Synthesis and crystal structures of two solvates of 1-{[2,6-bis-(hy-droxy-meth-yl)-4-methyl-phen-oxy]meth-yl}-3,5-bis-{[(4,6-di-methyl-pyridin-2-yl)amino]meth-yl}-2,4,6-tri-ethyl-benzene.

In the crystal structures of the formamide monosolvate (1a) and the n-propanol/H2O solvate/hydrate (1b) of the title compound, C38H50N4O3 (1), the tripodal host mol-ecule adopts a conformation in which the substituents attached to the central benzene ring are arranged in an alternating order above and below the ring plane. As a result of the different nature of the involved guest species, the crystal components in 1a create a three-dimensional supra-molecular architecture, while the crystal structure of 1b consists of two-dimensional supra-molecular aggregates extending parallel to the crystallographic ab plane.

[Patient group-specific data sovereignty needs and acceptance of electronic health records]. / Patientengruppenspezifische Datenhoheitsbedürfnisse und Akzeptanz der elektronischen Patientenakte.

BACKGROUND AND AIM: People in Germany are very sensitive about their health data. The electronic health record (ePA) also raises questions about the patient's need for data sovereignty and acceptance. The possibility of selectively withholding data stored in the ePA from physicians who continue to treat the patient (opt-out) and the patient's prior knowledge of the ePA could influence the need for data sovereignty and acceptance of the ePA. The aim of this explorative study is to investigate these influences for three patient groups: "acute patients," "diabetes type 2 patients," and "palliative patients," as differences are expected between these groups. MATERIALS AND METHODS: From August to October 2019, a quantitative survey was conducted among 140 patients in the abovementioned groups. RESULTS: Of the respondents, 76.0% supported the selective opt-out option and stated that this would increase their willingness to participate in the ePA. Specifically, 81.1% of acute care patients, 80.6% of palliative care patients, and 65.6% of type 2 diabetes patients made this statement. Differences between groups were not significant. A general prior knowledge of the ePA was related to a higher need for data sovereignty - 43.2% of those who had never heard of the ePA rollout would occasionally hide their health data from other physicians, compared with 54.5% who knew of the rollout. DISCUSSION: Consideration of the data sovereignty needs of patients in the further establishment of the ePA is recommended. The selective opt-out option can contribute to acceptance. Knowledge of the ePA should be expanded, especially in the doctor-patient discussion, to enable an informed decision.

Synthesis and crystal structure of 1,3-bis-{[N,N-bis-(2-hy-droxy-eth-yl)amino]-meth-yl}-5-{[(4,6-di-methyl-pyridin-2-yl)amino]-meth-yl}-2,4,6-tri-ethyl-benzene.

In the crystal structure of the title compound, C30H50N4O4, the two bis-(hy-droxy-eth-yl)amino moieties and the 2,4-di-methyl-pyridinyl-amino unit of the mol-ecule are located on one side of the central benzene ring, while the ethyl substituents are oriented in the opposite direction. The dihedral angle between the planes of the aromatic rings is 73.6 (1)°. The conformation of the mol-ecule is stabilized by intra-molecular O-H⋯O (1.86-2.12 Å) and C-H⋯N (2.40, 2.54 Å) hydrogen bonds. Dimers of inversion-related mol-ecules represent the basic supra-molecular entities of the crystal structure. They are further connected via O-H⋯O hydrogen bonding into undulating layers extending parallel to the crystallographic bc plane. Inter-layer inter-action is accomplished by weak C-H⋯π contacts.

Widespread mineralization of soft-bodied insects in Cretaceous amber.

