Reading the unique DNA methylation landscape of the brain: Non-CpG methylation,hydroxymethylation, and MeCP2

DNA methylation at CpG dinucleotides is an important epigenetic regulator common to virtually all mammalian cell types, but recent evidence indicates that during early postnatal development neuronal genomes also accumulate uniquely high levels of two alternative forms of methylation, non-CpG methylation and hydroxymethylation. Here we discuss the distinct landscape of DNA methylation in neurons, how it is established, and how it might affect the binding and function of protein readers of DNA methylation. We review studies of one critical reader of DNA methylation in the brain, the Rett syndrome protein methyl CpG-binding protein 2 (MeCP2), and discuss how differential binding affinity of MeCP2 for non-CpG and hydroxymethylation may affect the function of this methyl-binding protein in the nervous system.     

A new bioabsorbable sleeve for lung staple-line reinforcement (FOREseal™): report of a three-center phase II clinical trial

Objective: To investigate on the feasibility, safety, and effectiveness of a new bioabsorbable material for lung staple-line reinforcement. Methods: This prospective open trial included 66 patients (mean age of 56 ± 17 years) who underwent various types of lung resection using staplers with knitted calcium alginate sleeves for buttressing (FOREseal™, Laboratoires Brothier, Nanterre, France) at three academic centers: 29 lobectomies, 22 emphysema surgeries, 15 wedge resections or lung biopsies. Intraoperative air leakage was assessed at a mean respiratory peak pressure of 30 cmH₂O, and rated as grade 1, 2, or 3. Persistent air leakage in the postoperative course, as well as any relevant event, was assessed daily. The follow-up period was of 6 months. Results: No technical problem linked to the device occurred. Hemostasis of the cutting edges was completed in all patients. Fifty-six percent of the patients had no intraoperative air leak and 27.3% had grade 1 leaks. Mean postoperative air leaks and thoracic drainage times were 1.9 ± 2.3 days and 6 ± 5.3 days, respectively. In-hospital mortality was nil. There was no empyema. Mean hospital stay was 9.1 ± 6.6 days. At follow-up, one patient underwent lung transplantation, and pathology of the explanted specimen showed the absence of device-related foreign-body inflammation. One patient complained from metalloptysis, and another one, with a metastatic invasive aspergillosis, developed an infectious recurrence that required reoperation. Conclusions: FOREseal is an ergonomic, safe, and promising new material instead of nonabsorbable materials and xenomaterials for staple-line reinforcement. A randomized comparative study is now in progress.     

Intimal hyperplasia and coronary flow reserve after heart transplantation: association with big endothelin-1

BACKGROUND: Endothelin, a peptide with strong vasoconstrictive and mitogenic properties, has been found to increase after cardiac transplantation. We therefore assessed the association between its precursor peptide, big endothelin-1, and intimal hyperplasia and coronary flow reserve after heart transplantation. METHODS: Thirty-five patients without hemodynamically significant coronary artery disease after heart transplantation were investigated: Average peak flow velocity in the left anterior descending artery (LAD) was assessed by intracoronary Doppler at baseline as well as after injection of adenosine; coronary flow reserve was calculated as a ratio of both and was corrected for patient age and baseline average peak flow velocity. Lumen, intima + media and total vessel area were measured by intracoronary ultrasound. The plasma concentration of big endothelin-1 in venous blood was determined by radioimmunoassay. RESULTS: Patients with elevated big endothelin-1 levels (>2 fmol/ml) tended to have a decreased corrected coronary flow reserve (2.60 +/- 0.9 vs 3.21 +/- 1.0, p = 0.078). They also had a significantly larger intima + media area (5.82 +/- 2.9 vs 2.37 +/- 2.9 mm(2), p = 0.004) and total vessel area (18.36 +/- 5.8 vs 12.81 +/- 4.8 mm(2), p = 0.012) than those with normal plasma concentrations. CONCLUSIONS: Our study suggests an association between elevated big endothelin-1 plasma levels and the development of intimal hyperplasia and reduction of coronary flow reserve after cardiac transplantation.     

