Assessment of EGFR/HER2 dimerization by FRET-FLIM utilizing Alexa-conjugated secondary antibodies in relation to targeted therapies in cancers

The expression level of the HER family is unreliable as a predictive marker for targeted therapies in cancer. Thus, there is a need to develop other biomarkers, which can be used to accurately select responsive patients for targeted therapies. The HER dimerization status may be more important than HER receptor expression per se in determining sensitivity or resistance to a given therapeutic agent. The aim of the study is to develop a FRET assay using dye conjugated secondary antibodies to assess HER receptor dimerization. Using primary antibodies from different species in conjunction with Alexa488 and Alexa546 conjugated secondary antibodies, we validated our EGFR/HER2 dimerization assay in three cell lines, EGFR positive A431 cells as well as HER2 positive breast cell lines BT474 and SKBR3 cells. Finally, we applied our assay to assess EGFR/HER2 dimerization in paraffin embedded cell pellets. Our results show promise for the assay to be applied to tumor samples in order to assess the prognostic significance and predictive value of HER receptor dimerization in various cancers

Application of the penalty coupling method for the analysis of blood vessels

Due to the significant health and economic impact of blood vessel diseases on modern society, its analysis is becoming of increasing importance for the medical sciences. The complexity of the vascular system, its dynamics and material characteristics all make it an ideal candidate for analysis through fluid structure interaction (FSI) simulations. FSI is a relatively new approach in numerical analysis and enables the multi-physical analysis of problems, yielding a higher accuracy of results than could be possible when using a single physics code to analyse the same category of problems. This paper introduces the concepts behind the Arbitrary Lagrangian Eulerian (ALE) formulation using the penalty coupling method. It moves on to present a validation case and compares it to available simulation results from the literature using a different FSI method. Results were found to correspond well to the comparison case as well as basic theory

The effect of ketamine and D-cycloserine on the high frequency resting EEG spectrum in humans

Rationale Preclinical studies indicate that high-frequency oscillations, above 100 Hz (HFO:100–170 Hz), are a potential translatable biomarker for pharmacological studies, with the rapid acting antidepressant ketamine increasing both gamma (40–100 Hz) and HFO. Objectives To assess the effect of the uncompetitive NMDA antagonist ketamine, and of D-cycloserine (DCS), which acts at the glycine site on NMDA receptors on HFO in humans. Methods We carried out a partially double-blind, 4-way crossover study in 24 healthy male volunteers. Each participant received an oral tablet and an intravenous infusion on each of four study days. The oral treatment was either DCS (250 mg or 1000 mg) or placebo. The infusion contained 0.5 mg/kg ketamine or saline placebo. The four study conditions were therefore placebo-placebo, 250 mg DCS-placebo, 1000 mg DCS-placebo, or placebo-ketamine. Results Compared with placebo, frontal midline HFO magnitude was increased by ketamine (p = 0.00014) and 1000 mg DCS (p = 0.013). Frontal gamma magnitude was also increased by both these treatments. However, at a midline parietal location, only HFO were increased by DCS, and not gamma, whilst ketamine increased both gamma and HFO at this location. Ketamine induced psychomimetic effects, as measured by the PSI scale, whereas DCS did not increase the total PSI score. The perceptual distortion subscale scores correlated with the posterior low gamma to frontal high beta ratio. Conclusions Our results suggest that, at high doses, a partial NMDA agonist (DCS) has similar effects on fast neural oscillations as an NMDA antagonist (ketamine). As HFO were induced without psychomimetic effects, they may prove a useful drug development target

