[Ethical aspects of the fluoridation of water, salt, and milk]. / Ethik und die Fluoridierung von Salz, Milch und Trinkwasser.

The article discusses two ethical aspects of the fluoridation of water, salt, and milk. First, it considers whether fluoridation contradicts the right of self-determination. Second, it discusses the chances and risks of fluoridation. The answer to the first question depends on whether people can choose other options. Freedom of choice is not simply the right to choose between different options. It is a right which defends the moral integrity of persons. Nobody should be coerced to eat or drink something which he or she rejects morally. In the political sphere, personal rights of persons can be restricted if and only if it is necessary, if there is a public interest, and if the restriction of the right is reasonable. Regarding fluoridation, even in the best risk-chance scenario, some persons have to expect a net harm. Therefore, the reasoning in favor of fluoridation has to have a specific purpose. The proclaimed reasoning is that fluoridation will benefit the worst off and is therefore a demand of justice. But this argument fails as there are other options to benefit the worst off. Even in the best risk-chance scenario, only one option is morally permissible: the fluoridation of salt, which respects the freedom of choice.

Making contacts on a nucleic acid polymer.

The interaction of proteins bound at distant sites on a nucleic acid chain plays an important role in many molecular biological processes. Contact between the proteins is established by looping of the intervening polymer, which can comprise either double- or single-stranded DNA or RNA, or interphase or metaphase chromatin. The effectiveness of this process, as well as the optimal separation distance, is highly dependent on the flexibility and conformation of the linker. This article reviews how the probability of looping-mediated interactions is calculated for different nucleic acid polymers. In addition, the application of the equations to the analysis of experimental data is illustrated.

Action at a distance: DNA-looping and initiation of transcription.

Effective initiation of transcription, especially in eukaryotes, requires the specific assembly of large protein complexes at promoters. We ask here how activator proteins that are bound hundreds or thousands of base pairs away from the promoter might facilitate this process if protein-protein interactions occur via looping of the intervening DNA. We show that the local concentration at the promoter of activator proteins bound at vicinal DNA sites can be substantially regulated by intrinsic or protein-induced distortion of the regular DNA conformation.

GRUU-Net: Integrated convolutional and gated recurrent neural network for cell segmentation.

Cell segmentation in microscopy images is a common and challenging task. In recent years, deep neural networks achieved remarkable improvements in the field of computer vision. The dominant paradigm in segmentation is using convolutional neural networks, less common are recurrent neural networks. In this work, we propose a new deep learning method for cell segmentation, which integrates convolutional neural networks and gated recurrent neural networks over multiple image scales to exploit the strength of both types of networks. To increase the robustness of the training and improve segmentation, we introduce a novel focal loss function. We also present a distributed scheme for optimized training of the integrated neural network. We applied our proposed method to challenging data of glioblastoma cell nuclei and performed a quantitative comparison with state-of-the-art methods. Insights on how our extensions affect training and inference are also provided. Moreover, we benchmarked our method using a wide spectrum of all 22 real microscopy datasets of the Cell Tracking Challenge.

Pan-mutant-IDH1 inhibitor BAY1436032 is highly effective against human IDH1 mutant acute myeloid leukemia in vivo.

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are frequently found in several human cancer types including acute myeloid leukemia (AML) and lead to the production of high levels of the oncometabolite (R)-2-hydroxyglutarate (R-2HG). Here we report the characterization of BAY1436032, a novel pan-mutant IDH1 inhibitor, both in vitro and in vivo. BAY1436032 specifically inhibits R-2HG production and colony growth, and induces myeloid differentiation of AML cells carrying IDH1R132H, IDH1R132C, IDH1R132G, IDH1R132L and IDH1R132S mutations. In addition, the compound impacts on DNA methylation and attenuates histone hypermethylation. Oral administration of BAY1436032 led to leukemic blast clearance, myeloid differentiation, depletion of leukemic stem cells and prolonged survival in two independent patient-derived xenograft IDH1 mutant AML mouse models. Together, BAY1436032 is highly effective against all major types of IDH1 mutant AML.

Dimerization of signalling modules of the EvgAS and BvgAS phosphorelay systems.

