A GFP Inspired 8-Methoxyquinoline-Derived Fluorescent Molecular Sensor for the Detection of Zn2+ by Two-Photon Microscopy.

An effective, GFP-inspired fluorescent Zn2+ sensor is developed for two-photon microscopy and related biological application that features an 8-methoxyquinoline moiety. Excellent photophysical characteristics including a 37-fold fluorescence enhancement with excitation and emission maxima at 440 nm and 505 nm, respectively, as well as a high two-photon cross-section of 73 GM at 880 nm are reported. Based on the experimental data, the relationship between the structure and properties was elucidated and explained backed up by DFT calculations, particularly the observed PeT phenomenon for the turn-on process. Biological validation and detailed experimental and theoretical characterization of the free and the zinc-bound compounds are presented.

A Molecular Hybrid of the GFP Chromophore and 2,2'-Bipyridine: An Accessible Sensor for Zn2+ Detection with Fluorescence Microscopy.

The few commercially available chemosensors and published probes for in vitro Zn2+ detection in two-photon microscopy are compromised by their flawed spectroscopic properties, causing issues in selectivity or challenging multistep syntheses. Herein, we present the development of an effective small molecular GFP chromophore-based fluorescent chemosensor with a 2,2'-bipyridine chelator moiety (GFZnP BIPY) for Zn2+ detection that has straightforward synthesis and uncompromised properties. Detailed experimental characterizations of the free and the zinc-bound compounds within the physiologically relevant pH range are presented. Excellent photophysical characteristics are reported, including a 53-fold fluorescence enhancement with excitation and emission maxima at 422 nm and 492 nm, respectively. A high two-photon cross section of 3.0 GM at 840 nm as well as excellent metal ion selectivity are reported. In vitro experiments on HEK 293 cell culture were carried out using two-photon microscopy to demonstrate the applicability of the novel sensor for zinc bioimaging.

Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

Evolution of reproductive modes in sharks and rays.

The ecological and life history drivers of the diversification of reproductive modes in early vertebrates are not fully understood. Sharks, rays and chimaeras (group Chondrichthyes) have an unusually diverse variety of reproductive modes and are thus an ideal group to test the factors driving the evolution of reproductive complexity. Here, using 960 species representing all major Chondrichthyes taxa, we reconstruct the evolution of their reproduction modes and investigate the ecological and life history predictors of reproduction. We show that the ancestral Chondrichthyes state was egg-laying and find multiple independent transitions between egg-laying and live-bearing via an intermediate state of yolk-only live-bearing. Using phylogenetically informed analysis, we also show that live-bearing species have larger body size and larger offspring than egg-laying species. In addition, live-bearing species are distributed over shallow to intermediate depths, while egg-layers are typically found in deeper waters. This suggests that live-bearing is more closely associated with pelagic, rather than demersal habitats. Taken together, using a basal vertebrate group as a model, we demonstrat how reproductive mode co-evolves with environmental conditions and life-history traits.

Survivin prevents the polycomb repressor complex 2 from methylating histone 3 lysine 27.

This study investigates the role of survivin in epigenetic control of gene transcription through interaction with the polycomb repressive complex 2 (PRC2). PRC2 is responsible for silencing gene expression by trimethylating lysine 27 on histone 3. We observed differential expression of PRC2 subunits in CD4+ T cells with varying levels of survivin expression, and ChIP-seq results indicated that survivin colocalizes with PRC2 along DNA. Inhibition of survivin resulted in a significant increase in H3K27 trimethylation, implying that survivin prevents PRC2 from functioning. Peptide microarray showed that survivin interacts with peptides from PRC2 subunits, and machine learning revealed that amino acid composition contains relevant information for predicting survivin interaction. NMR and BLI experiments supported the interaction of survivin with PRC2 subunit EZH2. Finally, protein-protein docking revealed that the survivin-EZH2 interaction interface overlaps with catalytic residues of EZH2, potentially inhibiting its H3K27 methylation activity. These findings suggest that survivin inhibits PRC2 function.

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging.

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

Measuring and Modelling Structural Colours of Euphaedra neophron (Lepidoptera: Nymphalidae) Finely Tuned by Wing Scale Lower Lamina in Various Subspecies.

The nymphalid butterfly Euphaedra neophron (Hopffer, 1855) is the only structurally coloured species representing the genus along the Indian Ocean coast in East Africa and Southern Africa, with a distribution from southern Somalia to the Kwa-Zulu-Natal region of South Africa. The range of E. neophron is subdivided to several, geographically distinct populations, currently recognised as subspecies by taxonomists on the basis of violet, blue, and green-coloured morphs. We investigated the optical mechanism of all these morphs by various materials science techniques. We found that the structural colour is generated by the lower lamina of the cover scales and the different colours are tuned according to their thickness, which was also proved by modelling. The colour tuning of the different subspecies does not reflect any clinal pattern, be it geographical or altitudinal.