Zorgbalans 2014 : De prestaties van de Nederlandse gezondheidszorg

Dit rapport is in het Engels verschenen met nummer: 2015-0050 : Dutch Healthcare Performance Report De Zorgbalans geeft een beeld van de kwaliteit, de toegankelijkheid en de betaalbaarheid van de Nederlandse gezondheidszorg. Hieruit blijkt onder andere dat de toegankelijkheid van de gezondheidszorg een van de sterkste eigenschappen van de gezondheidszorg in Nederland is. De meeste eerstelijnszorgverleners zoals huisartsen, fysiotherapeuten of verloskundigen kunnen binnen een paar minuten worden bereikt. Een autorit naar een ziekenhuis duurt bijna nooit langer dan een half uur. Verder zijn wachttijden sinds 2008 voor de meeste behandelingen afgenomen. Er wacht nog wel een aanzienlijk aantal mensen op een plek in een verzorgingshuis of verpleeghuis, maar daardoor ontstaan zelden ernstige problemen. Het aantal mensen dat vanwege de kosten wel eens afziet van zorg is sinds 2010 toegenomen. Zo had 12 procent van de volwassen bevolking in 2013 wel eens afgezien van een bezoek aan een arts vanwege de kosten, tegenover 2 procent drie jaar daarvoor. Hiermee lijkt de financiële toegankelijkheid minder vanzelfsprekend dan voorheen; andere voorbeelden zijn medicijnen afhalen of een labonderzoek laten doen. Wat de kwaliteit van zorg betreft zijn er enkele gunstige ontwikkelingen te zien: het aantal mensen dat 30 dagen na een beroerte of hartinfarct stierf, nam af, evenals de (vermijdbare) sterfte in ziekenhuizen en het aantal ziekenhuisinfecties. Mensen met een gebroken heup worden sneller geopereerd, de vijfjaarsoverleving bij verschillende vormen van kanker steeg, en er zijn minder mensen in de langdurige zorg ondervoed. Ook internationaal scoort Nederland op veel punten bovengemiddeld. Zo worden veel minder antibiotica voorgeschreven in de eerste lijn dan in de meeste andere landen. Minder gunstig zijn het aantal sterfgevallen na een beroerte en de babysterfte. In de ouderenzorg is het tekort aan tijd en aandacht die worden besteed aan cliënten een veel voorkomend probleem. Meer dan de helft van de werknemers in verpleeghuizen gaf in 2013 aan dat er onvoldoende personeel is om goede kwaliteit van zorg te kunnen leveren. Verder blijkt de behandeling per zorgaanbieder sterk te kunnen verschillen. Een voorbeeld daarvan is de behandeling bij vrouwen die in een ziekenhuis bevallen van hun eerste kind zonder dat er sprake is van bijzonderheden (zoals een meerling of een stuitligging). In sommige ziekenhuizen wordt 40 procent van zulke bevallingen ingeleid, terwijl dit bij de meest ziekenhuizen in slechts 10 procent gedaan wordt. Vergelijkbare verschillen zijn waargenomen bij het uitvoeren van kunstverlossingen en keizersneden. De zorguitgaven vertonen na 2011 een opvallende trendbreuk. De uitgaven stegen tussen 2000 en 2013 gemiddeld met 5,5 procent per jaar, maar deze stijging vlakte de laatste drie jaar af. Binnen Europa hoort Nederland nog altijd tot de landen met de hoogste zorguitgaven als percentage van het Bruto Binnenlands Product, wat voornamelijk is toe te schrijven aan uitgaven aan de langdurige zorg. De Zorgbalans is nuttig voor iedereen die meer wil weten over de stand van zaken en ontwikkelingen binnen de Nederlandse gezondheidszorg. Aan de basis van de Zorgbalans ligt een schat aan informatie uit ruim 65 verschillende databronnen.The Dutch Health Care Performance Report reports on the quality, accessibility and affordability of Dutch health care. It shows, amongst others, that accessibility is one of the strong points of health care in the Netherlands. The majority of primary care professionals, such as general practitioners, physiotherapists and midwives, can be reached by car within a few minutes. A car journey to a hospital rarely takes more than half an hour. Waiting times have been reduced since 2008 for the vast majority of treatment modalities. Still, a considerable number of people are on a waiting list for a residential home or nursing home The number of people who occasionally forego care for financial reasons has been on the rise since 2010. Thus, 12 per cent of the adult population reported having decided against seeing a doctor in 2013 because of the anticipated costs against 2 per cent three years earlier. Financial access seems less a matter of course than it used to be; other examples are failure to undergo laboratory testing or to fill drug prescriptions. There are some favourable trends regarding the quality of care: the number of people that died within 30 days following stroke or acute myocardial infarction declined, and so did the rates of (avoidable) hospital mortality and hospital-acquired infections. People with a hip fracture were more promptly operated on, the five-year survival rates for several types of cancer increased, and less people in long-term care were undernourished. Internationally, the Netherlands scores also above average at various points. For example, far less antibiotics are prescribed in primary care in comparison with many other countries. Less positive are the rates for mortality following stroke and for neonatal mortality. In elderly care the lack of sufficient time and attention for clients, is a common problem. Over half of all care providers in nursing homes reported in 2013 that insufficient staff was available to enable good-quality care. Another finding pertains to the variation in practice between health care providers. An example is the treatment of women who give birth in a hospital to their first child who are not at a special risk (like multiple births or fetus in breech presentation). Some hospitals induce labour in 40 per cent of these women, as compared to only 10 per cent in most hospitals. Comparable disparities were found with regard to assisted and Caesarean deliveries. Dutch health care expenditures show a striking trend deviation after 2011. In the 2000-2013 period expenditures were mounting on average by 5.5% a year, but the increase slackened over the past three years. Within Europe, the Netherlands is still one of the countries with the highest health care spending as a percentage of the gross domestic product; this is attributable mainly to the costs of long-term care. The Dutch Health Care Performance Report is useful to everyone who wants to know more about the state of affairs and trends in Dutch health care. The report draws on a wealth of information from more than 65 data sources.Ministerie van VW