Comparison of 2-dimensional and 3-dimensional acquisition for 18F-FDG PET oncology studies performed on an LSO-based scanner.

Three-dimensional (3D) PET acquisition has the potential to reduce image noise but the advantage of 3D PET for studies outside the brain has not been well established. To compare the performance of 2-dimensional (2D) and 3D acquisition for whole-body (18)F-FDG applications, a series of patient studies were performed using a lutetium oxyorthosilicate (LSO)-based tomograph. METHODS: Comparative 2D and 3D images were acquired for 27 oncology patients using an LSO-based tomograph. Data acquisition (350-650 keV, 6 ns) started 99 +/- 12 min (mean +/- SD) after injection of 624 +/- 76 MBq (18)F-FDG. Bias caused by tracer redistribution and decay was eliminated by acquiring dynamic data over a single-bed position using a protocol that alternated between septa-in and septa-out modes (2D, 3D, 2D, 3D, 2D, 3D). Frames were combined to form 8 statistically independent sinograms: four 2D replicates (105 s) and four 3D replicates (90 s). The different frame durations in 2D and 3D compensated for the different number of overlapping bed positions required for an 85-cm whole-body study. Images were reconstructed with either 2D or fully 3D ordered-subsets expectation maximization (2 iterations and 8 subsets; 2D 6-mm gaussian, 3D 5- and 6-mm gaussian). Image target-to-background ratio was assessed by dividing the lesion maximum by the mean within a neighboring background region. Image noise was assessed by applying background regions of interest to the replicate images and calculating the within-patient coefficient of variation. RESULTS: The difference in target-to-background ratio between the 2D and 3D images, when they were filtered with 6-mm and 5-mm gaussian filters, respectively, was not highly statistically significant (P = 0.16). The mean ratio of 3D to 2D image values was 0.94 with 95% limits of agreement of 0.63-1.41. The within-patient coefficients of variation for the 2D and 3D images were 13% +/- 15% and 9% +/- 10%, respectively (P = 0.0005). CONCLUSION: Under conditions of matched target to-to-background ratios, the 3D mode was found to produce images with significantly less variability than the 2D mode. These data provide support for the use of 3D acquisition with LSO detectors to reduce scan times in whole-body (18)F-FDG applications.     

In vivo PET imaging of cardiac presynaptic sympathoneuronal mechanisms in the rat.

The sympathetic nervous system of the heart plays a key role in the pathophysiology of various cardiac diseases. Small-animal models are valuable for obtaining further insight into mechanisms of cardiac disease and therapy. To determine the translational potential of cardiac neuronal imaging from rodents to humans, we characterized the rat sympathetic nervous system using 3 radiotracers that reflect different subcellular mechanisms: (11)C-meta-hydroxyephedrine (HED), a tracer of neuronal transport showing stable uptake and no washout in healthy humans; (11)C-phenylephrine (PHEN), a tracer of vesicular leakage and intraneuronal metabolic degradation with initial uptake and subsequent washout in humans; and (11)C-epinephrine (EPI), a tracer of vesicular storage with stable uptake and no washout in humans. METHODS: We used a small-animal PET system to study healthy male Wistar rats at baseline, after desipramine (DMI) pretreatment (DMI block), and with DMI injection 15 min after tracer delivery (DMI chase). The rats were kept under general isoflurane anesthesia while dynamic emission scans of the heart were recorded for 60 min after radiotracer injection. A myocardial retention index was determined by normalizing uptake at 40 min to the integral under the arterial input curve. Washout rates were determined by monoexponential fitting of myocardial time-activity curves. RESULTS: At baseline, HED showed high myocardial uptake and sustained retention, EPI showed moderate uptake and significant biphasic washout, and PHEN showed moderate uptake and monoexponential washout. The average (+/- SD) left ventricular retention index for HED, PHEN, and EPI was 7.38% +/- 0.82%/min, 3.43% +/- 0.45%/min, and 4.24% +/- 0.59%/min, respectively; the washout rate for HED, PHEN, and EPI was 0.13% +/- 0.23%/min, 1.13% +/- 0.35%/min, and 0.50% +/- 0.24%/min, respectively. The DMI chase resulted in increased washout only for HED. DMI block decreased myocardial uptake of all tracers by less than 90%. CONCLUSION: Kinetic profiles of HED in the rat myocardium were similar to those of HED in humans, suggesting comparable neuronal transport density. Unlike in humans, however, significant washout of EPI and faster washout of PHEN were encountered, consistent with high intraneuronal metabolic activity, high catecholamine turnover, and reduced vesicular storage. This evidence of increased neuronal activity in rodents has implications for translational studies of cardiac neuronal biology in humans.     