Biophysical and biochemical properties of signalling proteins or domains derived from the unorthodox EvgAS and BvgAS two-component phosphorelay systems of Escherichia coli and Bordetella pertussis were investigated. Oligomerization of the effector proteins EvgA and BvgA and of truncated EvgS and BvgS derived signalling proteins containing the receiver and histidine containing phosphotransfer (HPt) domains or comprising only the HPt domains were characterized by native gel electrophoresis, gel permeation experiments and analytical ultracentrifugation. The results obtained by the different methods are consistent with non-phosphorylated EvgA and BvgA proteins being dimers in solution with a dissociation constant significantly below 1 microM. In contrast, all sensor derived domains of EvgS and BvgS were observed to be monomers in vitro. No indications for a phosphorylation induced stimulation of oligomerization of the C-terminal histidine kinase domains could be detected. In agreement with these data, surface plasmon resonance studies revealed a 2:1 stoichiometry in the interaction of EvgA with the immobilized EvgS HPt domain and an affinity constant of 1. 24x10(6) M(-1).

The effect of the DNA conformation on the rate of NtrC activated transcription of Escherichia coli RNA polymerase.sigma(54) holoenzyme.

The transcription activator protein NtrC (nitrogen regulatory protein C) can catalyze the transition of Escherichia coli RNA polymerase complexed with the sigma 54 factor (RNAP.sigma(54)) from the closed complex (RNAP.sigma(54) bound at the promoter) to the open complex (melting of the promoter DNA). This process involves phosphorylation of NtrC (NtrC-P), assembly of an octameric NtrC-P complex at the enhancer sequence, interaction of this complex with promoter-bound RNAP.sigma(54) via DNA looping, and hydrolysis of ATP. We have used this system to study the influence of the DNA conformation on the transcription activation rate in single-round transcription experiments with superhelical plasmids as well as linearized templates. Most of the templates had an intrinsically curved DNA sequence between the enhancer and the promoter and differed with respect to the location of the curvature and the distance between the two DNA sites. The following results were obtained: (i) a ten- to 60-fold higher activation rate was observed with the superhelical templates as compared to the linearized conformation; (ii) the presence of an intrinsically curved DNA sequence increased the activation rate of linear templates about five times; (iii) no systematic effect for the presence and/or location of the inserted curved sequence was observed for the superhelical templates. However, the transcription activation rate varied up to a factor of 10 between some of the constructs. (iv) Differences in the distance between enhancer and promoter had little effect for the superhelical templates studied. The results were compared with theoretical calculations for the dependence of the contact probability between enhancer and promoter expressed as the molar local concentration j(M). A correlation of j(M) with the transcription activation rate was observed for values of 10(-8) M

Association states of the transcription activator protein NtrC from E. coli determined by analytical ultracentrifugation.

The transcription activator protein NtrC (nitrogen regulatory protein C) can catalyze the transition of E. coli RNA polymerase complexed with the sigma54 factor (RNAP.sigma54) from the closed complex (RNAP.sigma54 bound at the promoter) to the open complex (melting of the promoter DNA). This process involves phosphorylation of NtrC, assembly of a multimeric NtrC complex at the enhancer DNA sequence, interaction of this complex with promoter bound RNAP. sigma54 via DNA looping, and hydrolysis of ATP. We have used analytical ultracentrifugation to study the different NtrC association states and to derive hydrodynamic models for the conformation of the various NtrC species. The following results were obtained. (i) The unphosphorylated wild-type protein formed a dimer with a measured molecular weight of 102(+/-3) kDa, which compares to a calculated molecular weight of 54 kDa for a monomer (concentration range studied 2 to 8 microM NtrC monomer). (ii) In the unphosphorylated state one NtrC dimer was bound to one binding site as determined with DNA oligonucleotide duplexes containing one or two binding sites (concentration range studied 50 to 1000 nM NtrC dimer). (iii) The data obtained at protein concentrations that were below the concentration of binding sites indicate that binding to the DNA duplex with two binding sites occurred with essentially no cooperativity. The experiments were conducted in the absence of ATP. (iv) The phosphorylated protein formed a specific complex at the DNA duplex with the enhancer sequence (two NtrC binding sites) that consisted of four dimers (concentration range studied 100 to 1000 nM NtrC dimer). (v) The formation of this octameric complex was highly cooperative, and the data suggest that two DNA strands could bind simultaneously to this complex. (vi) From the sedimentation data a model was derived in which the NtrC dimer adopts a V shaped structure with the DNA binding domains being located at the bottom and the two receiver domains at the top of the V. In this conformation higher order NtrC complexes can be stabilized by interaction between the phosphorylated receiver domain and the central activation domain of different NtrC dimers.