Temporal and spatial changes in wall shear stress during atherosclerotic plaque progression in mice

Wall shear stress (WSS) is involved in atherosclerotic plaque initiation, yet its role in plaque progression remains unclear. We aimed to study (i) the temporal and spatial changes in WSS over a growing plaque and (ii) the correlation between WSS and plaque composition, using animal-specific data in an atherosclerotic mouse model. Tapered casts were placed around the right common carotid arteries (RCCA) of ApoE−/− mice. At 5, 7 and 9 weeks after cast placement, RCCA geometry was reconstructed using contrast-enhanced micro-CT. Lumen narrowing was observed in all mice, indicating the progression of a lumen intruding plaque. Next, we determined the flow rate in the RCCA of each mouse using Doppler Ultrasound and computed WSS at all time points. Over time, as the plaque developed and further intruded into the lumen, absolute WSS significantly decreased. Finally at week 9, plaque composition was histologically characterized. The proximal part of the plaque was small and eccentric, exposed to relatively lower WSS. Close to the cast a larger and concentric plaque was present, exposed to relatively higher WSS. Lower WSS was significantly correlated to the accumulation of macrophages in the eccentric plaque. When pooling data of all animals, correlation between WSS and plaque composition was weak and no longer statistically significant. In conclusion, our data showed that in our mouse model absolute WSS strikingly decreased during disease progression, which was significantly correlated to plaque area and macrophage content. Besides, our study demonstrates the necessity to analyse individual animals and plaques when studying correlations between WSS and plaque composition

Contrast-enhanced micro-CT imaging in murine carotid arteries: A new protocol for computing wall shear stress

Background: Wall shear stress (WSS) is involved in the pathophysiology of atherosclerosis. The correlation between WSS and atherosclerosis can be investigated over time using a WSS-manipulated atheroscleroti

Post-exercise protein synthesis rates are only marginally higher in type I compared with type II muscle fibres following resistance-type exercise.

