Correlation of two-photon in vivo imaging and FIB/SEM microscopy.

Advances in the understanding of brain functions are closely linked to the technical developments in microscopy. In this study, we describe a correlative microscopy technique that offers a possibility of combining two-photon in vivo imaging with focus ion beam/scanning electron microscope (FIB/SEM) techniques. Long-term two-photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool for studying the dynamics of neurodegenerative diseases, such as Alzheimer's disease. However, light microscopy has important limitations in revealing alterations occurring at the synaptic level and when this is required, electron microscopy is mandatory. FIB/SEM microscopy is a novel tool for three-dimensional high-resolution reconstructions, since it acquires automated serial images at ultrastructural level. Using FIB/SEM imaging, we observed, at 10 nm isotropic resolution, the same dendrites that were imaged in vivo over 9 days. Thus, we analyzed their ultrastructure and monitored the dynamics of the neuropil around them. We found that stable spines (present during the 9 days of imaging) formed typical asymmetric contacts with axons, whereas transient spines (present only during one day of imaging) did not form a synaptic contact. Our data suggest that the morphological classification that was assigned to a dendritic spine according to the in vivo images did not fit with its ultrastructural morphology. The correlative technique described herein is likely to open opportunities for unravelling the earlier unrecognized complexity of the nervous system.

Human cardiac telocytes: 3D imaging by FIB-SEM tomography.

Telocyte (TC) is a newly identified type of cell in the cardiac interstitium (www.telocytes.com). TCs are described by classical transmission electron microscopy as cells with very thin and long telopodes (Tps; cellular prolongations) having podoms (dilations) and podomers (very thin segments). TCs' three-dimensional (3D) morphology is still unknown. Cardiac TCs seem to be particularly involved in long and short distance intercellular signalling and, therefore, their 3D architecture is important for understanding their spatial connections. Using focused ion beam scanning electron microscopy (FIB-SEM) we show, for the first time, the whole ultrastructural anatomy of cardiac TCs. 3D reconstruction of cardiac TCs by FIB-SEM tomography confirms that they have long, narrow but flattened (ribbon-like) telopodes, with humps generated by the podoms. FIB-SEM tomography also confirms the network made by TCs in the cardiac interstitium through adherens junctions. This study provides the first FIB-SEM tomography of a human cell type.

Mitochondrial DNA as a marker for treatment-response in post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a serious mental health condition thought to be mediated by a dysregulated stress response system. Stress, especially chronic stress, affects mitochondrial activity and their efficiency in duplicating their genomes. Human cells contain numerous mitochondria that harbor multiple copies of their own genome, which consist of a mixture of wild type and variant mtDNA - a condition known as mitochondrial heteroplasmy. Number of mitochondrial genomes in a cell and the degree of heteroplasmy may serve as an indicator of mitochondrial allostatic load. Changes in mtDNA copy number and the proportion of variant mtDNA may be related to mental disorders and symptom severity, suggesting an involvement of mitochondrial dysfunction also in PTSD. Therefore, we examined number and composition of mitochondrial DNA before and after six weeks of inpatient psychotherapy treatment in a cohort of 60 female PTSD patients. We extracted DNA from isolated monocytes before and after inpatient treatment and quantified cellular mtDNA using multiplex qPCR. We hypothesized that treatment would lead to changes in cellular mtDNA levels and that change in mtDNA level would be associated with PTSD symptom severity and treatment response. It could be shown that mtDNA copy number and the ratio of variant mtDNA decreased during therapy, however, this change did not correlate with treatment response. Our results suggest that inpatient treatment can reduce signs of mitochondrial allostatic load, which could have beneficial effects on mental health. The quantification of mtDNA and the determination of cellular heteroplasmy could represent valuable biomarkers for the molecular characterization of mental disorders in the future.

Dual-Layer Detector Cone-Beam CT Angiography for Stroke Assessment: First-in-Human Results (the Next Generation X-ray Imaging System Trial).