Scanning force microscopy of Escherichia coli RNA polymerase.sigma54 holoenzyme complexes with DNA in buffer and in air.

Scanning force microscopy (SFM) was used to visualize complexes of Escherichia coli RNA polymerase.sigma54 (RNAP.sigma54) and a 1036 base-pair linear DNA fragment containing the glnA promoter. In order to preserve the native hydration state of the protein-DNA complexes, the samples were injected directly into the SFM fluid cell and imaged in buffer. With this protocol, an apparent bending angle of 26(+/-34) degrees was determined for the specific complexes at the promoter. The bending angle of the unspecifically bound RNAP.sigma54 showed a somewhat broader distribution of 49(+/-48) degrees, indicating the existence of conformational differences as compared to the closed complex. In about two-thirds of the closed complexes, the RNA polymerase holoenzyme was located in a lateral position with respect to the DNA and the bend of the DNA was pointing away from the protein. This conformation was consistent with the finding that for the complexes at the promoter, the apparent contour length was reduced by only about 6 nm in buffer as compared to the free DNA. From these results we conclude that in the closed complex of RNAP. sigma54, the DNA was not wrapped around the polymerase, and we present a model for the trajectory of the DNA with respect to the RNA polymerase. The images acquired in buffer were compared to samples that were washed with water and then dried before imaging. Two artefacts of the washing and drying process were detected. First, extensive washing of the sample reduced the number of the specific complexes bound at the promoter (closed complex of RNAP.sigma54) from about 70% to 30%. This is likely to be a result of sliding of the RNAP.sigma54 holoenzyme along the DNA induced by the washing process. Second, the apparent DNA shortening of the contour length of RNAP.sigma54-DNA complexes at the promoter as compared to the contour length of the free DNA was 22 nm for the dried samples as opposed to only 6 nm for the undried samples imaged in buffer. This suggests an artefact of the drying process.

Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase x sigma 54 holoenzyme by scanning force microscopy.

Scanning force microscopy (SFM) has been used to study transcriptional activation of Escherichia coli RNA polymerase x sigma 54 (RNAP x sigma 54) at the glnA promoter by the constitutive mutant NtrC(D54E,S160F) of the NtrC Protein (nitrogen regulatory protein C). DNA-protein complexes were deposited on mica and images were recorded in air. The DNA template was a 726 bp linear fragment with two NtrC binding sites located at the end and about 460 bp away from the RNAP x sigma 54 glnA promoter. By choosing appropriate conditions the structure of various intermediates in the transcription process could be visualized and analyzed: (1) different multimeric complexes of NtrC(D54E,S160F) dimers bound to the DNA template; (2) the closed complex of RNAP x sigma 54 at the glnA promoter; (3) association between DNA bound RNAP x sigma 54 and NtrC(D54E,S160F) with the intervening DNA looped out; and (4) the activated open promoter complex of RNAP x sigma 54. Measurements of the DNA bending angle of RNAP x sigma 54 closed promoter complexes yielded an apparent bending angle of 49(+/-24) degrees. Under conditions that allowed the formation of the open promoter complex, the distribution of bending angles displayed two peaks at 50(+/-24) degrees and 114(+/-18) degrees, suggesting that the transition from the RNAP x sigma 54 closed complex to the open complex is accompanied by an increase of the DNA bending angle.

Rational design and molecular characterization of a chimaeric response regulator protein.