We examined the effect of an acute bout of resistance exercise on fractional muscle protein synthesis rates in human type I and type II muscle fibres. After a standardised breakfast (31 ± 1 kJ kg(−1) body weight, consisting of 52 Energy% (En%) carbohydrate, 34 En% protein and 14 En% fat), 9 untrained men completed a lower-limb resistance exercise bout (8 sets of 10 repetitions leg press and leg extension at 70% 1RM). A primed, continuous infusion of l-[ring-(13)C(6)]phenylalanine was combined with muscle biopsies collected from both legs immediately after exercise and after 6 h of post-exercise recovery. Single muscle fibres were dissected from freeze-dried biopsies and stained for ATPase activity with pre-incubation at a pH of 4.3. Type I and II fibres were separated under a light microscope and analysed for protein-bound l-[ring-(13)C(6)]phenylalanine labelling. Baseline (post-exercise) l-[ring-(13)C(6)]phenylalanine muscle tissue labelling, expressed as (∂(13)C/(12)C), averaged −32.09 ± 0.28, −32.53 ± 0.10 and −32.02 ± 0.16 in the type I and II muscle fibres and mixed muscle, respectively (P = 0.14). During post-exercise recovery, muscle protein synthesis rates were marginally (8 ± 2%) higher in the type I than type II muscle fibres, at 0.100 ± 0.005 versus 0.094 ± 0.005%/h, respectively (P < 0.05), whereby rates of mixed muscle protein were 0.091 ± 0.005%/h. Muscle protein synthesis rates following resistance-type exercise are only marginally higher in type I compared with type II muscle fibres

ADAM10 mediates trastuzumab resistance and is correlated with survival in HER2 positive breast cancer.

Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells (p ≤ 0.001 in BT474; p ≤ 0.01 in SKBR3) and in vivo (p ≤ 0.0001) compared to control, correlating with a decrease in PKB phosphorylation. ADAM10 inhibition or knockdown enhanced trastuzumab response in naïve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p ≤ 0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p ≤ 0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p ≤ 0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients

Inhibition of adipose tissue lipolysis increases intramuscular lipid use in type 2 diabetic patients

AIMS/HYPOTHESIS: In the present study, we investigated the consequences of adipose tissue lipolytic inhibition on skeletal muscle substrate use in type 2 diabetic patients. MATERIALS AND METHODS: We studied ten type 2 diabetic patients under the following conditions: (1) at rest; (2) during 60 min of cycling exercise at 50% of maximal workload capacity and subsequent recovery. Studies were done under normal, fasting conditions (control trial: CON) and following administration of a nicotinic acid analogue (low plasma non-esterified fatty acid trial: LFA). Continuous [U-13C]palmitate and [6,6 -2H2]glucose infusions were applied to quantify plasma NEFA and glucose oxidation rates, and to estimate intramuscular triacylglycerol (IMTG) and glycogen use. Muscle biopsies were collected before and after exercise to determine net changes in lipid and glycogen content specific to muscle fibre type. RESULTS: Following administration of the nicotinic acid analogue (Acipimox), the plasma NEFA rate of appearance was effectively reduced, resulting in lower NEFA concentrations in the LFA trial (p<0.001). Plasma NEFA oxidation rates were substantially reduced at rest, during exercise and subsequent recovery in the LFA trial. The lower plasma NEFA oxidation rates were compensated by an increase in IMTG and endogenous carbohydrate use (p<0.05). Plasma glucose disposal rates did not differ between trials. In accordance with the tracer data, a greater net decline in type I muscle fibre lipid content was observed following exercise in the LFA trial (p<0.05). CONCLUSIONS/INTERPRETATION: This study shows that plasma NEFA availability regulates IMTG use, and that adipose tissue lipolytic inhibition, in combination with exercise, could be an effective means of augmenting intramuscular lipid and glycogen use in type 2 diabetic patients in an overnight fasted state