Fossilized tree resin, or amber, commonly contains fossils of animals, plants and microorganisms. These inclusions have generally been interpreted as hollow moulds or mummified remains coated or filled with carbonaceous material. Here, we provide the first report of calcified and silicified insects in amber from the mid-Cretaceous Kachin (Burmese) amber. Data from light microscopy, scanning electron microscopy (SEM), energy-dispersive and wavelength-dispersive X-ray spectroscopy (EDX and WDX), X-ray micro-computed tomography (Micro-CT) and Raman spectroscopy show that these Kachin fossils owe their preservation to multiple diagenetic mineralization processes. The labile tissues (e.g. eyes, wings and trachea) mainly consist of calcite, chalcedony and quartz with minor amounts of carbonaceous material, pyrite, iron oxide and phyllosilicate minerals. Calcite, quartz and chalcedony also occur in cracks as void-filling cements, indicating that the minerals formed from chemical species that entered the fossil inclusions through cracks in the resin. The results demonstrate that resin and amber are not always closed systems. Fluids (e.g. sediment pore water, diagenetic fluid and ground water) at different burial stages have chances to interact with amber throughout its geological history and affect the preservational quality and morphological fidelity of its fossil inclusions.

The combination of Raman microscopy and electron microscopy - Practical considerations of the influence of vacuum on Raman microscopy.

Due to the specific vacuum requirements for scanning electron microscopy (SEM), the Raman microscope has to operate in vacuum in a correlative Raman-SEM, which is a type of microscope combination that has recently increased in popularity. This works considers the implications of conducting Raman microscopy under vacuum, as opposed to operating in ambient air, the standard working regime of this technique. We show that the performance of the optics of the Raman microscope are identical in both conditions, but laser beam-sample interactions, such as fluorescent bleaching and beam damage, might be different due to the lack of oxygen in vacuum. The bleaching of the fluorescent background appears to be mostly unaffected by the lack of oxygen, except when very low laser powers are used. Regarding laser-beam damage, organic samples are more sensitive in vacuum than in air, whereas no definite verdict is possible for inorganic samples. These findings have practical implications for the application of correlative Raman-SEM, as low laser powers, or in extreme cases cryo-methods, need to be used for organic samples that appear only moderately beam sensitive under usual ambient air.

Coupling SEM-EDS and confocal Raman-in-SEM imaging: A new method for identification and 3D morphology of asbestos-like fibers in a mineral matrix.

Asbestos consists in natural minerals crystallized in a specific habit and possessing in particular properties. In the case of Naturally Occurring Asbestos, usual methods applied to the identification of mineral fibers and the determination of their possible asbestiform nature seems not efficient, especially in the case of mineral fibers included in mineral matrix. We present a new in-situ method based on the use of confocal Raman-in-SEM imaging implemented in a Scanning Electron Microscope as an efficient method for in-situ mineralogy. The limitation of conventional methods is discussed. We applied 2D-Raman imaging to the identification of sub-micrometric fibers included in different mineral matrix. We were able to identify actinolite fibers down to 400 nm in diameter, included in feldspar, quartz and/or calcite matrix. Moreover, Confocal Raman allows the collection of 3D data that would provide access to critical information on the morphology of the amphibole fibers in the volume, such as aspect ratio, fibers distribution and amphibole volume fraction. We performed this method on various examples of rocks containing actinolite fibers of mean structural formula is: Na0,04-0,12 Mg2,79-3,73 Al0,29-0,58 K0,01 Ca1,79-1,98 Mn0,01-0,09 Fe2+0,99-1,91 Fe3+Si7,64-7,73 O22(OH)2. We demonstrated that coupling confocal Raman imaging and SEM is a new and efficient in-situ method for identification and morphological characterization of amphibole fibers.

Calcium silicate layer on titanium fabricated by electrospray deposition.

