Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships.

What Can Pharmacological Models of Retinal Degeneration Tell Us?

Animal models with pharmacologically induced retinal degeneration including sodium iodate (NaIO3) and N-methyl-N-nitrosourea (MNU) have been extensively used in ophthalmic research to investigate retinal degeneration. NaIO3 induces degeneration of the retinal pigment epithelium (RPE) followed by photoreceptor (PRC) cell death, mimicking features of age-related macular degeneration. In contrast, MNU leads to rapid destruction of the PRCs only, enabling the use of the MNU model to investigate degeneration induced in retinitis pigmentosa. It has been shown that multiple cell death pathways are involved in the cell-specific effects of the toxins. Necrosis has been identified as the cause of the NaIO3-induced RPE loss. PRC degeneration in the described models is mainly induced by programmed cell death, indicated by the upregulation of conventional apoptosis initiator and effector caspases. However, recent research points to the additional involvement of caspase-independent processes as endoplasmic reticulum stress and calpain activation. Since there is still a substantial amount of contradictory hypotheses concerning triggers of cell death, the use of pharmacological models is controversial. Thereby, the advantages of such models like the application reaching across species and strains as well as modulation of onset and severity of damage are not exploited to a full extent. Thus, the present review aims to give more insight into the involved cell death pathways and discusses recent findings in the most widely used retinal degeneration models. It might facilitate further studies aiming to develop putative therapeutic approaches for retinal degenerative diseases including combinatory treatment with cell death inhibitors and cell transplantation therapy.

The Nucleosome Remodeling and Deacetylase Complex NuRD Is Built from Preformed Catalytically Active Sub-modules.

The nucleosome remodeling deacetylase (NuRD) complex is a highly conserved regulator of chromatin structure and transcription. Structural studies have shed light on this and other chromatin modifying machines, but much less is known about how they assemble and whether stable and functional sub-modules exist that retain enzymatic activity. Purification of the endogenous Drosophila NuRD complex shows that it consists of a stable core of subunits, while others, in particular the chromatin remodeler CHD4, associate transiently. To dissect the assembly and activity of NuRD, we systematically produced all possible combinations of different components using the MultiBac system, and determined their activity and biophysical properties. We carried out single-molecule imaging of CHD4 in live mouse embryonic stem cells, in the presence and absence of one of core components (MBD3), to show how the core deacetylase and chromatin-remodeling sub-modules associate in vivo. Our experiments suggest a pathway for the assembly of NuRD via preformed and active sub-modules. These retain enzymatic activity and are present in both the nucleus and the cytosol, an outcome with important implications for understanding NuRD function.

Stimulation of cholesterol ester exchange by lipoprotein-free rabbit plasma.

A fraction in normal and hypercholesterolemic rabbit plasma of density greater than 1.25 stimulates the exchange of cholesterol esters between very low density and low density lipoproteins from hypercholesterolemic rabbit plasma. The exchange does not result from lecithin:cholesterol acyltransferase activity. The active factor appears to be a high molecular weight globulin with an isolelectric point of 5.2.

A robust characterization of retinoic acid response elements based on a comparison of sites in three species.

The availability of high-throughput genomic sequencing has allowed us to construct a more robust characterization of retinoic acid response elements than was possible in the past. We located human, mouse, and rat homologs for each of 51 well-documented, conserved retinoic acid response elements. Mathematical and statistical analyses of these 153 sites, 78 of which are new, shows that 92% of response elements have direct-repeat symmetry, but that only 76% exhibit canonical spacing attributes. While the familiar '(a/g)g(g/t)tca' hexamer motif is upheld, the more relaxed sequence, '(a/g)g(g/t)(g/t)(g/c)a', represents a 10% consensus. Sites are as likely to be on the coding strand as on the non-coding strand, and 86% of them are in upstream locations. From a statistical point of view, DR1 elements are fundamentally different from DR2 and DR5 elements, but this is only evident in the 5' hexamer. While there is considerable variation in core positions, and while no nucleotide can be considered forbidden at any position, variation among species at a fixed locus appears surprisingly constrained once a functional site has been attained.

[Stem cell therapy for retinal diseases]. / Stammzelltherapie für Netzhauterkrankungen.