BACKGROUND AND PURPOSE: In patients with stroke, IV cone-beam CTA in the angiography suite could be an alternative to CTA to shorten the door-to-thrombectomy time. However, image quality in cone-beam CTA is typically limited by artifacts. This study evaluated a prototype dual-layer detector cone-beam CT angiography versus CTA in patients with stroke. MATERIALS AND METHODS: A prospective, single-center trial enrolled consecutive patients with ischemic or hemorrhagic stroke on initial CT. Intracranial arterial segment vessel conspicuity and artifact presence were evaluated on dual-layer cone-beam CTA 70-keV virtual monoenergetic images and CTA. Eleven predetermined vessel segments were matched for every patient. Twelve patients were necessary to show noninferiority to CTA. Noninferiority was determined by the exact binomial test; the 1-sided lower performance boundary was prospectively set to 80% (98.75% CI). RESULTS: Twenty-one patients had matched image sets (mean age, 72 years). After excluding examinations with movement or contrast media injection issues, all readers individually considered dual-layer cone-beam CT angiography noninferior to CTA (CI boundary, 93%, 84%, 80%, respectively) when evaluating arteries relevant in candidates for intracranial thrombectomy. Artifacts were more prevalent compared with CTA. The majority assessment rated each individual segment except M1 as having noninferior conspicuity compared with CTA. CONCLUSIONS: In a single-center stroke setting, dual-layer detector cone-beam CTA virtual monoenergetic images are noninferior to CTA under certain conditions. Notably, the prototype is hampered by a long scan time and is not capable of contrast media bolus tracking. After excluding examinations with such scan issues, readers considered dual-layer detector cone-beam CTA noninferior to CTA, despite more artifacts.

Charon's size and an upper limit on its atmosphere from a stellar occultation.

Pluto and its satellite, Charon (discovered in 1978; ref. 1), appear to form a double planet, rather than a hierarchical planet/satellite couple. Charon is about half Pluto's size and about one-eighth its mass. The precise radii of Pluto and Charon have remained uncertain, leading to large uncertainties on their densities. Although stellar occultations by Charon are in principle a powerful way of measuring its size, they are rare, as the satellite subtends less than 0.3 microradians (0.06 arcsec) on the sky. One occultation (in 1980) yielded a lower limit of 600 km for the satellite's radius, which was later refined to 601.5 km (ref. 4). Here we report observations from a multi-station stellar occultation by Charon, which we use to derive a radius, R(C) = 603.6 +/- 1.4 km (1sigma), and a density of rho = 1.71 +/- 0.08 g cm(-3). This occultation also provides upper limits of 110 and 15 (3sigma) nanobar for an atmosphere around Charon, assuming respectively a pure nitrogen or pure methane atmosphere.

Targeted bisulfite sequencing: A novel tool for the assessment of DNA methylation with high sensitivity and increased coverage.

DNA methylation analysis is increasingly used in stress research. Available methods are expensive, laborious and often limited by either the analysis of short CpG stretches or low assay sensitivity. Here, we present a cost-efficient next generation sequencing-based strategy for the simultaneous investigation of multiple candidate genes in large cohorts. To illustrate the method, we present analysis of four candidate genes commonly assessed in psychoneuroendocrine research: Glucocorticoid receptor (NR3C1), Serotonin transporter (SLC6A4), FKBP Prolyl isomerase 5 (FKBP5), and the Oxytocin receptor (OXTR). DNA methylation standards (100 %; 75 %; 50 %; 25 % and 0 %) and DNA of a female and male donor were bisulfite treated in three independent trials and were used to generate sequencing libraries for 42 CpGs from the NR3C1 1 F promoter region, 84 CpGs of the SLC6A4 5' regulatory region, 5 CpGs located in FKBP5 intron 7, and additional 12 CpGs located in a potential enhancer element in intron 3 of the OXTR. In addition, DNA of 45 patients with borderline personality disorder (BPD) and 45 healthy controls was assayed. Multiplex libraries of all samples were sequenced on a MiSeq system and analyzed for mean methylation values of all CpG sites using amplikyzer2 software. Results indicated excellent accuracy of the assays when investigating replicates generated from the same bisulfite converted DNA, and very high linearity (R2 > 0.9) of the assays shown by the analysis of differentially methylated DNA standards. Comparing DNA methylation between BPD and healthy controls revealed no biologically relevant differences. The technical approach as described here facilitates targeted DNA methylation analysis and represents a highly sensitive, cost-efficient and high throughput tool to close the gap between coverage and precision in epigenetic research of stress-associated phenotypes.

