseline were referred for STDR (1.3%). Out of the 51 patients in this category referred only 1 required laser. The authors suggest that patients graded R0M0 could be screened biannually.

Is it safe to increase diabetic retinopathy screening intervals in patients with no background diabetic retinopathy?

Introduction. A 4 year retrospective follow up of 996 patients who pre-sented with no DR and 500 with background DR at baseline digital DR screening in 2006. Purpose. To evaluate the safety of increasing screening intervals in patients with no diabetic retinopathy (DR) or with background DR.Methods. A 4 year retrospective follow up of 996 patients who presented with no DR and 500 with background DR at baseline digital DR screening in 2006.results. Background DR Group: Of the 500 subjects that had back-ground DR in 2006, 231 were referred for DR, with an average DR routine referral rate of 12% (46 subjects) per year. nodrgrouP. Of the 996 patients who had no DR at baseline, 51 were referred over the 4 years for sight threatening DR (STDR), of these 45 patients have definite STDR confirmed by ophthalmological examination. 78% of these had type 2 diabetes and mean age at referral was 60 years (25-87). Mean diabetes duration was 10.7 years (3-32), with a mean HbA1c of 7.8% (5.7-11.3%). Eight patients (0.9%) were referred in the first year, 9 (0.9%) in the second year, 19 (1.9%) in the third year and 15 (1.5%) in the fourth year. 86% of referrals were for maculopathy, and all had observable retinopathy and none required ophthalmology clinic assessment or laser treatment.If biannual screening was adopted for patients with no DR at baseline, allowing for patients who subsequently develop background DR and would then revert to annual screening, a total of 7 (0.7%) patients would not have been appropriately referred for STDR and would have waited a further year for identification. None of the 51 referrals across the 4 years required laser treatment apart from just one patient who developed PDR in year 4 (2010) and had background since 2007.conclusIons. It could be recommended that it is safe to screen pa-tients with no DR biannually due to the low risk of developing STDR. However, patients who present with background DR should continue to be screened annually as there is a significant proportion developing STDR and would not be identified at an appropriate screening interval.

Oxidised LDL-lipids alter redox ratio, lipid raft formation and increase amyloid beta production by SHSY-5Y cells

Elevated cholesterol in mid-life has been associated with increased risk of dementia in later life. We have previously shown that low density lipoprotein (LDL) is more oxidised in the plasma of dementia patients although total cholesterol levels remained unchanged [1]. We have investigated the hypothesis that amyloid beta production and neurodegeneration can be driven by oxidised lipids derived from LDL following the loss of blood brain barrier integrity with ageing. Therefore, we have investigated amyloid beta formation in SHSY5Y cells treated with LDL, minimally modified (ox) LDL, and lipids extracted from both forms of LDL. LDL-treated SHSY-5Y cell viability was not significantly decreased with up to 8 μg LDL/2 × 104 cells compared to untreated cells. However, 8 μg oxLDL protein/2 × 104 cells decreased the cell viability significantly by 33.7% (P < 0.05). A more significant decrease in cell viability was observed when treating cells with extracted lipids from 8 μg of LDL (by 32.7%; P < 0.01) and oxLDL (by 41%; P < 0.01). In parallel, the ratio of reduced to oxidised GSH was decreased; GSH concentrations were significantly decreased following treatment with 0.8 μg/ml oxLD-L (7.35 ± 0.58;P < 0.01), 1.6 μg/ml (5.27 ± 0.23; P < 0.001) and 4 μg/ml (5.31 ± 0.31; P < 0.001). This decrease in redox potential was associated with an increase acid sphingomyelinase activity and lipid raft formation which could be inhibited by desipramine; SHSY5Y cells treated with oxLDL, and lipids from LDL and oxLDL for 16 h showed significantly increased acid sphingomyelinase activity (5.32 ± 0.35; P < 0.05, 5.21 ± 0.6; P < 0.05, and 5.58 ± 0.44; P < 0.01, respectively) compared to control cells (2.96 ± 0.34). As amyloid beta production is driven by the activity of beta secretase and its association with lipid rafts, we investigated whether lipids from ox-LDL can influence amyloid beta by SHSY-5Y cells in the presence of oxLDL. Using ELISA and Western blot, we confirmed that secretion of amyloid beta oligomers is increased by SHSY-5Y cells in the presence of oxLDL lipids. These data suggest a mechanism whereby LDL, and more significantly oxLDL lipids, can drive amyloid beta production and cytotoxicity in neuronal cells. [1] Li L, Willets RS, Polidori MC, Stahl W, Nelles G, Sies H, Griffiths HR. Oxidative LDL modification is increased in vascular dementia and is inversely associated with cognitive performance. Free Radic Res. 2010 Mar; 44(3): 241–8.

