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.

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.

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.

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.

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.

Assembly and mobility of exon-exon junction complexes in living cells.

The exon-exon junction complex (EJC) forms via association of proteins during splicing of mRNA in a defined manner. Its organization provides a link between biogenesis, nuclear export, and translation of the transcripts. The EJC proteins accumulate in nuclear speckles alongside most other splicing-related factors. We followed the establishment of the EJC on mRNA by investigating the mobility and interactions of a representative set of EJC factors in vivo using a complementary analysis with different fluorescence fluctuation microscopy techniques. Our observations are compatible with cotranscriptional binding of the EJC protein UAP56 confirming that it is involved in the initial phase of EJC formation. RNPS1, REF/Aly, Y14/Magoh, and NXF1 showed a reduction in their nuclear mobility when complexed with RNA. They interacted with nuclear speckles, in which both transiently and long-term immobilized factors were identified. The location- and RNA-dependent differences in the mobility between factors of the so-called outer shell and inner core of the EJC suggest a hypothetical model, in which mRNA is retained in speckles when EJC outer-shell factors are missing.

Thrombogenic collagen-mimetic peptides: Self-assembly of triple helix-based fibrils driven by hydrophobic interactions.

Collagens are integral structural proteins in animal tissues and play key functional roles in cellular modulation. We sought to discover collagen model peptides (CMPs) that would form triple helices and self-assemble into supramolecular fibrils exhibiting collagen-like biological activity without preorganizing the peptide chains by covalent linkages. This challenging objective was accomplished by placing aromatic groups on the ends of a representative 30-mer CMP, (GPO)(10), as with l-phenylalanine and l-pentafluorophenylalanine in 32-mer 1a. Computational studies on homologous 29-mers 1a'-d' (one less GPO), as pairs of triple helices interacting head-to-tail, yielded stabilization energies in the order 1a' > 1b' > 1c' > 1d', supporting the hypothesis that hydrophobic aromatic groups can drive CMP self-assembly. Peptides 1a-d were studied comparatively relative to structural properties and ability to stimulate human platelets. Although each 32-mer formed stable triple helices (CD) spectroscopy, only 1a and 1b self-assembled into micrometer-scale fibrils. Light microscopy images for 1a depicted long collagen-like fibrils, whereas images for 1d did not. Atomic force microscopy topographical images indicated that 1a and 1b self-organize into microfibrillar species, whereas 1c and 1d do not. Peptides 1a and 1b induced the aggregation of human blood platelets with a potency similar to type I collagen, whereas 1c was much less effective, and 1d was inactive (EC(50) potency: 1a/1b >> 1c > 1d). Thus, 1a and 1b spontaneously self-assemble into thrombogenic collagen-mimetic materials because of hydrophobic aromatic interactions provided by the special end-groups. These findings have important implications for the design of biofunctional CMPs.

In vivo BiFC analysis of Y14 and NXF1 mRNA export complexes: preferential localization within and around SC35 domains.

The bimolecular fluorescence complementation (BiFC) assay, which allows the investigation of interacting molecules in vivo, was applied to study complex formation between the splicing factor Y14 and nuclear export factor 1 (NXF1), which evidence indicates are functionally associated with nuclear mRNA. Y14 linked to the COOH terminus of yellow fluorescent protein (YFP; YC-Y14), and NXF1 fused to the NH2 terminus of YFP (YN-NXF1) expressed in MCF7 cells yielded BiFC upon specific binding. Fluorescence accumulated within and around nuclear speckles, suggesting the involvement of speckles in mRNA processing and export. Accordingly, BiFC depended on transcription and full-length NXF1. Coimmunoprecipitation of YC-Y14 with YN-NXF1, NXF1, Y14, and RNA indicated that YC-Y14 and YN-NXF1 functionally associate with RNA. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching revealed that roughly half of the accumulated BiFC complexes were immobile in vivo. This immobile fraction was readily depleted by adenosine triphosphate (ATP) administration in permeabilized cells. These results suggest that a fraction of RNA, which remains in the nucleus for several hours despite its association with splicing and export proteins, accumulates in speckles because of an ATP-dependent mechanism.