Cell-free DNA release under psychosocial and physical stress conditions.

The understanding of mechanisms linking psychological stress to disease risk depend on reliable stress biomarkers. Circulating cell-free DNA (cfDNA) has emerged as a potential biomarker of cellular stress, aging, inflammatory processes, and cell death. Recent studies indicated that psychosocial stress and physical exercise might also influence its release. We compared the effects of acute psychosocial and physical exercise stress on cfDNA release by exposing 20 young, healthy men to both an acute psychosocial laboratory stressor and an acute physical exercise stressor. Venous blood and saliva samples were collected before and after stress exposure. Cell-free DNA was extracted from plasma and quantified by qPCR. Furthermore, cfDNA fragment length was analyzed and cfDNA methylation patterns were assayed across time. In addition, release of stress hormones and subjective stress responses were measured. Results showed a twofold increase of cfDNA after TSST and fivefold increase after exhaustive treadmill exercise, with an overabundance of shorter cfDNA fragments after physical exhaustion. Interestingly, cell-free mitochondrial DNA showed similar increase after both stress paradigms. Furthermore, cfDNA methylation signatures-used here as a marker for diverse cellular origin-were significantly different post stress tests. While DNA methylation decreased immediately after psychosocial stress, it increased after physical stress, suggesting different cellular sources of active DNA release. In summary, our results suggest stimulus and cell-specific regulation of cfDNA release. Whereas the functional role of stress-associated cfDNA release remains elusive, it might serve as a valuable biomarker in molecular stress research as a part of the psychophysiological stress response.

Brefeldin A action and recovery in Chlamydomonas are rapid and involve fusion and fission of Golgi cisternae.

CHLAMYDOMONAS NOCTIGAMA has a non-motile Golgi apparatus consisting of several Golgi stacks adjacent to transitional ER. These domains are characterized by vesicle-budding profiles and the lack of ribosomes on the side of the ER proximal to the Golgi stacks. Immunogold labelling confirms the presence of COPI-proteins at the periphery of the Golgi stacks, and COPII-proteins at the ER-Golgi interface. After addition of BFA (10 microg/ml) a marked increase in the number of vesicular profiles lying between the ER and the Golgi stacks is seen. Serial sections of cells do not provide any evidence for the existence of tubular connections between the ER and the Golgi stacks, supporting the notion that COPI- but not COPII-vesicle production is affected by BFA. The fusion of COPII-vesicles at the CIS-Golgi apparatus apparently requires the presence of retrograde COPI-vesicles. After 15 min the cisternae of neighbouring Golgi stacks begin to fuse forming "mega-Golgis", which gradually curl before fragmenting into clusters of vesicles and tubules. These are surrounded by the transitional ER on which vesicle-budding profiles are still occasionally visible. Golgi remnants continue to survive for several hours and do not completely disappear. Washing out BFA leads to a very rapid reassembly of Golgi cisternae. At first, clusters of vesicles are seen adjacent to transitional ER, then "mini Golgis" are seen whose cisternae grow in length and number to produce "mega Golgis". These structures then divide by vertical fission to produce Golgi stacks of normal size and morphology roughly 60 min after drug wash-out.

Stability and cellular studies of [rac-1,2-bis(4-fluorophenyl)-ethylenediamine][cyclobutane-1,1- dicarboxylato]platinum(II), a novel, highly active carboplatin derivative.

The synthesis of the diastereomeric [1,2-bis(4-fluorophenyl)ethylenediamine][cyclobutane-1, 1-dicarboxylato]platinum(II) complexes, rac- and meso-4F-Pt(CBDC), the evaluation of their structures, their tumor-inhibiting properties and their stability in physiological environment are described (reference complexes: the dichloro- and sulfatoplatinum(II) analogues, carboplatin and cisplatin). The most interesting diastereomer, rac-4F-Pt(CBDC), equals cisplatin and surpasses carboplatin in its effect on human breast cancer cell lines (MCF-7 and MDA-MB-231). Rac-4F-Pt(CBDC) is largely insensitive against attack of nucleophiles e.g. Cl-, a prerequisite for sufficient stability in vivo and for fewer side effects. In accordance with this, in vitro studies on the binding of rac-4F-Pt(CBDC) to albumin, the main plasma protein, show that the free, non-protein-bound fraction is relatively high, coming close to that of carboplatin. These properties are of importance for the transferability of the promising effects found in the cell culture experiments to in vivo conditions. The distinctly better anti-breast cancer activity of rac-4F-Pt(CBDC) than of carboplatin has been attributed to its ability to accumulate in the tumor cells. The human ovarian cancer cell line NIH-OVCAR-3 is also strongly inhibited by rac-4F-Pt(CBDC).