Modified lipids from LDL in the blood during mid-life increase blood brain barrier permeability

Low density lipoprotein levels (LDL) are consistently elevated in cardiovascular disease. It has been suggested that those with high circulating LDL levels in mid-life may be susceptible to develop neurodegenerative diseases in later life. In the circulation, high levels of LDL are associated with increased oxidative modification (oxLDL) and nitration. We have investigated the hypothesis that disruption of blood brain barrier function by oxLDL and their lipids may increase risk of neurodegeneration in later life and that statin intervention in mid-life can mitigate the neurodegenerative effects of hyperlipidaemia. Blood from statin-naïve, normo- and hyperlipidaemic subjects (n=10/group) was collected at baseline. Hyperlipidaemic subjects received statin-intervention whereas normolipidaemic subjects did not prior to a second blood sampling, taken after 3 months. The intervention will be completed in June 2013. Plasma was separated by centrifugation (200g, 30min) and LDL was isolated by potassium bromide density gradient ultracentrifugation. Total homocysteine, LDL cholesterol, 8-isoprostane F2α levels were measured in plasma using commercial kits. LDL were analysed by agarose gel electrophoresis. LDL-lipids were extracted by partitioning in 1:1 chloroform:methanol (v/v) and conjugated to fatty acid free-BSA in serum-free EGM-2 medium (4hrs, 370C) for co-culture with human microvascular endothelial cells (HMVEC). HMVEC were maintained on polycarbonate inserts for two weeks to create a microvascular barrier. Change in barrier permeability was measured by trans-endothelial electrical resistance (TER), FITC-dextran permeability and immunohistochemistry. HMVEC glutathione (GSH) levels were measured after 2 hours by GSH-glo assay. LDL isolated from statin-naïve hyperlipidaemic subjects had higher mobility by agarose gel electrophoresis (Rf;0.53±0.06) and plasma 8-isoprostane F2α (43.5±8.42 pg/ml) compared to control subjects (0.46±0.05 and 24.2±5.37 pg/ml; p<0.05). Compared to HMVEC treatment with the LDL-lipids (5μM) from normolipidaemic subjects, LDL-lipids from hyperlipidaemic subjects increased barrier permeability (103.4±12.5 Ωcm2 v 66.7±7.3 Ωcm2,P<0.01) and decreased GSH (18.5 nmol/mg v 12.3 nmol/mg; untreated cells 26.2±3.6 nmol/mg).

Diverse antiepileptic drugs increase the ratio of background synaptic inhibition to excitation and decrease neuronal excitability in neurones of the rat entorhinal cortex in vitro

Although most anti-epileptic drugs are considered to have a primary molecular target, it is clear that their actions are unlikely to be limited to effects on a single aspect of inhibitory synaptic transmission, excitatory transmission or voltage-gated ion channels. Systemically administered drugs can obviously simultaneously access all possible targets, so we have attempted to determine the overall effect of diverse agents on the balance between GABAergic inhibition, glutamatergic excitation and cellular excitability in neurones of the rat entorhinal cortex in vitro. We used an approach developed for estimating global background synaptic excitation and inhibition from fluctuations in membrane potential obtained by intracellular recordings. We have previously validated this approach in entorhinal cortical neurones [. Greenhill and Jones (2007a) Neuroscience 147:884-892]. Using this approach, we found that, despite their differing pharmacology, the drugs tested (phenytoin, lamotrigine, valproate, gabapentin, felbamate, tiagabine) were unified in their ability to increase the ratio of background GABAergic inhibition to glutamatergic excitation. This could occur as a result of decreased excitation concurrent with increased inhibition (phenytoin, lamotrigine, valproate), a decrease in excitation alone (gabapentin, felbamate), or even with a differential increase in both (tiagabine). Additionally, we found that the effects on global synaptic conductances agreed well with whole cell patch recordings of spontaneous glutamate and GABA release (our previous studies and further data presented here). The consistency with which the synaptic inhibition:excitation ratio was increased by the antiepileptic drugs tested was matched by an ability of all drugs to concurrently reduce intrinsic neuronal excitability. Thus, it seems possible that specific molecular targets among antiepileptic drugs are less important than the ability to increase the inhibition:excitation ratio and reduce overall neuronal and network excitability. © 2010 IBRO.

