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Plant phytochromes perceive red and far-red light to elicit adaptations to the changing environment. Downstream physiological responses revolve around red-light-induced interactions with phytochrome-interacting factors (PIF). Phytochromes double as thermoreceptors, owing to the pronounced temperature dependence of thermal reversion from the light-adapted Pfr to the dark-adapted Pr state. Here, we assess whether thermoreception may extend to the phytochrome:PIF interactions. While the association between Arabidopsis (Arabidopsis thaliana) PHYTOCHROME B (PhyB) and several PHYTOCHROME-INTERACTING FACTOR (PIF) variants moderately accelerates with temperature, the dissociation does more so, thus causing net destabilization of the phytochrome:PIF complex. Markedly different temperature profiles of PIF3 and PIF6 might underlie stratified temperature responses in plants. Accidentally, we identify a photoreception mechanism under strong continuous light, where the extent of phytochrome:PIF complexation decreases with red-light intensity rather than increases. Mathematical modeling rationalizes this attenuation mechanism and ties it to rapid red-light-driven Pr⇄Pfr interconversion and complex dissociation out of Pr. Varying phytochrome abundance, e.g., during diurnal and developmental cycles, and interaction dynamics, e.g., across different PIFs, modify the nature and extent of attenuation, thus permitting light-response profiles more malleable than possible for the phytochrome Pr⇄Pfr interconversion alone. Our data and analyses reveal a photoreception mechanism with implications for plant physiology, optogenetics, and biotechnological applications., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Sensory photoreceptors mediate numerous light-dependent adaptations across organisms. In optogenetics, photoreceptors achieve the reversible, non-invasive, and spatiotemporally precise control by light of gene expression and other cellular processes. The light-oxygen-voltage receptor PAL binds to small RNA aptamers with sequence specificity upon blue-light illumination. By embedding the responsive aptamer in the ribosome-binding sequence of genes of interest, their expression can be downregulated by light. We developed the pCrepusculo and pAurora optogenetic systems that are based on PAL and allow to down- and upregulate, respectively, bacterial gene expression using blue light. Both systems are realized as compact, single plasmids that exhibit stringent blue-light responses with low basal activity and up to several 10-fold dynamic range. As PAL exerts light-dependent control at the RNA level, it can be combined with other optogenetic circuits that control transcription initiation. By integrating regulatory mechanisms operating at the DNA and mRNA levels, optogenetic circuits with emergent properties can thus be devised. As a case in point, the pEnumbra setup permits to upregulate gene expression under moderate blue light whereas strong blue light shuts off expression again. Beyond providing novel signal-responsive expression systems for diverse applications in biotechnology and synthetic biology, our work also illustrates how the light-dependent PAL-aptamer interaction can be harnessed for the control and interrogation of RNA-based processes.

In optogenetics, as in nature, sensory photoreceptors serve to control cellular processes by light. Bacteriophytochrome (BphP) photoreceptors sense red and far-red light via a biliverdin chromophore and, in response, cycle between the spectroscopically, structurally, and functionally distinct Pr and Pfr states. BphPs commonly belong to two-component systems that control the phosphorylation of cognate response regulators and downstream gene expression through histidine kinase modules. We recently demonstrated that the paradigm BphP from Deinococcus radiodurans exclusively acts as a phosphatase but that its photosensory module can control the histidine kinase activity of homologous receptors. Here, we apply this insight to reprogram two widely used setups for bacterial gene expression from blue-light to red-light control. The resultant pREDusk and pREDawn systems allow gene expression to be regulated down and up, respectively, uniformly under red light by 100-fold or more. Both setups are realized as portable, single plasmids that encode all necessary components including the biliverdin-producing machinery. The triggering by red light affords high spatial resolution down to the single-cell level. As pREDusk and pREDawn respond sensitively to red light, they support multiplexing with optogenetic systems sensitive to other light colors. Owing to the superior tissue penetration of red light, the pREDawn system can be triggered at therapeutically safe light intensities through material layers, replicating the optical properties of the skin and skull. Given these advantages, pREDusk and pREDawn enable red-light-regulated expression for diverse use cases in bacteria.

Objectives: Atrophic resorption of the maxillary alveolar ridge is a complication that makes implantological rehabilitation critical. Our aim was to develop a novel computer aided procedure for the accurate quantitative assessment of maxillary residual ridge resorption including pneumatisation of the maxillary sinus that goes beyond previously described approaches and to apply it to a large dataset., Materials and Methods: To develop and refine the method, we performed a retrospective analysis using computed tomography data from 405 patients to generate segmented, three-dimensional models of zygomaticomaxillary bones and maxillary sinuses. Using anatomical landmarks and orientation lines or planes, all models were aligned automatically to subsequently generate cross-sectional images (n = 2835), enabling the classification of atrophy as well as the quantification of volumes and caudal extensions of the maxillary sinuses., Results: We developed and implemented an accurate and reproducible workflow for the semi-automated analysis of volumetric maxillary images. Comprehensive statistical analysis of the large quantitative dataset revealed various correlations of maxillary process heights and sinus volumes with atrophy class, age and region and identified conjectural trends over the patient group., Conclusions: The method was used successfully to process a large dataset to classify atrophy, to measure alveolar height parameters, and to quantify maxillary sinus volume, bottom volume and pneumatisation., Clinical Relevance: Apart from the anthropometric value of the generated dataset, the method could be applied to provide additional and more accurate data to assess the necessity of bone augmentation in the context of three-dimensional planning before implantation.

Plant phytochromes enable vital adaptations to red and far-red light. At the molecular level, these responses are mediated by light-regulated interactions between phytochromes and partner proteins, foremost the phytochrome-interacting factors (PIF). Although known for decades, quantitative analyses of these interactions have long been sparse. To address this deficit, we here studied by an integrated fluorescence-spectroscopic approach the equilibrium and kinetics of Arabidopsis thaliana phytochrome B binding to a tetramerized PIF6 variant. Several readouts consistently showed the stringently light-regulated interaction to be little affected by PIF tetramerization. Analysis of the binding kinetics allowed the determination of bimolecular association and unimolecular dissociation rate constants as a function of light. Unexpectedly, the stronger affinity of A. thaliana phytochrome B under red light relative to far-red light is entirely due to accelerated association rather than decelerated dissociation. The association reaction under red light is highly efficient and only 3-fold slower than the diffusion limit. The present findings pertain equally to the analysis of signal transduction in plants and to the biotechnological application of phytochromes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nanoporous media are of great importance for drug delivery or filtration. Typically the pore structure of such media is characterized using high-resolution techniques such as electron microscopy or atomic force microscopy. However, these techniques are restricted to the surface of the material and/or are highly invasive. In a proof-of-concept experiment we have employed three-dimensional single-particle orbit tracking for testing the three-dimensional pore structure of a liquid filled nanoporous polystyrene-block-polyisoprene-block-poly(N-isopropylacrylamide) (PS-b-PI-b-PNiPAAm) triblock terpolymer membrane. Using fluorescent tracers with a diameter of about 10% of the relevant void structures, the tracking experiments yielded results that were comparable to those obtained from reference experiments using environmental scanning electron microscopy (eSEM). This testifies that single-particle orbit tracking can serve as a useful non-invasive alternative for characterising the structure of nanoporous materials.

Based on a sample of the Hannover registration office this project analyses the health and prevention behaviour of Russian- and Turkish-language migrants compared to natives. The project analysis revealed considerable differences, particularly concerning addiction behaviour and physical activities., (© Georg Thieme Verlag KG Stuttgart · New York.)

Phenoxy radical coupling reactions are involved in the biosynthesis of lignans in planta. Interestingly, the reaction can be guided by dirigent proteins, which mediate the stereoselective formation of either (+) or (-)-pinoresinol from coniferyl alcohol. So far, the mechanism is poorly understood, and for detailed mechanistic studies, a heterologous expression platform which allows the cost-effective, fast, and robust expression in high yields is needed. We established a reliable, high-yield fed-batch fermentation process with Pichia pastoris resulting in 47 mg L⁻¹ of the dirigent protein AtDIR6, which represents a more than 250-fold increase compared to previous studies. Biochemical characterization of AtDIR6 produced with P. pastoris showed an overall agreement in protein structure, N-glycosylation sites, and dirigent activity compared to AtDIR6 produced by plant cell cultures of Solanum peruvianum. CD spectroscopy verified the β-barrel structure proposed by earlier studies and bioconversion experiments revealed similar activities to plant-derived protein, validating P. pastoris as a suitable expression system for dirigent proteins. Compared to the complex glycan structures of most plant cells, proteins produced with P. pastoris have the advantage that they can be enzymatically deglycosylated under non-denaturating conditions. With this study, we demonstrate that the glycan structures of AtDIR6 are essential for structure, solubility, and function of the protein as deglycosylation induced conformational changes leading to the complete loss in dirigent activity and subsequent protein aggregation.

Membrane protein translocation and insertion is a central issue in biology. Here we focus on a minimal system, the membrane insertase YidC of Escherichia coli that inserts small proteins into the cytoplasmic membrane. In a reconstituted system individual insertion processes were followed by single-pair fluorescence resonance energy transfer (FRET), with a pair of fluorophores on YidC and the substrate Pf3 coat protein. After addition of N-terminally labeled Pf3 coat protein a close contact to YidC at its cytoplasmic label was observed. This allowed to monitor the translocation of the N-terminal domain of Pf3 coat protein across the membrane in real time. Translocation occurred within milliseconds as the label on the N-terminal domain rapidly approached the fluorophore on the periplasmic domain of YidC at the trans side of the membrane. After the close contact, the two fluorophores separated, reflecting the release of the translocated Pf3 coat protein from YidC into the membrane bilayer. When the Pf3 coat protein was labeled C-terminally, no translocation of the label was observed although efficient binding to the cytoplasmic positions of YidC occurred.

Nuclear pore complexes (NPCs) fuse the two membranes of the nuclear envelope (NE) to a pore, connecting cytoplasm and nucleoplasm and allowing exchange of macromolecules between these compartments. Most NPC proteins do not contain integral membrane domains and thus it is largely unclear how NPCs are embedded and anchored in the NE. Here, we show that the evolutionary conserved nuclear pore protein Nup53 binds independently of other proteins to membranes, a property that is crucial for NPC assembly and conserved between yeast and vertebrates. The vertebrate protein comprises two membrane binding sites, of which the C-terminal domain has membrane deforming capabilities, and is specifically required for de novo NPC assembly and insertion into the intact NE during interphase. Dimerization of Nup53 contributes to its membrane interaction and is crucial for its function in NPC assembly.

Dirigent proteins impart stereoselectivity on the phenoxy radical-coupling reaction, yielding optically active lignans from two molecules of coniferyl alcohol. By an unknown mechanism, they direct the coupling of two phenoxy radicals toward the formation of optically active (+)- or (-)-pinoresinol. We show here that the dirigent protein AtDIR6 from Arabidopsis thaliana is a homodimeric all-beta protein in the superfamily of calycins. Based on its homology with calycins, the structure of AtDIR6 was modeled using allene oxide cyclase as template. The structural model of AtDIR6 was supported experimentally by confirmation of a predicted disulfide bridge and by the characterization of two N-linked glycans at the solvent-exposed protein surface. The model shows AtDIR6 as an eight-stranded antiparallel β-barrel with a central hydrophobic cavity for substrate binding, suggesting that dirigent proteins evolved from hydrophobic ligand-binding proteins. The data are fully consistent with the current view of the dirigent protein mode of action, according to which each subunit of the homodimer captures one of the substrate radicals and orients them in a way that precludes undesired reaction channels, thus favoring the formation of the optically pure coupling product., (© 2012 The Authors Journal compilation © 2012 FEBS.)

In line with the National Action Plan "IN FORM" the German Federal Ministry of Health funds the establishment of 11 regional community health promotion networks (alliances). To meet quality development standards, an external evaluation project has been established in addition to the alliances' internal evaluation. Scientific monitoring focuses on alliance-spanning investigation of quality of planning, structures and processes and uses different methods and instruments (e.g., guided interviews, analytical framework, Goal Attainment Scaling). Regional networks also receive support for their efforts to quality development. Up to now concluding analysis can be done on aspects of planning quality based on findings of the analytical framework developed within the project. 5 selected results presented in the article reveal a heterogeneous picture: on one hand the standard on which the alliances' rationales on demands justifying the project's strategy are written at is very encouraging. The situation is similar with the descriptions of the work planning and statements concerning the sustainability at which the implemented activities are aimed. On the other hand, in explaination criteria like consideration of target group's needs or definition of goals the possibilities are not exhausted yet considering the state of the debate. Hence in similar future projects there is a clear necessity of assistance in approaching these aspects, provided preferably in an early stage of the planning phase., (© Georg Thieme Verlag KG Stuttgart · New York.)

Na(+) is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) as the first complex in its respiratory chain. The Na(+)-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na(+) translocation by the Na(+)-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na(+)-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na(+)-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA.

The binding of Pf3 coat protein to the membrane insertase YidC from Escherichia coli induces a conformational change in the tertiary structure of the insertase, resulting in a quenching of the intrinsic tryptophan (Trp) fluorescence. Tryptophan mutants of YidC were generated to examine such conformational movements in detail with time-resolved and steady-state fluorescence spectroscopy. Ten of the 11 Trp residues within YidC were substituted to phenylalanines generating single Trp mutants either at position 354, 454, or 508. In addition, a double mutant with Trp residues at 332 and 334 was studied. Purified YidC mutants were reconstituted into DOPC/DOPG vesicles and titrated with a Trp-free mutant of Pf3 coat, enabling a detailed conformational study of the periplasmic P1, P2, and P3 domains of YidC before and after binding of substrate. Time-resolved fluorescence anisotropy revealed that the mobility of the residues W332/W334 and W508 was considerably increased after binding of Pf3 coat to the insertase. Furthermore, analysis of the fluorescence emission spectra and the decay times showed that all Trp residues are embedded in an equivalent environment that is a membrane/water interface.

The membrane insertase YidC from Escherichia coli reversibly binds its substrate Pf3 coat protein. The effect of this initial binding process was examined in vitro by fluorescence quenching of the tryptophan (Trp) residues of YidC which are highly sensitive fluorescent probes for changes of the protein's tertiary structure. Membrane-reconstituted (in DOPC or DOPE/DOPG vesicles) as well as detergent-solubilized (C(12)PC) YidC was titrated with a Trp-free Pf3 coat mutant. Quenching of the intrinsic Trp fluorescence after titration indicates a change in the tertiary structure of YidC upon binding to the Pf3 coat substrate. Analysis of the binding curves taken from the fluorescence data yielded values for the dissociation constant (K(D)) in the range of 0.5-1.8 microM. Titration experiments with two Trp mutants reveal that the change in the tertiary structure involves mainly the membrane-spanning domain. The influence of the different environments on the secondary structure of YidC as well as of the YidC large periplasmic domain (P1) was investigated by circular dichroism (CD). The CD data show that the YidC secondary structure changes upon reconstitution into a membrane environment when compared to the detergent-solubilized state. In particular, the P1 domain of YidC is considerably affected by the detergent C(12)PC. This underlines the importance to study conformational changes with membrane-inserted proteins.

The binding of the inner membrane insertase YidC from Escherichia coli to its substrate, the Pf3 coat protein, was examined in vitro by fluorescence spectroscopy. Purified YidC protein was solubilized with the lipid-like detergent n-dodecylphosphocholine and noncovalently labeled with 1-anilino-naphthalene-8-sulfonate (ANS), whereas the Pf3 coat protein was kept in solution by the addition of 10% (v/v) isopropanol to the buffer. The binding of Pf3 coat protein was analyzed by fluorescence quenching of ANS bound to YidC. All binding curves showed a strict hyperbolic form at pH values between 9.0 and 5.0, indicating a reversible and noncooperative binding between YidC and its substrate. Analysis of the data revealed a dissociation constant K D for the binding process in the range of 1 microM. The pH profile of the K D values suggests that the binding of the Pf3 coat protein is dominated by hydrophobic interactions. The titration experiments provide strong evidence for a conformational change of the insertase upon binding a Pf3 coat protein molecule.

The effect of the interaction of the reaction center (RC) upon the geometrical arrangement of the bacteriochlorophyll a (BChla) pigments in the light-harvesting 1 complex (LH1) from Rhodospirillum rubrum has been examined using single molecule spectroscopy. Fluorescence excitation spectra at 1.8 K obtained from single detergent-solubilized as well as single membrane-reconstituted LH1-RC complexes showed predominantly (>70%) a single broad absorption maximum at 880-900 nm corresponding to the Q(y) transition of the LH1 complex. This absorption band was independent of the polarization direction of the excitation light. The remaining complexes showed two mutually orthogonal absorption bands in the same wavelength region with moderate splittings in the range of DeltaE = 30-85 cm(-1). Our observations are in agreement with simulated spectra of an array of 32 strongly coupled BChla dipoles arranged in perfect circular symmetry possessing only a diagonal disorder of

Spectroscopic and polarization properties of single light-harvesting complexes of higher plants (LHC-II) were studied at both room temperature and T < 5 K. Monomeric complexes emit roughly linearly polarized fluorescence light thus indicating the existence of only one emitting state. Most probably this observation is explained by efficient triplet quenching restricted to one chlorophyll a (Chl a) molecule or by rather irreversible energy transfer within the pool of Chl a molecules. LHC-II complexes in the trimeric (native) arrangement bleach in a number of steps, suggesting localization of excitations within the monomeric subunits. Interpretation of the fluorescence polarization properties of trimers requires the assumption of transition dipole moments tilted out of the symmetry plane of the complex. Low-temperature fluorescence emission of trimers is characterized by several narrow spectral lines. Even at lowest excitation intensities, we observed considerable spectral diffusion most probably due to low temperature protein dynamics. These results also indicate weak interaction between Chls belonging to different monomeric subunits within the trimer thus leading to a localization of excitations within the monomer. The experimental results demonstrate the feasibility of polarization sensitive studies on single LHC-II complexes and suggest an application for determination of the Chl transition-dipole moment orientations, a key issue in understanding the structure-function relationships.

Two patterns of transforming growth factor-beta1 (TGF-beta1) expression were identified in brains of normotensive rats following permanent occlusion of the middle cerebral artery (MCAO). First, a relative increase of TGF-beta1 mRNA by 37% was found at 12 h after MCAO in the ipsilateral cingulate cortex as compared to the homotopic contralateral region. The cingulate cortex is located distant from the ischemic territory. Treatment with the glutamate receptor antagonists MK-801 and NBQX did not reduce this expression (34% and 26% increase, respectively). Therefore, peri-infarct depolarization waves were probably not responsible for induction. Secondly, an increase of TGF-beta1 mRNA by 116% was found at 7 days after MCAO within infarcted tissue. This expression was not reduced by the glutamate receptor antagonists MK-801 (increase 140%) and NBQX (increase 137%), either. TGF-beta1 mRNA expression in the cingulate cortex at 12 h after MCAO is possibly mediated by neurons and astroglia and may support cell survival. Expression in the infarcted tissue at 7 days after MCAO is most likely related to the invasion of monocytes and may be involved in the downregulation of inflammatory events, in neoangiogenesis, and in formation of a glial scar around the infarct., (Copyright 1999 Elsevier Science B.V.)

The function of membrane phospholipids (PL) in the regulation of gamma-aminobutyric acid (GABA) transport and GABA carrier binding has been investigated in organized cultures of rat cerebral cortex. The cellular lipid composition has been changed by growing the cells in a delipidated nutrient solution or by short-term exposure of the cells to PL emulsions. Introduction of PL into the cellular matrix was monitored by analysis of biologically active fluorescently labeled phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Parinaroyl and dansyl derivatives were used. Conditions of maintenance as well as exogenously given PL affected the transport of GABA. Two transport systems were observed, one first-order system and one cooperative system. Saturated species of PC or PE reduced first-order GABA uptake with increase in chain length of the fatty acid residues. The effects of unsaturated PL were dependent upon the polar head. Unsaturated PC enhanced the capacity of the first-order transport of the amino acid. In comparison to cultures grown in lipid-free medium, introduction of diarachinoyl-PC into the cells increased the density of the first-order active transport sites by a factor of 8 and the affinity constant by a factor of 17. Diarachinoyl-PE reduced both kinetic parameters. GABA uptake via the cooperative system was enhanced by the unsaturated PE, not by PC. The role of endogenous PL and their asymmetric distribution was studied by application of phospholipase A2, C, and D. Stimulation of carrier activity was induced by hydrolysis of PL on the external leaflet. Inhibition occurred upon enzymatic degradation of external and cytoplasmic PL. Lipolysis also affected GABA receptor binding, suggesting that the effects observed represent the activity of both classes of binding sites, the carrier and the receptor. However the latter accounted for a small fraction of the binding. Transport of the amino acid was temperature sensitive. The temperature curve was shifted within two discontinuities, appearing in the Arrhenius plot as a function of membrane lipids. The results suggest a partitioning of the proteins between fluid and ordered lipid domains. Displacement of the protein may govern the rate constants and/or the effective protein concentration.

Studies on the interaction between cortex grey matter (CC), diencephalon (DE), mesencephalon (ME) and pure glial cells free of neurons were undertaken in explant cultures. The culture system provided the formation of neuritic bridges between individual tissue pieces. Cocultivation of different brain tissues resulted in changes of leucine and glycerol-3-phosphate incorporation characteristic for individual brain areas. Glial cell (GC) did not respond to innervation by neurites of various sources. Glycerol-3-phosphate uptake rates were substantially altered in cocultures containing neurons and glia.