BACKGROUND: Due to an ageing population the incidence and prevalence of retinal diseases and visual disabilities will continue to grow. A great number of patients would principally be able to benefit from a stem cell-based therapy. OBJECTIVES: To introduce readers to the terminology and current concepts associated with stem cell therapy in ocular research and to provide an overview of the current status of preclinical and clinical research. MATERIAL AND METHODS: We performed a systematic review of relevant entries on ocular stem cell therapy for retinal diseases in PubMed and ClinicalTrials.gov. Differences between various stem cell types are displayed systematically, followed by a discussion of preclinical studies. Translational aspects are highlighted leading to the first clinical trials, including surgical and ethical facets. RESULTS: In preclinical studies, photoreceptor cell precursors and retinal pigment epithelium (RPE) cells were differentiated and subretinally transplanted into animal models. Besides exclusion of a teratoma formation, some functional improvements were also observed. Intraocular transplantation of stem cell-derived RPE cells was the first successful clinical application of pluripotent stem cells in man. CONCLUSION: Promising results of preclinical and clinical studies have identified important challenges and confirmed the potential of stem cell therapy for ophthalmology.

Molecular and functional analysis of tumor-suppressor genes by transfection.

The transformed and tumorigenic phenotype of H-ras transfected rat FE-8 cells can be suppressed by cell fusion with normal rat embryo fibroblasts. Transfection into FE-8 cells of DNA prepared from normal human placenta followed by selective elimination of tumorigenic transfected cell clones resulted in the isolation of phenotypically normal revertants. These cells exhibited a fibroblastlike, normal morphology; were anchorage-dependent; and were unable to proliferate in medium with reduced serum concentrations. Their tumorigenicity was also reduced. The suppressed phenotype has been transferred in a second transfection cycle. Human repetitive DNA sequences were detected in secondary transfectant DNA. A putative human suppressor gene, designated NTS-1, has been molecularly cloned. Reintroduction of cloned NTS-1 sequences into FE-8 cells resulted in suppression of the neoplastic phenotype in spite of a high ras expression.

Reliability of an air-braked ergometer to record peak power during a maximal cycling test.

PURPOSE: To assess the reliability of the Kingcycle ergometer, this study compared peak power recorded using a Kingcycle and SRMTM power meters during Kingcycle maximal aerobic power tests. METHODS: The study was completed in two parts: for part 1, nine subjects completed three maximal tests with a stabilizing kit attached to the Kingcycle rig and calibration of the Kingcycle checked against SRM (MAP(C)); and for part 2, nine subjects completed two maximal tests without the stabilizing kit and the Kingcycle calibrated using the standard procedure (MAP(S)). Each MAP(C) test was separated by 1 wk; however, MAPs tests were separated by 54 +/- 32 d, (mean +/- SD). Testing procedure was repeated for each MAP and peak power output was calculated as the highest average power output recorded during any 60-s period of the MAP test using the Kingcycle (King(PPO)) and SRM (SRM(PPO)). RESULTS: Coefficient of variations (CVs) for King(PPO) were larger than those of SRM(PPO); 2.0% (95%CI = 1.5-3.0) versus 1.3% (95%CI = 1.0-2.0) and 4.6% (95%CI = 2.7-7.6) versus 3.6% (95%CI = 2.1-6.0) for MAPC( and MAP(S), respectively. During all tests, King(PPO) was higher than SRM(PPO) by an average of approximately 10% (P < 0.001). CONCLUSIONS: Investigators should be aware of the discrepancy between the two systems when assessing peak power and that SRM cranks provide a more reproducible measure of peak power than the Kingcycle ergometer.

Peak power predicts performance power during an outdoor 16.1-km cycling time trial.