Vertikális korlátozások - növelik vagy csökkentik a jólétet? Érvek az irodalomból = Vertical constraints - do they increase or reduce welfare? Arguments in the literature

A vertikális korlátozások témaköre a versenypolitika és -szabályozás egyik legfontosabb, de mindenképpen legtöbb vitát generáló területe. A téma magyar irodalma elsősorban elméleti kérdésekkel foglalkozik, mellőzve a konkrét iránymutatásokat a probléma kezelésére. Először röviden bemutatjuk a vertikális korlátozások indokait és hatásmechanizmusát magyarázó teoretikus irodalmat, felsorolva azok hatékonyságnövelő hatását hangsúlyozó érvrendszereket, illetve a versenykorlátozás lehetőségét tárgyaló modelleket is. Ezután bemutatjuk, hogy milyen fontos empirikus vizsgálatokat végeztek ezen elméletek vizsgálatára, és milyen következtetések vonhatóak le a tapasztalatokból. _________ The field of vertical constraints is one of the most important in competition policy and regulation, but one that generates the most debate. Hungarian literature on the subject deals primarily with theoretical questions, refraining from offering specific guidelines for handling the problem. The paper first covers briefly the causes of the vertical constraints and the Hungarian theoretical literature on its effective mechanism, listing the system of arguments that point to its effect of increasing efficiency and the models that discuss the possibility of limiting competition. The paper then presents the major empirical examinations made of these theories and the conclusions drawn from that experience.

A közalkalmazotti béremelés hatása a tanárok pályaelhagyási döntéseire (The effect of a public-sector pay increase on teachers attrition)

A tanárok pályaelhagyási döntését vizsgálva, a tanulmány a következő két kérdésre keresi a választ. Milyen szerepet játszanak e döntésekben a keresetek, alternatív kereseti lehetőségek? Hogyan hatott a tanárok pályaelhagyására a 2002. évi közalkalmazotti béremelés? Az elemzéshez az OEP-ONYF-FH összekapcsolt nagymintás adatbázis felhasználásával kétféle modellt becsült a szerző: 1. két lehetőséget megkülönböztetve (elhagyja a tanári pályát/nem hagyja el) Cox-féle arányos hazárdfüggvényeket, 2. a pályaelhagyás okai között a más állásba kerülést és az egyéb pályaelhagyási okokat megkülönböztetve versengő kockázati modelleket. Az eredmények azt mutatják, hogy a kereseti lehetőségek hatnak a pályaelhagyási döntésekre. A magasabb jövedelem és magasabb relatív kereset csökkenti annak valószínűségét, hogy egy tanár elhagyja a pályát, és más pályán helyezkedjen el, vagy nem foglalkoztatotti státusba kerüljön. A közalkalmazotti béremelés átmenetileg csökkentette a pályaelhagyás valószínűségét a fiatal tanárok körében, de a hatás egy-két év alatt eltűnt. Az 51 évesnél idősebb tanárokat pedig inkább a pályán tartotta a béremelés, csökkentette annak valószínűségét is, hogy más pályán helyezkedjenek el, vagy hogy nem foglalkoztatotti státusba kerüljenek. ______ The paper investigates teachers decisions to leave the profession. It first examines the role in such decisions of pay compared with earnings in alternative occupations, and then discusses how the public-sector pay increase of 2002 af-fected exit decisions by teachers. Duration models were estimated using large merged administrative data sets. First binary-choice Cox proportional hazard models (leaving teaching profession or not), then competing risk models that distinguish exits to another occupation and exits to a non-working state. Results show that earnings matter. Higher wages reduce the probability of exiting teacher profession to go to another occupation or to non-employment. The public-sector pay increase decreased the probability of inexperienced teachers leaving the teacher profession temporarily, but the effect disappeared after one or two years. For experienced teachers over 51 years old, the wage increase was found to reduce attrition.

Mindmapping as an adult learning notetaking strategy to increase the encoding of information in a corporate training course

A study was conducted to investigate the effectiveness, as measured by performance on course posttests, of mindmapping versus traditional notetaking in a corporate training class. The purpose of this study was to increase knowledge concerning the effectiveness of mindmapping as an information encoding tool to enhance the effectiveness of learning. Corporations invest billions of dollars, annually, in training programs. Given this increased demand for effective and efficient workplace learning, continual reliance on traditional notetaking is questionable for the high-speed and continual learning required on workers.^ An experimental, posttest-only control group design was used to test the following hypotheses: (1) there is no significant difference in posttest scores on an achievement test, administered immediately after the course, between adult learners using mindmapping versus traditional notetaking methods in a training lecture, and (2) there is no significant difference in posttest scores on an achievement test, administered 30 days after the course, between adult learners using mindmapping versus traditional notetaking methods in a training lecture. After a 1.5 hour instruction on mindmapping, the treatment group used mindmapping throughout the course. The control group used traditional notetaking. T-tests were used to determine if there were significant differences between mean posttest scores between the two groups. In addition, an attitudinal survey, brain hemisphere dominance survey, course dynamics observations, and course evaluations were used to investigate preference for mindmapping, its perceived effect on test performance, and the effectiveness of mindmapping instruction.^ This study's principal finding was that although the mindmapping group did not perform significantly higher on posttests administered immediately and 30 days after the course, than the traditional notetaking group, the mindmapping group did score higher on both posttests and reported higher ratings of the course on every evaluation criteria. Lower educated, right brain dominant learners reported a significantly positive learning experience. These results suggest that mindmapping enhances and reinforces the preconditions of learning. Recommendations for future study are provided. ^