Titanium and its alloys have been used as implant materials. Non-ideal osseointegration of the implant materials has facilitated the development of the bioactive coatings on the implant surfaces. In this work, the bioactive calcium silicate (CaSi) powder prepared in a green synthesis route was used to cover the surface of Ti implants by a facile electrospray deposition method. Post annealing in air was also applied to form the oxidation layer on the Ti surface with the aim of increasing the bond strength between the CaSi coating layer and Ti substrate. For the characterization of the coatings several analytical methods such as X-ray diffraction, scanning electron microscopy, secondary neutral mass spectrometry, and Raman-spectroscopy were used, in addition to the measurement of bond strength and corrosion resistance. The results indicated a uniform CaSi layer with a thickness of about 1 µm deposited on the Ti substrate. Annealing in the range of 700-900 °C in air resulted in the formation of rutile phase of TiO2; more importantly, annealing at 800 °C did not significantly affect the composition of the CaSi layer consisting of ß-Ca2SiO4. The bond strength between the coating layer and Ti substrate can be remarkably enhanced at an annealing temperature of 700 or 800 °C compared with the as-prepared coating without annealing. The annealed coatings had a better corrosion resistance than the as-prepared coating. It is concluded that the electrospray method associated with the post-annealing can be successfully used for the deposition of a CaSi layer with a defined structure and composition on titanium implants.

Correlative Raman spectroscopy and focused ion beam for targeted phase boundary analysis of titania polymorphs.

Site-specific preparation of specimens using focused ion beam instruments for transmission electron microscopy is at the forefront of targeting regions of interest for nanoscale characterization. Typical methods of pinpointing desired features include electron backscatter diffraction for differentiating crystal structures and energy-dispersive X-Ray spectroscopy for probing compositional variations. Yet there are situations, notably in the titanium dioxide system, where these techniques can fail. Differentiating between the brookite and anatase polymorphs of titania is either excessively laborious or impossible with the aforementioned techniques. However, due to differences in bonding structure, Raman spectroscopy serves as an ideal candidate for polymorph differentiation. In this work, a correlative approach utilizing Raman spectroscopy for targeted focused ion beam specimen preparation was employed. Dark field imaging and diffraction in the transmission electron microscope confirmed the region of interest located via Raman spectroscopy and demonstrated the validity of this new method. Correlative Raman spectroscopy, scanning electron microscopy, and focused ion beam is shown to be a promising new technique for identifying site-specific preparation of nanoscale specimens in cases where conventional approaches do not suffice.

The in vivo dynamics of TCERG1, a factor that couples transcriptional elongation with splicing.

Coupling between transcription and RNA processing is key for gene regulation. Using live-cell photobleaching techniques, we investigated the factor TCERG1, which coordinates transcriptional elongation with splicing. We demonstrate that TCERG1 is highly mobile in the nucleoplasm and that this mobility is slightly decreased when it is associated with speckles. Dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) but not α-amanitin treatment reduced the mobility of TCERG1, which suggests interaction with paused transcription elongation complexes. We found that TCERG1 mobility is rapid at the transcription site (TS) of a reporter that splices post-transcriptionally and that TCERG1 is recruited to the active TS independent of the CTD of RNAPII, thus excluding phosphorylated CTD as a requirement for recruiting this factor to the TS. Importantly, the mobility of TCERG1 is reduced when the reporter splices cotranscriptionally, which suggests that TCERG1 forms new macromolecular complexes when splicing occurs cotranscriptionally. In this condition, spliceostatin A has no effect, indicating that TCERG1 rapidly binds and dissociates from stalled spliceosomal complexes and that the mobility properties of TCERG1 do not depend on events occurring after the initial spliceosome formation. Taken together, these data suggest that TCERG1 binds independently to elongation and splicing complexes, thus performing their coupling by transient interactions rather than by stable association with one or the other complexes. This finding has conceptual implications for understanding the coupling between transcription and RNA processing.

A microspectroscopic insight into the resistivity switching of individual Ag-TCNQ nanocrystals.

We investigate the resistivity switching in individual Ag-TCNQ wires with on/off-ratios of up to 10(3). Raman and soft X-ray absorption microspectroscopy studies disclose reverse charge transfer. Quantifying of the fraction of neutral TCNQ within the switched material yields values up to 22.3%. These findings expedite the understanding of the switching process in Ag-TCNQ nanowires.

Event detection by feature unpredictability in phase-contrast videos of cell cultures.