Survivin promotes a glycolytic switch in CD4+ T cells by suppressing the transcription of PFKFB3 in rheumatoid arthritis.

In this study, we explore the role of nuclear survivin in maintaining the effector phenotype of IFNγ-producing T cells acting through the transcriptional control of glucose utilization. High expression of survivin in CD4+T cells was associated with IFNγ-dependent phenotype and anaerobic glycolysis. Transcriptome of CD4+ cells and sequencing of survivin-bound chromatin showed that nuclear survivin had a genome-wide and motif-specific binding to regulatory regions of the genes controlling cell metabolism. Survivin coprecipitates with transcription factors IRF1 and SMAD3, which repressed the transcription of the metabolic check-point enzyme phosphofructokinase 2 gene PFKFB3 and promoted anaerobic glycolysis. Combining transcriptome analyses of CD4+ cells and functional studies in glucose metabolism, we demonstrated that the inhibition of survivin reverted PFKFB3 production, inhibited glucose uptake, and reduces interferon effects in CD4+ cells. These results present a survivin-dependent mechanism in coordinating the metabolic adaptation of CD4+T cells and propose an attractive strategy to counteract IFNγ-dependent inflammation in autoimmunity.

Cortex-wide response mode of VIP-expressing inhibitory neurons by reward and punishment.

Neocortex is classically divided into distinct areas, each specializing in different function, but all could benefit from reinforcement feedback to inform and update local processing. Yet it remains elusive how global signals like reward and punishment are represented in local cortical computations. Previously, we identified a cortical neuron type, vasoactive intestinal polypeptide (VIP)-expressing interneurons, in auditory cortex that is recruited by behavioral reinforcers and mediates disinhibitory control by inhibiting other inhibitory neurons. As the same disinhibitory cortical circuit is present virtually throughout cortex, we wondered whether VIP neurons are likewise recruited by reinforcers throughout cortex. We monitored VIP neural activity in dozens of cortical regions using three-dimensional random access two-photon microscopy and fiber photometry while mice learned an auditory discrimination task. We found that reward and punishment during initial learning produce rapid, cortex-wide activation of most VIP interneurons. This global recruitment mode showed variations in temporal dynamics in individual neurons and across areas. Neither the weak sensory tuning of VIP interneurons in visual cortex nor their arousal state modulation was fully predictive of reinforcer responses. We suggest that the global response mode of cortical VIP interneurons supports a cell-type-specific circuit mechanism by which organism-level information about reinforcers regulates local circuit processing and plasticity.

Sharp-wave ripple doublets induce complex dendritic spikes in parvalbumin interneurons in vivo.

Neuronal plasticity has been shown to be causally linked to coincidence detection through dendritic spikes (dSpikes). We demonstrate the existence of SPW-R-associated, branch-specific, local dSpikes and their computational role in basal dendrites of hippocampal PV+ interneurons in awake animals. To measure the entire dendritic arbor of long thin dendrites during SPW-Rs, we used fast 3D acousto-optical imaging through an eccentric deep-brain adapter and ipsilateral local field potential recording. The regenerative calcium spike started at variable, NMDA-AMPA-dependent, hot spots and propagated in both direction with a high amplitude beyond a critical distance threshold (~150 µm) involving voltage-gated calcium channels. A supralinear dendritic summation emerged during SPW-R doublets when two successive SPW-R events coincide within a short temporal window (~150 ms), e.g., during more complex association tasks, and generated large dSpikes with an about 2.5-3-fold amplitude increase which propagated down to the soma. Our results suggest that these doublet-associated dSpikes can work as a dendritic-level temporal and spatial coincidence detector during SPW-R-related network computation in awake mice.

The evolution of parental care in salamanders.

Complex parenting has been proposed to contribute to the evolutionary success of vertebrates. However, the evolutionary routes to complex parenting and the role of parenting in vertebrate diversity are still contentious. Although basal vertebrates provide clues to complex reproduction, these are often understudied. Using 181 species that represent all major lineages of an early vertebrate group, the salamanders and newts (Caudata, salamanders henceforth) here we show that fertilisation mode is tied to parental care: male-only care occurs in external fertilisers, whereas female-only care exclusively occurs in internal fertilisers. Importantly, internal fertilisation opens the way to terrestrial reproduction, because fertilised females are able to deposit their eggs on land, and with maternal care provision, the eggs could potentially develop outside the aquatic environment. Taken together, our results of a semi-aquatic early vertebrate group propose that the diversity and follow-up radiation of terrestrial vertebrates are inherently associated with a complex social behaviour, parenting.