BvgA and EvgA are closely related response regulators from Bordetella pertussis and Escherichia coli. To analyze the domain borders and linker sequences of these proteins, we used limited proteolysis and matrix-assisted laser desorption/ionization-mass spectrometry analysis of the in-gel-digested proteolytic fragments. The thermolysin-sensitive linker regions were found to extend from Leu130 to Thr144 for BvgA and from Leu127 to Ser133 for EvgA. These data provided the rationale for the construction of the chimaeric protein HA. HA carries the EvgA receiver and BvgA output domains, fused in the central part of the linker sequences of the parent proteins. Thermolysin-sensitive sites of HA were found at positions identical with those in the EvgA and BvgA linker sequences, indicating intact folding of its receiver and output domains. Consistent with this, the chimaera showed virtually unchanged phosphorylation and dimerization properties. However, BvgA and HA differed in the effect of phosphorylation on their DNA-binding activities. In the case of BvgA, phosphorylation resulted in an increased affinity and specificity in DNA binding, whereas the DNA-binding properties of HA were not affected by phosphorylation. The chimaera HA was unable to activate transcription of the BvgA-dependent fha promoter, either in vivo or in vitro. These results indicate that the phosphorylation-induced activation of BvgA requires specific interactions between the receiver and output domains that are disturbed in the chimaera.

Specificity mechanisms in the control of transcription.

In this overview we analyze and illustrate the principles underlying some of the specificity mechanisms that control the initiation, elongation, and termination phases of transcription. Thermodynamic mechanisms dominate in the first steps of initiation, where promoters at various levels of activation can be considered to be in competition for a limiting supply of core RNA polymerase. In the later stages of initiation, as well as in elongation and termination, the regulatory mechanisms that control specificity are largely kinetic, involving rate competition between branching reaction pathways where the outcome depends on the rates (and equilibria) of reaction and interconversion of different forms of the transcription complex. Elongation complexes are very stable at most positions along the DNA template, meaning that only RNA chain elongation (and editing) can occur at these positions. However, the stability of transcription complexes decreases abruptly when termination sequences are encountered, and here the outcome can be easily switched between elongation and termination (RNA release) by minor changes in the relative rates of these competing processes. Cis effectors, defined as sites at which regulatory proteins bind to upstream activation loci on either the DNA or the nascent RNA, play important roles in the control of both initiation and of the elongation-termination decision. Examples, drawn from studies of phage lambda N-dependent antitermination and E. coli rho-dependent termination processes, illustrate the flexibility and additivity of regulatory components within control mechanisms in transcription that involve multiple determinants. The generality of such regulatory principles are stressed.

A parallel stranded linear DNA duplex incorporating dG.dC base pairs.

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.

Silencing the Singer. Antibioethics in Germany.

"Die Gedanken sind frei," in the words of the old song. But in Germany, thoughts are no longer free. Peter Singer, the "death ethicist," has become a special target for activists attempting to silence bioethical debate in Germany. In the context of the trauma inflicted by National Socialism, a profound unease over issues at the end of life is accompanied by an insistence that these issues are not to be discussed.

[Therapy or euthanasia? How far may (and should) the veterinarian go in the treatment of an animal?]. / Therapieren oder euthanasieren? Wie weit darf (und soll) der Tierarzt in der Behandlung eines Tieres gehen?

Veterinarians are often faced with the question of whether they should treat or--in accordance with the animal owner--to end the life of a pet animal. This situation can be described as a moral dilemma. Can this moral dilemma be solved? The author argues that we have special moral obligations to care for pet animals. The special, quasi-personal relationships between pet animal and animal owners provide veterinarians in most cases with moral reasons to carry out the treatment.

Simultaneous binding of two DNA duplexes to the NtrC-enhancer complex studied by two-color fluorescence cross-correlation spectroscopy.

The transcription activator protein NtrC (nitrogen regulatory protein C, also termed NR(I)) can catalyze the transition of Escherichia coli RNA polymerase complexed with the sigma(54) factor (RNAP x sigma(54)) from the closed complex (RNAP x sigma(54) bound at the promoter) to the open complex (melting of the promoter DNA). This process involves phosphorylation of NtrC (NtrC-P), assembly of an octameric NtrC-P complex at the enhancer DNA sequence, interaction of this complex with promoter-bound RNAP x sigma(54) via DNA looping, and hydrolysis of ATP. Here it is demonstrated by two-color fluorescence cross-correlation spectroscopy measurements of 6-carboxyfluorescein and 6-carboxy-X-rhodamine-labeled DNA oligonucleotide duplexes that the NtrC-P complex can bind two DNA duplexes simultaneously. This suggests a model for the conformation of the looped intermediate that is formed between NtrC-P and RNAP. sigma(54) at the glnAp2 promoter during the activation process.

Substrate properties of 25-nt parallel-stranded linear DNA duplexes.