In this work we propose a novel framework for generic event monitoring in live cell culture videos, built on the assumption that unpredictable observations should correspond to biological events. We use a small set of event-free data to train a multioutput multikernel Gaussian process model that operates as an event predictor by performing autoregression on a bank of heterogeneous features extracted from consecutive frames of a video sequence. We show that the prediction error of this model can be used as a probability measure of the presence of relevant events, that can enable users to perform further analysis or monitoring of large-scale non-annotated data. We validate our approach in two phase-contrast sequence data sets containing mitosis and apoptosis events: a new private dataset of human bone cancer (osteosarcoma) cells and a benchmark dataset of stem cells.

Stable assembly of HIV-1 export complexes occurs cotranscriptionally.

The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post- or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest that cotranscriptional formation of a stable export complex serves as a means to ensure efficient export of unspliced viral RNAs.

Diffusion and binding analyzed with combined point FRAP and FCS.

To quantify more precisely and more reliably diffusion and reaction properties of biomolecules in living cells, a novel closed description in 3D of both the bleach and the post-bleach segment of fluorescence recovery after photobleaching (FRAP) data acquired at a point, i.e., a diffraction-limited observation area, termed point FRAP, is presented. It covers a complete coupled reaction-diffusion scheme for mobile molecules undergoing transient or long-term immobilization because of binding. We assess and confirm the feasibility with numerical solutions of the differential equations. By applying this model to free EYFP expressed in HeLa cells using a customized confocal laser scanning microscope that integrates point FRAP and fluorescence correlation spectroscopy (FCS), the applicability is validated by comparison with results from FCS. We show that by taking diffusion during bleaching into consideration and/or by employing a global analysis of series of bleach times, the results can be improved significantly. As the point FRAP approach allows to obtain data with diffraction-limited positioning accuracy, diffusion and binding properties of the exon-exon junction complex (EJC) components REF2-II and Magoh are obtained at different localizations in the nucleus of MCF7 cells and refine our view on the position-dependent association of the EJC factors with a maturating mRNP complex. Our findings corroborate the concept of combining point FRAP and FCS for a better understanding of the underlying diffusion and binding processes.

Chitosan-coated triangular silver nanoparticles as a novel class of biocompatible, highly sensitive plasmonic platforms for intracellular SERS sensing and imaging.

There is a need for new strategies for noninvasive imaging of pathological conditions within the human body. The approach of combining the unique physical properties of noble-metal nanoparticles with their chemical specificity and an easy way of conjugation open up new routes toward building bio-nano-objects for biomedical tracking and imaging. This work reports the design and assessment of a novel class of biocompatible, highly sensitive SERS nanotags based on chitosan-coated silver nanotriangles (Chit-AgNTs) labeled with para-aminothiophenol (p-ATP). The triangular nanoparticles are used as Raman scattering enhancers and have proved to yield a reproducible and strong SERS signal. When tested inside lung cancer cells (A549) this class of SERS nanotags presents low in vitro toxicity, without interfering with cell proliferation. Easily internalized by the cells, as demonstrated by imaging using both reflected bright-light optical microscopy and SERS spectroscopy, the particles are proved to be detectable inside cells under a wide window of excitation wavelengths, ranging from visible to near infrared (NIR). Their high sensitivity and NIR availability make this class of SERS nanotags a promising candidate for noninvasive imaging of cancer cells.

Perispeckles are major assembly sites for the exon junction core complex.

The exon junction complex (EJC) is loaded onto mRNAs as a consequence of splicing and regulates multiple posttranscriptional events. MLN51, Magoh, Y14, and eIF4A3 form a highly stable EJC core, but where this tetrameric complex is assembled in the cell remains unclear. Here we show that EJC factors are enriched in domains that we term perispeckles and are visible as doughnuts around nuclear speckles. Fluorescence resonance energy transfer analyses and EJC assembly mutants show that perispeckles do not store free subunits, but instead are enriched for assembled cores. At the ultrastructural level, perispeckles are distinct from interchromatin granule clusters that may function as storage sites for splicing factors and intermingle with perichromatin fibrils, where nascent RNAs and active RNA Pol II are present. These results support a model in which perispeckles are major assembly sites for the tetrameric EJC core. This subnuclear territory thus represents an intermediate region important for mRNA maturation, between transcription sites and splicing factor reservoirs and assembly sites.