Cohesin-Mediated Chromatin Interactions and Autoimmunity.

Proper physiological functioning of any cell type requires ordered chromatin organization. In this context, cohesin complex performs important functions preventing premature separation of sister chromatids after DNA replication. In partnership with CCCTC-binding factor, it ensures insulator activity to organize enhancers and promoters within regulatory chromatin. Homozygous mutations and dysfunction of individual cohesin proteins are embryonically lethal in humans and mice, which limits in vivo research work to embryonic stem cells and progenitors. Conditional alleles of cohesin complex proteins have been generated to investigate their functional roles in greater detail at later developmental stages. Thus, genome regulation enabled by action of cohesin proteins is potentially crucial in lineage cell development, including immune homeostasis. In this review, we provide current knowledge on the role of cohesin complex in leukocyte maturation and adaptive immunity. Conditional knockout and shRNA-mediated inhibition of individual cohesin proteins in mice demonstrated their importance in haematopoiesis, adipogenesis and inflammation. Notably, these effects occur rather through changes in transcriptional gene regulation than through expected cell cycle defects. This positions cohesin at the crossroad of immune pathways including NF-kB, IL-6, and IFNγ signaling. Cohesin proteins emerged as vital regulators at early developmental stages of thymocytes and B cells and after antigen challenge. Human genome-wide association studies are remarkably concordant with these findings and present associations between cohesin and rheumatoid arthritis, multiple sclerosis and HLA-B27 related chronic inflammatory conditions. Furthermore, bioinformatic prediction based on protein-protein interactions reveal a tight connection between the cohesin complex and immune relevant processes supporting the notion that cohesin will unearth new clues in regulation of autoimmunity.

Chemical Mapping Exposes the Importance of Active Site Interactions in Governing the Temperature Dependence of Enzyme Turnover.

Uncovering the role of global protein dynamics in enzyme turnover is needed to fully understand enzyme catalysis. Recently, we have demonstrated that the heat capacity of catalysis, ΔC P ‡, can reveal links between the protein free energy landscape, global protein dynamics, and enzyme turnover, suggesting that subtle changes in molecular interactions at the active site can affect long-range protein dynamics and link to enzyme temperature activity. Here, we use a model promiscuous enzyme (glucose dehydrogenase from Sulfolobus solfataricus) to chemically map how individual substrate interactions affect the temperature dependence of enzyme activity and the network of motions throughout the protein. Utilizing a combination of kinetics, red edge excitation shift (REES) spectroscopy, and computational simulation, we explore the complex relationship between enzyme-substrate interactions and the global dynamics of the protein. We find that changes in ΔC P ‡ and protein dynamics can be mapped to specific substrate-enzyme interactions. Our study reveals how subtle changes in substrate binding affect global changes in motion and flexibility extending throughout the protein.

Theoretical Design, Synthesis, and In Vitro Neurobiological Applications of a Highly Efficient Two-Photon Caged GABA Validated on an Epileptic Case.

In this paper, we present an additional, new cage-GABA compound, called 4-amino-1-(4'-dimethylaminoisopropoxy-5',7'-dinitro-2',3'-dihydro-indol-1-yl)-1-oxobutane-γ-aminobutyric acid (iDMPO-DNI-GABA), and currently, this compound is the only photoreagent, which can be applied for GABA uncaging without experimental compromises. By a systematic theoretical design and successful synthesis of several compounds, the best reagent exhibits a high two-photon efficiency within the 700-760 nm range with excellent pharmacological behavior, which proved to be suitable for a complex epileptic study. Quantum chemical design showed that the optimal length of the cationic side chain enhances the two-photon absorption by 1 order of magnitude due to the cooperating internal hydrogen bonding to the extra nitro group on the core. This feature increased solubility while suppressing membrane permeability. The efficiency was demonstrated in a systematic, wide range of in vitro single-cell neurophysiological experiments by electrophysiological as well as calcium imaging techniques. Scalable inhibitory ion currents were elicited by iDMPO-DNI-GABA with appropriate spatial-temporal precision, blocking both spontaneous and evoked cell activity with excellent efficiency. Additionally, to demonstrate its applicability in a real neurobiological study, we could smoothly and selectively modulate neuronal activities during artificial epileptic rhythms first time in a neural network of GCaMP6f transgenic mouse brain slices.

Are evolutionary transitions in sexual size dimorphism related to sex determination in reptiles?