Synthesis of aromatic monothiols and aromatic dithiols to increase the folding rate and yield of disulfide containing proteins

Most pharmaceutically relevant proteins and many extracellular proteins contain disulfide bonds. Formation of the correct disulfide bonds is essential for stability in almost all cases. Disulfide containing proteins can be rapidly and inexpensively overexpressed in bacteria. However, the overexpressed proteins usually form aggregates inside the bacteria, called inclusion bodies, which contains inactive and non-native protein. To obtain native protein, inclusion bodies need to be isolated and resolubilized, and then the resulting protein refolded in vitro. In vitro protein folding is aided by the addition of a redox buffer, which is composed of a small molecule disulfide and/or a small molecule thiol. The most commonly used redox buffer contains reduced and oxidized glutathione. Recently, aliphatic dithiols and aromatic monothiols have been employed as redox buffers. Aliphatic dithiols improved the yield of native protein as compared to the aliphatic thiol, glutathione. Dithiols mimic the in vivo protein folding catalyst, protein disulfide isomerase, which has two thiols per active site. Furthermore, aromatic monothiols increased the folding rate and yield of lysozyme and RNase A relative to glutathione. By combining the beneficial properties of aliphatic dithiols and aromatic monothiols, aromatic dithiols were designed and were expected to increase in vitro protein folding rates and yields. Aromatic monothiols (1-4) and their corresponding disulfides (5-8), two series of ortho- and para-substituted ethylene glycol dithiols (9-15), and a series of aromatic quaternary ammonium salt dithiols (16-17) were synthesized on a multigram scale. Monothiols and disulfides (1-8) were utilized to fold lysozyme and bovine pancreatic trypsin inhibitor. Dithiols (11-17) were tested for their ability to fold lysozyme. At pH 7.0 and pH 8.0, and high protein concentration (1 mg/mL), aromatic dithiols (16, 17) and a monothiol (3) significantly enhanced the in vitro folding rate and yield of lysozyme relative to the aliphatic thiol, glutathione. Additionally, aromatic dithiols (16, 17) significantly enhance the folding yield as compared to the corresponding aromatic monothiol (3). Thus, the folding rate and yield enhancements achieved in in vitro protein folding at high protein concentration will decrease the volume of renaturation solution required for large scale processes and consequently reduce processing time and cost.

Does management capacity increase organizational performance? An empirical analysis of public housing agencies

Since the 1990s, scholars have paid special attention to public management’s role in theory and research under the assumption that effective management is one of the primary means for achieving superior performance. To some extent, this was influenced by popular business writings of the 1980s as well as the reinventing literature of the 1990s. A number of case studies but limited quantitative research papers have been published showing that management matters in the performance of public organizations. ^ My study examined whether or not management capacity increased organizational performance using quantitative techniques. The specific research problem analyzed was whether significant differences existed between high and average performing public housing agencies on select criteria identified in the Government Performance Project (GPP) management capacity model, and whether this model could predict outcome performance measures in a statistically significant manner, while controlling for exogenous influences. My model included two of four GPP management subsystems (human resources and information technology), integration and alignment of subsystems, and an overall managing for results framework. It also included environmental and client control variables that were hypothesized to affect performance independent of management action. ^ Descriptive results of survey responses showed high performing agencies with better scores on most high performance dimensions of individual criteria, suggesting support for the model; however, quantitative analysis found limited statistically significant differences between high and average performers and limited predictive power of the model. My analysis led to the following major conclusions: past performance was the strongest predictor of present performance; high unionization hurt performance; and budget related criterion mattered more for high performance than other model factors. As to the specific research question, management capacity may be necessary but it is not sufficient to increase performance. ^ The research suggested managers may benefit by implementing best practices identified through the GPP model. The usefulness of the model could be improved by adding direct service delivery to the model, which may also improve its predictive power. Finally, there are abundant tested concepts and tools designed to improve system performance that are available for practitioners designed to improve management subsystem support of direct service delivery.^