PURPOSE: To assess i) the reproducibility of peak power output recorded during a maximal aerobic power test (MAP), and ii) its validity to predict endurance performance during a field based 16.1-km time trial (16.1-km TT). METHODS: Two studies were completed: for part I, nine subjects performed three MAP tests; for part II, 16 subjects completed a MAP test and 16.1-km TT. Power output was recorded using an SRM power meter and was calculated as peak power output (PPO) recorded during 60 s of MAP and mean power output for the 16.1-km TT (16.1-km TT(PO)). RESULTS: There was no difference between PPO recorded during the three MAP trials, mean coefficient of variation for individual cyclists was 1.32% (95%CI = 0.97-2.03), and test-retest reliability expressed as an intraclass correlation coefficient was 0.99 (95%CI = 0.96-1.00). A highly significant relationship was found between PPO and 16.1-km TT(PO) (r = 0.99, P < 0.001) but not for PPO and 16.1-km TT time (r = 0.46. P > 0.05). CONCLUSION: The results show that PPO affords a valid and reliable measure of endurance performance which can be used to predict average power during a 16.1-km TT but not performance time.

Assessment of blood lactate: practical evaluation of the Biosen 5030 lactate analyzer.

PURPOSE: The aim of this study was to assess the validity and reliability of the Biosen 5030 lactate analyzer compared with a YSI 2300 lactate analyzer and a Kodak Ektachem DTII in a practical laboratory study context. METHODS: To assess validity, 144 triplicate capillarized blood samples, across a range of values, were analyzed using the three analyzers. To assess reliability a further 665 samples were repeat analyzed. Temporal stability was determined by the reanalysis of resting and maximal exercise blood samples, after a period of storage ranging from 7 to 20 h, at room temperature. To measure inter- and intra-investigator reliability, 20 resting samples were taken from three different subjects by different investigators and a coefficient of variation was determined. RESULTS: There were strong relationships between the Biosen, the YSI (r2 = 0.97), and the Kodak Ektachem (r2 = 0.91). An analysis of Biosen compared with YSI revealed a positive bias of 0.37 mmol x L(-1) (95% limits of agreement, -0.85 to 1.59 mmol x L(-1)). The test-retest reliability correlation was significant (r2 = 0.99, P < 0.05), but a paired t-test revealed a small (0.03 mmol x L(-1), P < 0.05) significant difference. The coefficient of variation from the three investigators across the 20 samples ranged from 1.3 to 3%. Blood lactate concentration in resting blood samples did significantly increase in value (0.2 mmol x L(-1), P < 0.05) after 7-h exposure to the air, whereas there was no change in maximal exercise blood lactate values after 20-h exposure to the air. CONCLUSIONS: In a practical context, the Biosen 5030 lactate analyzer was comparable to the other analyzers giving fast reliable measures of blood lactate concentrations over the full range of values, which remained stable over extended periods at room temperature.

Modified Stryker frame for intraoperative positioning of the patient with burns.

Positioning large patients with burns for prone burn procedures can be very difficult, requiring operating room personnel to use a gang-lift technique. We have used the upper portion of a Stryker frame mounted to a standard operating room table base in more than 200 burn procedures in adults to move patients from the supine to the prone position, and back, without complication.

Combined aerobic and resistance training in breast cancer survivors: A randomized, controlled pilot trial.

The purpose of this pilot study was to examine the effects of a combined cardiorespiratory and resistance exercise training program of short duration on the cardiorespiratory fitness, strength endurance, task specific functional muscle capacity, body composition and quality of life (QOL) in women breast cancer survivors. Sixteen subjects were randomly assigned to either a training (n = 8; age: 50 +/- 5 yrs) or control non-exercising group (n = 8; age: 51 +/- 10 yrs). The training group followed an 8-week exercise program consisting of 3 weekly sessions of 90-min duration, supervised by an experienced investigator and divided into resistance exercises and aerobic training. Before and after the intervention period, all of the subjects performed a cardiorespiratory test to measure peak oxygen uptake (VO2peak), a dynamic strength endurance test (maximum number of repetitions for chest and leg press exercise at 30 - 35 % and 100 - 110 % of body mass, respectively) and a sit-stand test. Quality of life was assessed using the European Organization for Research and Treatment of Cancer QLQ-C30 (EORTC-C30) questionnaire. In response to training, QOL, VO2peak (mean 3.9 ml/kg/min; 95 % CI, 0.93, 6.90) performance in leg press (17.9 kg; 95 % CI, 12.8, 22.4) and sit-stand test (- 0.67 s; 95 % CI, - 0.52, - 1.2) improved (p < or = 0.05). We observed no significant changes in the control group. Combined cardiorespiratory and resistance training, even of very brief duration, improves the QOL and the overall physical fitness of women breast cancer survivors.