Sex determination systems are highly variable in vertebrates, although neither the causes nor the implications of this diversity are fully understood. Theory suggests that sex determination is expected to relate to sexual size dimorphism, because environmental sex determination promotes sex-specific developmental bias in embryonic growth rates. Furthermore, selection for larger size in one sex or the other has been proposed to drive the evolution of different genetic sex determination systems. Here, we investigate whether sex determination systems relate to adult sexual size dimorphism, using 250 species of reptiles (Squamata, Testudines and Crocodylia) representing 26 families. Using phylogenetically informed analyses, we find that sexual size dimorphism is associated with sex determination: species with TSDIa sex determination (i.e. in which the proportion of female offspring increases with incubation temperature) have more female-biased size dimorphism than species with TSDII (i.e. species in which males are produced at mid temperatures). We also found a trend that species with TSD ancestors had more male-biased size dimorphism in XY sex chromosome systems than in ZW sex chromosome systems. Taken together, our results support the prediction that sexual size dimorphism is linked to sex-dependent developmental variations caused by environmental factors and also by sex chromosomes. Since the extent of size dimorphism is related to various behavioural, ecological and life-history differences between sexes, our results imply profound impacts of sex determination systems for vertebrate diversity.

High-resolution macromolecular crystallography at the FemtoMAX beamline with time-over-threshold photon detection.

Protein dynamics contribute to protein function on different time scales. Ultrafast X-ray diffraction snapshots can visualize the location and amplitude of atom displacements after perturbation. Since amplitudes of ultrafast motions are small, high-quality X-ray diffraction data is necessary for detection. Diffraction from bovine trypsin crystals using single femtosecond X-ray pulses was recorded at FemtoMAX, which is a versatile beamline of the MAX IV synchrotron. The time-over-threshold detection made it possible that single photons are distinguishable even under short-pulse low-repetition-rate conditions. The diffraction data quality from FemtoMAX beamline enables atomic resolution investigation of protein structures. This evaluation is based on the shape of the Wilson plot, cumulative intensity distribution compared with theoretical distribution, I/σ, Rmerge/Rmeas and CC1/2 statistics versus resolution. The FemtoMAX beamline provides an interesting alternative to X-ray free-electron lasers when studying reversible processes in protein crystals.

Long-term temporal patterns in flight activities of a migrant diurnal butterfly.

Recent studies demonstrated that the Painted Lady (Vanessa cardui), a cosmopolitan diurnal butterfly performs long-range migration between subtropical Africa and north-western Europe, covered by individuals belonging to up to six generations. Here we analyze temporal patterns of complete annual migratory activity of the Painted Lady in Hungary, located in its Central European migratory route, almost completely unstudied before. To do so, we used field occurrence data collected between 2000 and 2019 and estimated temporal patterns in migratory activity by fitting kernel density functions on the daily mean number of individuals and observation frequency. The temporal distributions of kernel density estimates were analyzed as a function of time and key climatic predictors of the study area. We found that (i) the timing of spring arrivals has been advancing; (ii) the relative intensity of the first and last migratory peaks of the Painted Lady significantly increased during the past decades; and (iii) intensity of the last migratory peak is related to the mean temperature of the previous month, inferring that the migration is shifting to earlier dates and their volume of the migration has substantially intensified, evoking mutually nonexclusive, competing hypotheses. Our study indicates the strengthening migration activities of a southerly distributed, long-distance migrant diurnal butterfly, most probably linked to the northward shift of wintering areas induced by warming trends of the southern parts of Europe. However, the complexity of the likely processes leading to changing migratory strategies calls up for further research in both breeding and wintering areas.

Ultrafast structural changes within a photosynthetic reaction centre.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

A tool for visualizing protein motions in time-resolved crystallography.

Time-resolved serial femtosecond crystallography (TR-SFX) at an x-ray free electron laser enables protein structural changes to be imaged on time-scales from femtoseconds to seconds. It can, however, be difficult to grasp the nature and timescale of global protein motions when structural changes are not isolated near a single active site. New tools are, therefore, needed to represent the global nature of electron density changes and their correlation with modeled protein structural changes. Here, we use TR-SFX data from bacteriorhodopsin to develop and validate a method for quantifying time-dependent electron density changes and correlating them throughout the protein. We define a spherical volume of difference electron density about selected atoms, average separately the positive and negative electron difference densities within each volume, and walk this spherical volume through all atoms within the protein. By correlating the resulting difference electron density amplitudes with time, our approach facilitates an initial assessment of the number and timescale of structural intermediates and highlights quake-like motions on the sub-picosecond timescale. This tool also allows structural models to be compared with experimental data using theoretical difference electron density changes calculated from refined resting and photo-activated structures.