Microprocessor dynamics and interactions at endogenous imprinted C19MC microRNA genes.

Nuclear primary microRNA (pri-miRNA) processing catalyzed by the DGCR8-Drosha (Microprocessor) complex is highly regulated. Little is known, however, about how microRNA biogenesis is spatially organized within the mammalian nucleus. Here, we image for the first time, in living cells and at the level of a single microRNA cluster, the intranuclear distribution of untagged, endogenously-expressed pri-miRNAs generated at the human imprinted chromosome 19 microRNA cluster (C19MC), from the environment of transcription sites to single molecules of fully released DGCR8-bound pri-miRNAs dispersed throughout the nucleoplasm. We report that a large fraction of Microprocessor concentrates onto unspliced C19MC pri-miRNA deposited in close proximity to their genes. Our live-cell imaging studies provide direct visual evidence that DGCR8 and Drosha are targeted post-transcriptionally to C19MC pri-miRNAs as a preformed complex but dissociate separately. These dynamics support the view that, upon pri-miRNA loading and most probably concomitantly with Drosha-mediated cleavages, Microprocessor undergoes conformational changes that trigger the release of Drosha while DGCR8 remains stably bound to pri-miRNA.

Retroviral GAG proteins recruit AGO2 on viral RNAs without affecting RNA accumulation and translation.

Cellular micro(mi)RNAs are able to recognize viral RNAs through imperfect micro-homologies. Similar to the miRNA-mediated repression of cellular translation, this recognition is thought to tether the RNAi machinery, in particular Argonaute 2 (AGO2) on viral messengers and eventually to modulate virus replication. Here, we unveil another pathway by which AGO2 can interact with retroviral mRNAs. We show that AGO2 interacts with the retroviral Group Specific Antigen (GAG) core proteins and preferentially binds unspliced RNAs through the RNA packaging sequences without affecting RNA stability or eliciting translation repression. Using RNAi experiments, we provide evidences that these interactions, observed with both the human immunodeficiency virus 1 (HIV-1) and the primate foamy virus 1 (PFV-1), are required for retroviral replication. Taken together, our results place AGO2 at the core of the retroviral life cycle and reveal original AGO2 functions that are not related to miRNAs and translation repression.

Coding RNAs with a non-coding function: maintenance of open chromatin structure.

The multi-layered organization of the genome in a large nucleoprotein complex termed chromatin regulates nuclear functions by establishing subcompartments with distinct DNA-associated activities. Here, we demonstrate that RNA plays an important role in maintaining a decondensed and biologically active interphase chromatin conformation in human and mouse cell lines. As shown by RNase A microinjection and fluorescence microscopy imaging, digestion of single-stranded RNAs induced a distinct micrometer scale chromatin aggregation of these decondensed regions. In contrast, pericentric heterochromatin was more resistant to RNase A treatment. We identified a class of coding RNA transcripts that are responsible for this activity, and thus termed these 'chromatin-interlinking' RNAs or ciRNAs. The initial chromatin distribution could be restored after RNase A treatment with a purified nuclear RNA fraction that was analyzed by high-throughput sequencing. It comprised long > 500 nucleotides (nt) RNA polymerase II (RNAP II) transcripts that were spliced, depleted of polyadenylation and was enriched with long 3'-untranslated regions (3'-UTRs) above ~800 nt in length. Furthermore, similar reversible changes of the chromatin conformation and the RNAP II distribution were induced by either RNA depletion or RNAP II inhibition. Based on these results we propose that ciRNAs could act as genome organizing architectural factors of actively transcribed chromatin compartments.

Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation.

Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2-GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules.