Extensive Structural Changes Research Articles

Image-based identification and isolation of micronucleated cells to dissect cellular consequences.

Recent advances in isolating cells based on visual phenotypes have transformed our ability to identify the mechanisms and consequences of complex traits. Micronucleus (MN) formation is a frequent outcome of genome instability, triggers extensive disease-associated changes in genome structure and signaling coincident with MN rupture, and is almost exclusively defined by visual analysis. Automated MN detection in microscopy images has proved extremely challenging, limiting unbiased discovery of the mechanisms and consequences of MN formation and rupture. In this study we describe two new MN segmentation modules: a rapid and precise model for classifying micronucleated cells and their rupture status (VCS MN), and a robust model for accurate MN segmentation (MNFinder) from a broad range of microscopy images. As a proof-of-concept, we define the transcriptome of non-transformed human cells with intact or ruptured MN after inducing chromosome missegregation by combining VCS MN with photoactivation-based cell isolation and RNASeq. Surprisingly, we find that neither MN formation nor rupture triggers a unique transcriptional response. Instead, transcriptional changes are correlated with increased aneuploidy in these cell classes. Our MN segmentation modules overcome a significant challenge to reproducible MN quantification, and, joined with visual cell sorting, enable the application of powerful functional genomics assays, including pooled CRISPR screens and time-resolved analyses of cellular and genetic consequences, to a wide-range of questions in MN biology.

Alterations in cortical volume and complexity in Parkinson's disease with depression.

The aim of this study is to investigate differences in gray matter volume and cortical complexity between Parkinson's disease with depression (PDD) patients and Parkinson's disease without depression (PDND) patients. A total of 41 PDND patients, 36 PDD patients, and 38 healthy controls (HC) were recruited and analyzed by Voxel-based morphometry (VBM) and surface-based morphometry (SBM). Differences in gray matter volume and cortical complexity were compared using the one-way analysis of variance (ANOVA) and correlated with the Hamilton Depression Scale-17 (HAMD-17) scores. PDD patients exhibited significant cortical atrophy in various regions, including bilateral medial parietal-occipital-temporal lobes, right dorsolateral temporal lobes, bilateral parahippocampal gyrus, and bilateral hippocampus, compared to HC and PDND groups. A negative correlation between the GMV of left precuneus and HAMD-17 scores in the PDD group tended to be significant (r = -0.318, p = 0.059). Decreased gyrification index was observed in the bilateral insular and dorsolateral temporal cortex. However, there were no significant differences found in fractal dimension and sulcal depth. Our research shows extensive cortical structural changes in the insular cortex, parietal-occipital-temporal lobes, and hippocampal regions in PDD. This provides a morphological perspective for understanding the pathophysiological mechanism underlying depression in Parkinson's disease.

Aging of Amateur Singers and Non-singers: From Behavior to Resting-state Connectivity.

Healthy aging is associated with extensive changes in brain structure and physiology, with impacts on cognition and communication. The "mental exercise hypothesis" proposes that certain lifestyle factors such as singing-perhaps the most universal and accessible music-making activity-can affect cognitive functioning and reduce cognitive decline in aging, but the neuroplastic mechanisms involved remain unclear. To address this question, we examined the association between age and resting-state functional connectivity (RSFC) in 84 healthy singers and nonsingers in five networks, auditory, speech, language, default mode, and dorsal attention, and its relationship to auditory cognitive aging. Participants underwent cognitive testing and fMRI. Our results show that RSFC is not systematically lower with aging and that connectivity patterns vary between singers and nonsingers. Furthermore, our results show that RSFC of the precuneus in the default mode network was associated with auditory cognition. In these regions, lower RSFC was associated with better auditory cognitive performance for both singers and nonsingers. Our results show, for the first time, that basic brain physiology differs in singers and nonsingers and that some of these differences are associated with cognitive performance.

Development of white matter fiber covariance networks supports executive function in youth

During adolescence, the brain undergoes extensive changes in white matter structure that support cognition. Data-driven approaches applied to cortical surface properties have led the field to understand brain development as a spatially and temporally coordinated mechanism that follows hierarchically organized gradients of change. Although white matter development also appears asynchronous, previous studies have relied largely on anatomical tract-based atlases, precluding a direct assessment of how white matter structure is spatially and temporally coordinated. Harnessing advances in diffusion modeling and machine learning, we identified 14 data-driven patterns of covarying white matter structure in a large sample of youth. Fiber covariance networks aligned with known major tracts, while also capturing distinct patterns of spatial covariance across distributed white matter locations. Most networks showed age-related increases in fiber network properties, which were also related to developmental changes in executive function. This study delineates data-driven patterns of white matter development that support cognition.

Two-photon Absorption and Photoionization of a Bacterial Phytochrome

Phytochromes constitute a family of photosensory proteins that are utilized by various organisms to regulate several physiological processes. Phytochromes bind a bilin pigment that switches its isomeric state upon absorption of red or far-red photons, resulting in protein conformational changes that are sensed by the organism. Previously, the ultrafast dynamics in bacterial phytochrome was resolved to atomic resolution by time-resolved serial femtosecond X-ray diffraction (TR-SFX), showing extensive changes in its molecular conformation at 1 picosecond delay time. However, the large excitation fluence of mJ/mm2 used in TR-SFX questions the validity of the observed dynamics. In this work, we present an excitation-dependent ultrafast transient absorption study to test the response of a related bacterial phytochrome to excitation fluence. We observe excitation power-dependent sub-picosecond dynamics, assigned to the population of high-lying excited state Sn through resonantly enhanced two-photon absorption, followed by rapid internal conversion to the low-lying S1 state. Inspection of the long-lived spectrum under high fluence shows that in addition to the primary intermediate Lumi-R, spectroscopic signatures of solvated electrons and ionized chromophore radicals are observed. Supported by numerical modelling, we propose that under excitation fluences of tens of μJ/mm2 and higher, bacterial phytochrome partly undergoes photoionization from the Sn state in competition with internal conversion to the S1 state in 300 fs. We suggest that the extensive structural changes of related, shorter bacterial phytochrome, lacking the PHY domain, resolved from TR-SFX may have been affected by the ionized species. We propose approaches to minimize the two-photon absorption process by tuning the excitation spectrum away from the S1 absorption or using phytochromes exhibiting minimized or shifted S1 absorption.

KDM6A/UTX promotes spermatogenic gene expression across generations and is not required for male fertility†.

Paternal chromatin undergoes extensive structural and epigenetic changes during mammalian spermatogenesis, producing sperm with an epigenome optimized for the transition to embryogenesis. Lysine demethylase 6a (KDM6A, also called UTX) promotes gene activation in part via demethylation of H3K27me3, a developmentally important repressive modification abundant throughout the epigenome of spermatogenic cells and sperm. We previously demonstrated increased cancer risk in genetically wild-type mice derived from a paternal germ line lacking Kdm6a (Kdm6a cKO), indicating a role for KDM6A in regulating heritable epigenetic states. However, the regulatory function of KDM6A during spermatogenesis is not known. Here, we show that Kdm6a is transiently expressed in spermatogenesis, with RNA and protein expression largely limited to late spermatogonia and early meiotic prophase. Kdm6a cKO males do not have defects in fertility or the overall progression of spermatogenesis. However, hundreds of genes are deregulated upon loss of Kdm6a in spermatogenic cells, with a strong bias toward downregulation coinciding with the time when Kdm6a is expressed. Misregulated genes encode factors involved in chromatin organization and regulation of repetitive elements, and a subset of these genes was persistently deregulated in the male germ line across two generations of offspring of Kdm6a cKO males. Genome-wide epigenetic profiling revealed broadening of H3K27me3 peaks in differentiating spermatogonia of Kdm6a cKO mice, suggesting that KDM6A demarcates H3K27me3 domains in the male germ line. Our findings highlight KDM6A as a transcriptional activator in the mammalian male germ line that is dispensable for spermatogenesis but important for safeguarding gene regulatory state intergenerationally.

Pro-ecological and conservation activities are not always beneficial to nature: a case study of two lowland streams in Central Europe

Since 1990 and in particular, after the implementation of the Water Frame Directive, many positive effects of pro-ecological projects are evident; unfortunately, examples of adverse effects have also been observed. This study aims to indicate how some ill-considered actions, called “pro-ecological”, may lead to habitat degradation and the disappearance of valuable hydrobiont species. Two watercourses, representing the lowland gravel stream and sandy stream type, were selected for the study. Literature indicated that in the past, these watercourses were characterized by an excellent ecological status and the presence of valuable rheophilic fauna and flora. Environmental parameters were recorded, macroinvertebrates and ichthyofauna were sampled and analyzed, and finally, indexes were calculated. The results were compared with literature data. In the course of studies conducted in 2011–2015, drastic habitat deterioration and extensive changes in the species structure of ichthyofauna and aquatic invertebrates were observed. Changes in the Smolnica stream have been caused by the three retention basins constructed in 2000, along the lower and middle course; while in Kiszewko, however, the factor for habitat deterioration was connected with the excessive expansion of the Eurasian beaver (Castor fiber), which created a beaver pond 20 m in width, with impoundment elevations of up to 2 m.

Predicting Mutation-Induced Changes in the Electronic Properties of Photosynthetic Proteins from First Principles: The Fenna-Matthews-Olson Complex Example.

Multiscale molecular modeling is utilized to predict optical absorption and circular dichroism spectra of two single-point mutants of the Fenna-Matthews-Olson photosynthetic pigment-protein complex. The modeling approach combines classical molecular dynamics simulations with structural refinement of photosynthetic pigments and calculations of their excited states in a polarizable protein environment. The only experimental input to the modeling protocol is the X-ray structure of the wild-type protein. The first-principles modeling reproduces changes in the experimental optical spectra of the considered mutants, Y16F and Q198V. Interestingly, the Q198V mutation has a negligible effect on the electronic properties of the targeted bacteriochlorophyll a pigment. Instead, the electronic properties of several other pigments respond to this mutation. The molecular modeling demonstrates that a single-point mutation can induce long-range effects on the protein structure, while extensive structural changes near a pigment do not necessarily lead to significant changes in the electronic properties of that pigment.

Oxidation-driven lignin removal by Agaricus bisporus from wheat straw-based compost at industrial scale

Fungi are main lignin degraders and the edible white button mushroom, Agaricus bisporus, inhabits lignocellulose-rich environments. Previous research hinted at delignification when A. bisporus colonized pre-composted wheat straw-based substrate in an industrial setting, assumed to aid subsequent release of monosaccharides from (hemi-)cellulose to form fruiting bodies. Yet, structural changes and specific quantification of lignin throughout A. bisporus mycelial growth remain largely unresolved. To elucidate A. bisporus routes of delignification, at six timepoints throughout mycelial growth (15 days), substrate was collected, fractionated, and analyzed by quantitative pyrolysis-GC–MS, 2D-HSQC NMR, and SEC. Lignin decrease was highest between day 6 and day 10 and reached in total 42 % (w/w). The substantial delignification was accompanied by extensive structural changes of residual lignin, including increased syringyl to guaiacyl (S/G) ratios, accumulated oxidized moieties, and depleted intact interunit linkages. Hydroxypropiovanillone and hydroxypropiosyringone (HPV/S) subunits accumulated, which are indicative for β-|O-4′ ether cleavage and imply a laccase-driven ligninolysis. We provide compelling evidence that A. bisporus is capable of extensive lignin removal, have obtained insights into mechanisms at play and susceptibilities of various substructures, thus we were contributing to understanding fungal lignin conversion.

Insight in changes in starch and proteins molecular structure of non-wheat cereal flours influenced by roasting and extrusion treatments

Barley, rye, triticale, oat, sorghum and millet flours, subjected to roasting or extrusion treatment, were analyzed by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) and Raman spectroscopy with aim to determine structural changes within starch and protein molecules that caused specific rheological properties of heat treated flours. Mixolab was used to determine how changes in starch and protein structure are reflected in rheological properties of dough. Extrusion treatment caused more extensive changes both in starch and protein structure than roasting treatment. Changes of protein secondary structure from α-helix to β-turn aggregated by hydrogen bonds, formed mainly between tyrosyl groups, as well as change in disulphide bond conformation, were more pronounced in extruded flour samples. Changes in the starch structure were also more extensive in extruded flours samples, including complete gelatinization and formation of amylose-lipid complexes. The results obtained by Mixolab showed that all extruded doughs showed resistance at C2 point, which was shifted to approximately 30 min, indicating higher mechanical resistance. It was supposed that this change in dough behaviour is related to protein polymerization during extrusion treatment and starch aggregation during roasting treatment.

Study of gray matter atrophy pattern with subcortical ischemic vascular disease-vascular cognitive impairment no dementia based on structural magnetic resonance imaging.

This study explored the structural imaging changes in patients with subcortical ischemic vascular disease (SIVD)-vascular cognitive impairment no dementia (VCIND) and the correlation between the changes in gray matter volume and the field of cognitive impairment to provide new targets for early diagnosis and treatment. Our study included 15 patients with SIVD-normal cognitive impairment (SIVD-NCI), 63 with SIVD-VCIND, 26 with SIVD-vascular dementia (SIVD-VD), and 14 normal controls (NC). T1-weighted images of all participants were collected, and DPABI and SPM12 software were used to process the gray matter of the four groups based on voxels. Fisher's exact test, one-way ANOVA and Kruskal-Wallis H test were used to evaluate all clinical and demographic data and compare the characteristics of diencephalic gray matter atrophy in each group. Finally, the region of interest (ROI) of the SIVD-VCIND was extracted, and Pearson correlation analysis was performed between the ROI and the results of the neuropsychological scale. Compared to the NC, changes in gray matter atrophy were observed in the bilateral orbitofrontal gyrus, right middle temporal gyrus, superior temporal gyrus, and precuneus in the SIVD-VCIND. Gray matter atrophy was observed in the left cerebellar region 6, cerebellar crural region 1, bilateral thalamus, right precuneus, and calcarine in the SIVD-VD. Compared with the SIVD-VCIND, gray matter atrophy changes were observed in the bilateral thalamus in the SIVD-VD (p < 0.05, family-wise error corrected). In the SIVD-VCIND, the total gray matter volume, bilateral medial orbital superior frontal gyrus, right superior temporal gyrus, middle temporal gyrus, and precuneus were positively correlated with Boston Naming Test score, whereas the total gray matter volume, right superior temporal gyrus, and middle temporal gyrus were positively correlated with overall cognition. Structural magnetic resonance imaging can detect extensive and subtle structural changes in the gray matter of patients with SIVD-VCIND and SIVD-VD, providing valuable evidences to explain the pathogenesis of subcortical vascular cognitive impairment and contributing to the early diagnosis of SIVD-VCIND and early warning of SIVD-VD.

Neural correlates of control over pain in fibromyalgia patients

The perceived lack of control over the experience of pain is arguably-one major cause of agony and impaired life quality in patients with chronic pain disorders as fibromyalgia (FM). The way perceived control affects subjective pain as well as the underlying neural mechanisms have so far not been investigated in chronic pain. We used functional magnetic resonance imaging (fMRI) to examine the neural correlates of self-controlled compared to computer-controlled heat pain in healthy controls (HC, n = 21) and FM patients (n = 23). Contrary to HC, FM failed to activate brain areas usually involved in pain modulation as well as reappraisal processes (right ventrolateral (VLPFC), dorsolateral prefrontal cortex (DLPFC) and dorsal anterior cingulate cortex (dACC)). Computer-controlled (compared to self-controlled) heat revealed significant activations of the orbitofrontal cortex (OFC) in HC, whereas FM activated structures that are typically involved in neural emotion processing (amygdala, parahippocampal gyrus). Additionally, FM displayed disrupted functional connectivity (FC) of the VLPFC, DLPFC and dACC with somatosensory and pain (inhibition)-related areas during self-controlled heat stimulation as well as significantly decreased gray matter (GM) volumes compared to HC in DLPFC and dACC. The described functional and structural changes provide evidence for far-reaching impairments concerning pain-modulatory processes in FM. Our investigation represents a first demonstration of dysfunctional neural pain modulation through experienced control in FM according to the extensive functional and structural changes in relevant sensory, limbic and associative brain areas. These areas may be targeted in clinical pain therapeutic methods involving TMS, neurofeedback or cognitive behavioral trainings.

New insights into the effect of mutations on affibody-Fc interaction, a molecular dynamics simulation approach

Staphylococcal protein A (SpA) domain B (the basis of affibody) has been widely used in affinity chromatography and found therapeutic applications against inflammatory diseases through targeting the Fc part of immunoglobulin G (IgG). We have performed extensive molecular dynamics simulation of 41 SpA mutants and compared their dynamics and conformations to wild type. The simulations revealed the molecular details of structural and dynamics changes that occurred due to introducing point mutations and helped to explain the SPR results. It was observed in some variants a point mutation caused extensive structural changes far from the mutation site, while an effect of some other mutations was limited to the site of the mutated residue. Also, the pattern of hydrogen bond networks and hydrophobic core arrangements were investigated. We figured out mutations that occurred at positions 128, 136, 150 and 153, affected two hydrophobic cores at the interface as well as mutations introduced at positions 129 and 154 interrupted two hydrogen bond networks of the interface, SPR data showed all of these mutations reduced binding affinity significantly. Overall, by scanning the SpA-Fc interface through the large numbers of introduced mutations, the new insights have been gained which would help to design high- affinity ligands of IgG.

Structural evolution of Delta lineage of SARS-CoV-2

One of the main obstacles in prevention and treatment of COVID-19 is the rapid evolution of the SARS-CoV-2 Spike protein. Given that Spike is the main target of common treatments of COVID-19, mutations occurring at this virulent factor can affect the effectiveness of treatments. The B.1.617.2 lineage of SARS-CoV-2, being characterized by many Spike mutations inside and outside of its receptor-binding domain (RBD), shows high infectivity and relative resistance to existing cures. Here, utilizing a wide range of computational biology approaches, such as immunoinformatics, molecular dynamics (MD), analysis of intrinsically disordered regions (IDRs), protein-protein interaction analyses, residue scanning, and free energy calculations, we examine the structural and biological attributes of the B.1.617.2 Spike protein. Furthermore, the antibody design protocol of Rosetta was implemented for evaluation the stability and affinity improvement of the Bamlanivimab (LY-CoV55) antibody, which is not capable of interactions with the B.1.617.2 Spike. We observed that the detected mutations in the Spike of the B1.617.2 variant of concern can cause extensive structural changes compatible with the described variation in immunogenicity, secondary and tertiary structure, oligomerization potency, Furin cleavability, and drug targetability. Compared to the Spike of Wuhan lineage, the B.1.617.2 Spike is more stable and binds to the Angiotensin-converting enzyme 2 (ACE2) with higher affinity.

Site-Directed Spin Labeling EPR Spectroscopy for Determination of Albumin Structural Damage and Hypoalbuminemia in Critical COVID-19.

The main factors in the COVID-19 pathology, which can initiate extensive structural changes at the cellular and molecular levels, are the generation of free radicals in abnormal amounts, and oxidative stress. Under "oxidative shock" conditions, the proteins undergo various modifications that affect their function and activity, and as a result distribute malfunctioning protein derivatives in the body. Human serum albumin is a small globular protein characterized by a high overall binding capacity for neutral lipophilic and acidic dosage forms. The albumin concentration is crucial for the maintenance of plasma oncotic pressure, the transport of nutrients, amino acids, and drugs, the effectiveness of drug therapy, and the prevention of drug toxicity. Hypoalbuminemia and structural defects molecule in the protein suggest a risk of changed metabolism and increased plasma concentration of unbound drugs. Therefore, the albumin structural and functional changes accompanied by low protein levels can be a serious prerequisite for ineffective therapy, frequent complications, and high mortality in patients with SARS-CoV-2 infection. The current opinion aims the research community the application of Site-Directed Spin Labeling Electron Paramagnetic Resonance spectroscopy (SDSL-EPR) and 3-Maleimido-PROXYL radical in determining abnormalities of the albumin dynamics and protein concentrations in COVID-19 critical patients.

Tourist accommodation, consumption and platforms

AbstractThe expansion of mediated accommodation on peer to peer (P2P) platforms, such as Airbnb, has generated extensive economic impact and structural changes in all the destinations involved. This study proposes an innovative analysis which estimates the economic impact associated with the expenditure of tourists staying in traditional hotel establishments in comparison to the impact of those staying in tourist housing mediated through P2P platforms. This research analyses fieldwork based on 1343 surveys carried out in the city of Granada, one of the main tourist destinations in Spain. Through the application of the input–output methodology we found that tourists staying in tourist housing mediated through P2P platforms generate a greater impact as a consequence of longer average stays and more heterogeneously distributed consumption. Their average expenditure is similar to that of tourists in hotels, but the indirect impact generated is greater. Consequently, we can better comprehend the economic impact associated with these platforms and their real effect. Public planners have to consider this information as part of the regulation and restriction of this activity.

NSP4 and ORF9b of SARS-CoV-2 Induce Pro-Inflammatory Mitochondrial DNA Release in Inner Membrane-Derived Vesicles.

Circulating cell-free mitochondrial DNA (cf-mtDNA) has been found in the plasma of severely ill COVID-19 patients and is now known as a strong predictor of mortality. However, the underlying mechanism of mtDNA release is unexplored. Here, we show a novel mechanism of SARS-CoV-2-mediated pro-inflammatory/pro-apoptotic mtDNA release and a rational therapeutic stem cell-based approach to mitigate these effects. We systematically screened the effects of 29 SARS-CoV-2 proteins on mitochondrial damage and cell death and found that NSP4 and ORF9b caused extensive mitochondrial structural changes, outer membrane macropore formation, and the release of inner membrane vesicles loaded with mtDNA. The macropore-forming ability of NSP4 was mediated through its interaction with BCL2 antagonist/killer (BAK), whereas ORF9b was found to inhibit the anti-apoptotic member of the BCL2 family protein myeloid cell leukemia-1 (MCL1) and induce inner membrane vesicle formation containing mtDNA. Knockdown of BAK and/or overexpression of MCL1 significantly reversed SARS-CoV-2-mediated mitochondrial damage. Therapeutically, we engineered human mesenchymal stem cells (MSCs) with a simultaneous knockdown of BAK and overexpression of MCL1 (MSCshBAK+MCL1) and named these cells IMAT-MSCs (intercellular mitochondrial transfer-assisted therapeutic MSCs). Upon co-culture with SARS-CoV-2-infected or NSP4/ORF9b-transduced airway epithelial cells, IMAT-MSCs displayed functional intercellular mitochondrial transfer (IMT) via tunneling nanotubes (TNTs). The mitochondrial donation by IMAT-MSCs attenuated the pro-inflammatory and pro-apoptotic mtDNA release from co-cultured epithelial cells. Our findings thus provide a new mechanistic basis for SARS-CoV-2-induced cell death and a novel therapeutic approach to engineering MSCs for the treatment of COVID-19.

Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH

The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface-bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.

The Rhenish Coal-Mining Area—Assessing the Transformational Talents and Challenges of a Region in Fundamental Structural Change

This paper addresses the extensive structural changes of the Rhenish coal-mining area in Germany. Coal mining was and still is a relevant economic activity throughout Europe and is the focus of many political and societal debates, as well as research activities in the Rhenish coal-mining area. The project DAZWISCHEN followed the concept of evidence-based planning and therefore identified, by means of a GIS-based analysis, the structural changes within the Rhenish coal-mining area for the conflicting thematic clusters for settlement development and open space. Moreover, we investigated the complex multi-level governance that the region is characterized by. The results suggest an increased pressure on blue and green infrastructure by new urban development, especially in the northern part of the Rhenish coal-mining area. On the other hand, the southern part of the Rhenish coal-mining area will be more likely to undergo a process towards an increase in green infrastructure. Thus, the future development of the whole area is segregated in two different development trends in the north and south parts. The complex governance structure in the Rhenish coal-mining area requires an in-depth view of the ongoing working processes in the development of ideas and visions for regional mission statements of different planning areas, levels, and network partners in a real-life laboratory.

Local Conformational Constraint of Firefly Luciferase Can Affect the Energy of Bioluminescence and Enzyme Stability.

Conformational dynamics contribute importantly to enzyme catalysis, such that targeted conformational constraint may affect catalysis. Firefly luciferases undergo extensive structural change during catalysis; key residues form a hydrophobic pocket, excluding water and enabling maximally energetic light production. Point mutants almost always luminesce at longer wavelengths (lower energy) than the wild type. Conformational constraint, using dipeptide analogue 3 at a position critical for optimized excited state structure, produced luciferase emission at a shorter wavelength by ~10 nm. In comparison, introduction of conformationally constrained analogues 4, 5, or 7 afforded luciferases emitting at longer wavelengths, while a related unconstrained luciferase (analogue 6) exhibited wild-type emission. The constrained luciferases tested were more stable than the wild type. Protein modeling demonstrated that the "inside" or "outside" orientation of the conformationally constrained dipeptide led to the shorter or longer emission wavelength, respectively. More broadly, these results suggest that local conformational constraint can control specific elements of enzyme behavior, both in vitro and in vivo. This represents the first example of studying enzyme function by introducing conformationally constrained dipeptides at a specific protein position. The principles discovered here in luciferase modification will enable studies to control the wavelength emission and photophysical properties of modified luciferases.