Basic protein enhances the incorporation of DNA into lipid vesicles: model for the formation of primordial cells.

DNA can be encapsulated into lipid vesicles formed by sonication. The presence of a basic protein, lysozyme, enhances the incorporation 100-fold above the level expected by random trapping. This is demonstrated by the ability of the lipid vesicles to protect DNA from digestion with DNase. Such an enhancement of nuclei acid incorporation into vesicles by basic polypeptides and the sharply increased concentration of these macromolecules in the internal volume may have been advantageous in prebiotic evolution.     

Immunohistological identification of cell subsets in human gingival after local treatment for gingivitis or periodontitis

The cellular infiltrate present in human diseased gingiva was analyzed in biopsies from 12 patients with gingivitis or periodontitis. The samples studied had been obtained in the course of surgery at inflammatory sites remaining after institution of periodontal treatment. Histological and immunological techniques were used to identify macrophages, B-cells, plasma-cells, T-cells and T cell subsets, as well as cells expressing class II HLA membrane antigens. T-cells appeared as the predominant population, but plasma-cells were also visualized in nearly all samples. Both OKT4+ and OKT8+ cells were seen in all cases, the latter being more numerous in periodontitis patients. Interdigitating-like cells were observed, positively labelled for class II antigens, as well as macrophages which were more numerous in periodontitis patients. These results suggest the participation of all components of the immune response in gingival disease, in a way resembling chronic recurrent inflammatory diseases.     

Inhibition and reversal of platelet-rich arterial thrombus in vivo: direct vs. indirect factor Xa inhibition

BACKGROUND/OBJECTIVE: The efficacy of a direct factor (F)Xa inhibitor, ZK-807834, was compared with indirect inhibition by enoxaparin for inhibition and deaggregation of acute platelet-rich thrombi in a well-characterized porcine carotid injury model. METHODS: A crush injury was performed on a randomly chosen carotid artery and the thrombus allowed to propagate for 30 min. Pigs then received intravenous drug for 35 min: ZK-807834-Dose 1 (40 microg kg(-1) bolus + 1.5 microg kg(-1) min(-1) infusion, n=6); ZK-807834-Dose 2 (20 microg kg(-1) bolus + 0.75 microg kg(-1) min(-1) infusion; n=6); enoxaparin (1 mg kg(-1) bolus; n=6); or saline (n=6). Five minutes after drug initiation, the contralateral artery was injured. Thrombus size was monitored by scintillation detection of autologous 111In-platelets. RESULTS: The prothrombin time ratio was 2.2 +/- 0.1; 1.4 +/- 0.3; 1.2 +/- 0.9 and 1.1 +/- 0.2, respectively. ZK-807834-Dose 1 significantly inhibited carotid platelet deposition (525 +/- 226 x 10(6) cm(-2); P = 0.008), whereas ZK-807834-Dose 2 (2325 +/- 768) and enoxaparin (1236 +/- 383) were not different from saline (2776 +/- 642). Thrombus deaggregation was greatest for animals receiving ZK-807834-Dose 1 (473 +/- 185). Neither ZK-807834-Dose 2 (1588 +/- 480) nor enoxaparin (1618 +/- 686) was different from saline control (2222 +/- 598). CONCLUSIONS: Direct FXa inhibition with ZK-807834, at a prothrombin time ratio of 2.2, effectively inhibits thrombosis and promptly deaggregates thrombi induced by arterial injury. In contrast, indirect FXa inhibition with enoxaparin was ineffective.