Four 25-nt oligonucleotides consisting of sequences of dA and dT (D1-4) have been synthesized. As shown in a companion paper (Rippe et al., 1989), the two combinations D1.D3 and D2.D4 form normal antiparallel duplexes, whereas the pairs D1.D2 and D3.D4 constitute duplexes with the same sequences, but with the two strands parallel to each other. The activities of the following DNA processing enzymes and chemical reagents on the parallel stranded (ps) and antiparallel stranded (aps) duplexes were tested. (i) The restriction endonucleases DraI, SspI, and MseI do not cut the ps duplexes. (ii) DNase I and exonuclease III exhibit a much lower activity with the ps duplexes. (iii) The nuclease activities of S 1 nuclease, micrococcal nuclease (S 7), phage lambda 5'-exonuclease, and the 3'-5' nuclease activity of Escherichia coli DNA polymerase I and its large fragment are higher with the ps than with the aps substrates. (iv) Bal 31 nuclease and the chemical nuclease 1,10-phenanthroline-copper ion [(OP)2Cu+] degrade ps-DNA and aps-DNA at approximately the same rate but show preferred cutting sites only with the aps molecules. (v) The iron(II)-EDTA complex has equivalent nuclease activities with the ps and the aps molecules. (vi) The ps duplex is not a substrate for blunt-end ligation with phage T4 DNA ligase.

Superhelix dimensions of a 1868 base pair plasmid determined by scanning force microscopy in air and in aqueous solution.

We have used scanning force microscopy (SFM) to study the conformation of a 1868 base pair plasmid (p1868) in its open circular form and at a superhelical density of sigma= -0.034. The samples were deposited on a mica surface in the presence of MgCl2. DNA images were obtained both in air and in aqueous solutions, and the dimensions of the DNA superhelix were analysed. Evaluation of the whole plasmid yielded average superhelix dimensions of 27 +/- 9 nm (outer superhelix diameter D), 107 +/- 51 nm (superhelix pitch P), and 54 +/-8 degrees (superhelix pitch angle alpha). We also analysed compact superhelical regions within the plasmid separately, and determined values of D = 9.2 +/- 3.3 nm, P = 42 +/- 13 nm and alpha= 63 +/- 20 degrees for samples scanned in air or rehydrated in water. These results indicate relatively large conformation changes between superhelical and more open regions of the plasmid. In addition to the analysis of the DNA superhelix dimensions, we have followed the deposition process of open circular p1868 to mica in real time. These experiments show that it is possible to image DNA samples by SFM without prior drying, and that the surface bound DNA molecules retain some ability to change their position on the surface.

Helix-coil transition of parallel-stranded DNA. Thermodynamics of hairpin and linear duplex oligonucleotides.

The stabilities have been determined of different DNA double helices constructed with the two constituent strands in a parallel orientation. These molecules incorporate polarity-inverting loop structures (hairpins) or nucleotide sequences (duplexes) which impose the desired polarity on the two strands constituting the sugar-phosphate backbone. The hairpins consisted of d(A.T)n stems (n = 8 or 10) and either a 5'-p-5' linkage in a d(C)4 loop (ps-C8 and ps-C10) or a 3'-p-3' linkage in a d(G)4 loop (ps-G10). The linear duplexes had 21-nt (ps-C2.C3) and 25-nt (ps-D1.D2, ps-D3.D4) mixed A,T sequences and normal chemical linkages throughout. Reference molecules with normal antiparallel helical orientations (hairpins aps-C8, aps-C10, and aps-G10 and duplexes aps-C3.C7, aps-D1.D3, and aps-D2.D4) were also synthesized and studied. Hydrogen bonding in ps-DNA is via reverse Watson-Crick base pairs, and the various constructs display spectroscopic, chemical, biochemical, and electrophoretic properties distinct from those of their aps counterparts. For example, both the ps and aps molecules show a pronounced UV absorption hyperchromicity upon melting, but the spectral distribution is not the same. Thus, the difference spectra (ps-aps) in the native state are characterized by a positive peak at 252 nm, an isosbestic point at 267 nm, and a negative peak at 282 nm. Temperature-dependent absorbances were recorded at selected wavelengths and in the form of complete spectra to derive the thermodynamic parameters for the helix-coil transitions.(ABSTRACT TRUNCATED AT 250 WORDS)