Three-day therapy of vulvovaginal candidiasis with econazole: a multicentric study comprising 996 cases.

Within the scope of three open multicentric studies, effect and tolerability of 150 mg. econazole vaginal suppositories were tested in a total of 996 women, 13 to 74 years old (mean age, 29 years). Vaginal candidiasis had been mycologically demonstrated in every patient prior to therapy, and the total cure rate of 93.4 per cent in Studies I and II (880 patients) was also mycologically confirmed. The tolerability was tested by checking of 30 laboratory variables before and after the treatment of 116 patients (Study III); no therapy-related adverse changes were recorded. Connections between therapeutic results, age distribution, initial hormonal situation, and conception of the therapy are pointed out.

1053-nm-wavelength selection in a diode-laser-pumped Nd:YLF laser.

We report on a Nd:YLF laser that operates at 1053 nm without optical intracavity elements for the suppression of the stronger 1047-nm transition. The Nd:YLF crystal is end pumped by a fiber optically coupled 10-W diode-laser bar. The different thermal-lensing focal lengths of the two main lasing wavelengths in a plane-parallel resonator were used to achieve the selection by tilting the end mirror slightly from its optimum position for maximum output power. With 9.8-W cw diode-laser-pumping power the 1053-nm Nd:YLF laser produces a maximum output power of 1.9 W in cw operation and nearly 1 W of average power at a Q-switch repetition rate of 15 kHz. The highest slope efficiency of 47% achieved in cw operation.

Q-switched Nd:YLF laser end pumped by a diode-laser bar.

We describe 20-kW operation of a Q-switched Nd:YLF laser end pumped by a 5-mJ, 25-W quasi-cw diode-laser bar. A fiber coupler was used to adapt the 1-cm-wide active area of the diode-laser bar to the circular shape of the TEM(00) mode. The maximum 1047-nm TEM(00) output energy was 0.5 mJ in a 22-nsec Q-switched pulse. This corresponds to a peak power in excess of 20 kW and was obtained at an optical slope efficiency of 13%.

Carbohydrate ingestion improves endurance performance during a 1 h simulated cycling time trial.

This study examined the effect of carbohydrate ingestion on metabolic and performance-related responses during and after a simulated 1 h cycling time trial. Eight trained male cyclists (VO2 peak = 66.5 ml kg-1 min-1) rode their own bicycles mounted on a windload simulator to imitate real riding conditions. At a self-selected maximal pace, the cyclists performed two 1 h rides (separated by 7 days) and were fed either an 8% carbohydrate or placebo solution. The beverages were administered 25 min before (4.5 ml kg-1) and at the end (4.5 ml kg-1) of the ride. With carbohydrate feeding, plasma glucose tended (P = 0.21) to rise before the time trial. Compared with rest, the plasma glucose concentration decreased significantly (P < 0.05) at the end of both rides, with no statistically significant difference being observed between treatments. Thereafter, plasma glucose increased significantly (P < 0.05) at 15 and 30 min into recovery and was significantly higher at 30 min during the carbohydrate trial compared with the placebo trial. No significant changes in plasma free fatty acids were observed during the ride. However, a significant increase (P < 0.05) in free fatty acids was found at 15 and 30 min into recovery, with no difference between trials. Mean power output was significantly (P < 0.05) greater during the carbohydrate compared with the placebo trial (mean +/- S.E.: 277 +/- 3 and 269 +/- 3 W, respectively). The greater distance covered in the carbohydrate compared with the placebo trial (41.5 +/- 1.06 and 41.0 +/- 1.06 km, respectively; P < 0.05) was equivalent to a 44 s improvement. We conclude that pre-exercise carbohydrate ingestion significantly increases endurance performance in trained cyclists during a 1 h simulated time trial. Although the mechanism for this enhancement in performance with carbohydrate ingestion cannot be surmised from the present results, it could be related to a higher rate of carbohydrate oxidation, or to favourable effects of carbohydrate ingestion on the central component of fatigue.