Titres of anti-neutrophil cytoplasmic antibodies in inflammatory bowel disease are not related to disease activity.

In the systemic vasculitides, serial measurement of titres of anti-neutrophil cytoplasmic autoantibodies (ANCA) is useful for follow-up of disease activity and prediction of relapses. ANCA have been detected in patients with inflammatory bowel disease, but their relation to disease activity in these diseases is unclear. We analysed the relation between disease activity and ANCA titres as determined by indirect immunofluorescence in paired samples obtained during active disease and at remission from individual patients with ulcerative colitis (n=60) and Crohn's disease (n=101). In addition, patients were followed prospectively, to study the fluctuations of ANCA with time in relation to disease activity. We did not detect a correlation between disease activity and ANCA titres, either in paired samples from active disease and remission, or in serial samples, either in ulcerative colitis or in Crohn's disease. In contrast to the ANCA-associated systemic vasculitides, serial measurement of ANCA titres is not useful in the monitoring of disease activity in patients with inflammatory bowel disease.

Synthesis of (S)-3,4-diaminobutanenitriles as precursors for 3-amino-GABA derivatives.

Starting from natural asparagine (1) a synthesis of the protected (S)-3,4-diaminobutanenitriles 5 and 8a-c via the beta-homoserine derivative 2 is described. The amino function in position 4 was introduced by Mitsunobu-coupling or by reductive amination when a strange deformylation of the amino aldehyde 7 was observed as a side reaction. The Mitsunobu-product 5 was converted into the dibenzylamine substituted GABA 6b which was investigated for its affinity at the GABA-A receptor.

Rises in anti-double stranded DNA antibody levels prior to exacerbations of systemic lupus erythematosus are not merely due to polyclonal B cell activation.

We investigated whether rises in anti-double stranded DNA (anti-ds DNA) antibody levels prior to disease exacerbations of systemic lupus erythematosus (SLE) are part of a restricted immune response or merely the consequence of polyclonal B cell activation. As possible inducers of polyclonal B cell activation, we analyzed, in addition, the role of clinically apparent infections in relation to changes in levels of anti-ds DNA and disease exacerbations. We prospectively followed 72 lupus patients who were examined for disease activity and infections at least every 3 months by history and physical examination according to a protocol. Once a month, we measured levels of IgG-class antibodies to an unrelated recall antigen (tetanus toxoid), levels of IgG-class antibodies to a viral antigen (cytomegalovirus late antigens [CMV-LA]), levels of total immunoglobulins G and M, and levels of anti-ds DNA (by ELISA and Farr assay). Thirty-three exacerbations and 31 infections were observed during a follow-up period of an average of 18.5 patient months. Twenty-four out of the 27 exacerbations accompanied by a positive test for anti-ds DNA were preceded by a significant rise in anti-ds DNA: in 17 cases by ELISA and in 22 cases by Farr assay. These 24 rises in levels of anti-ds DNA prior to the exacerbation were paralleled by a significant rise in levels of IgG-class anti-tetanus antibodies in 8 cases, anti-CMV-LA antibodies in 9 cases, total IgG in 7 cases, and total IgM in 15 cases. Median rise in anti-ds DNA, as measured both by ELISA and Farr assay, exceeded the median rise in anti-tetanus antibodies, anti-CMV-LA antibodies, and total IgG and IgM (P less than 0.0001). Only one infection was recorded within a period of 3 months prior to an exacerbation, whereas infections never occurred within a period of 3 months prior to a rise in anti-ds DNA. We conclude that rises in anti-ds DNA prior to exacerbations of SLE are largely due to preferential activation of anti-ds DNA-specific B cells and not merely to polyclonal B cell hyperactivity. Clinically significant infections are not related to rises in levels of anti-ds DNA nor to the induction of exacerbations in SLE.