Rhizophagus intraradices and Azospirillum brasilense improve growth of herbaceous plants and soil biological activity in revegetation of a recovering coal-mining area.

We assessed, in a field experiment, the effects of arbuscular mycorrhizal fungi (Rhizophagus intraradices) and plant growth-promoting bacteria (Azospirillum brasilense) on the soil biological activity and the growth of key pioneer species used in the revegetation of coal-mining areas undergoing recovery. We applied four inoculation treatments to the pioneer plant species (Lablab purpureus, Paspalum notatum, Crotalaria juncea, Neonotonia wightii, Stylosanthes guianensis, Andropogon gayanus and Trifolium repens) used in the recovery process: NI (Control - Non-inoculated), AZO (A. brasilense), AMF (R. intraradices), and co-inoculation of AZO and AMF. On the 75th and 180th days, we measured plant dry mass, mycorrhizal colonization, N and P concentration, and accumulation in plant tissue. We collected soil to quantify glomalin content and soil enzyme activity. After 180 days, we did a phytosociological characterization of the remaining spontaneous plants.The both microorganisms, singly or co-inoculated, promoted increases in different fractions of soil glomalin, acid phosphatase activity, and fluorescein diacetate activity at 75 and 180 days. The inoculation was linked to higher plant biomass production (62-89%) and increased plant P and N accumulation by 34-75% and 70-85% at 180 days, compared with the non-inoculated treatment. Among the pioneer species sown Crotalaria juncea produced the highest biomass at the 75th and 180th days (67% and 76% of all biomass), followed by Lablab purpureus (3% and 0.5%), while the other species failed to establish. At 180 days, we observed twenty spontaneous plant species growing in the area, primarily from the Poaceae family (74%). That suggests that the pioneer species present in the area do not hinder the ecological succession process. Inoculation of R. intraradices and A. brasilense, isolated or combined, increases soil biological activity, growth, and nutrient accumulation in key pioneer plant species, indicating the potential of that technique for the recovery of lands degraded by coal mining.

Int&in: A machine learning-based web server for active split site identification in inteins.

Inteins are proteins that excise themselves out of host proteins and ligate the flanking polypeptides in an auto-catalytic process called protein splicing. In nature, inteins are either contiguous or split. In the case of split inteins, the two fragments must first form a complex for the splicing to occur. Contiguous inteins have previously been artificially split in two fragments because split inteins allow for distinct applications than contiguous ones. Even naturally split inteins have been split at unnatural split sites to obtain fragments with reduced affinity for one another, which are useful to create conditional inteins or to study protein-protein interactions. So far, split sites in inteins have been heuristically identified. We developed Int&in, a web server freely available for academic research (https://intein.biologie.uni-freiburg.de) that runs a machine learning model using logistic regression to predict active and inactive split sites in inteins with high accuracy. The model was trained on a dataset of 126 split sites generated using the gp41-1, Npu DnaE and CL inteins and validated using 97 split sites extracted from the literature. Despite the limited data size, the model, which uses various protein structural features, as well as sequence conservation information, achieves an accuracy of 0.79 and 0.78 for the training and testing sets, respectively. We envision Int&in will facilitate the engineering of novel split inteins for applications in synthetic and cell biology.

Modulation of the Excited States of Ruthenium(II)-perylene Dyad to Access Near-IR Luminescence, Long-Lived Perylene Triplet State and Singlet Oxygen Photosensitization.

Herein, we present a novel ruthenium(II)-perylene dyad (RuPDI-Py) that combines the photophysical properties of pyrrolidine-substituted perylene diimide (PDI-Py) and the ruthenium(II) polypyridine complex [Ru(phen)3]2+. A comprehensive study of excited-state dynamics was carried out using time-resolved and steady-state methods in a dimethyl sulfoxide solution. The RuPDI-Py dyad demonstrated excitation wavelength-dependent photophysical behavior. Upon photoexcitation above 600 nm, the dyad exclusively exhibits the near-infrared (NIR) fluorescence of the 1PDI-Py state at 785 nm (τfl = 1.50 ns). In contrast, upon photoexcitation between 350 and 450 nm, the dyad also exhibits a photoinduced electron transfer from the {[Ru(phen)3]2+} moiety to PDI-Py, generating the charge-separated intermediate state {Ru(III)-(PDI-Py)•-} (4 µs). This state subsequently decays to the long-lived triplet excited state 3PDI-Py (36 µs), which is able to sensitize singlet oxygen (1O2). Overall, tuning 1O2 photoactivation or NIR fluorescence makes RuPDI-Py a promising candidate for using absorbed light energy to perform the desired functions in theranostic applications.

iNClusive: a database collecting useful information on non-canonical amino acids and their incorporation into proteins for easier genetic code expansion implementation.

The incorporation of non-canonical amino acids (ncAAs) into proteins is a powerful technique used in various research fields. Genetic code expansion (GCE) is the most common way to achieve this: a specific codon is selected to be decoded by a dedicated tRNA orthogonal to the endogenous ones. In the past 30 years, great progress has been made to obtain novel tRNA synthetases (aaRSs) accepting a variety of ncAAs with distinct physicochemical properties, to develop robust in vitro assays or approaches for codon reassignment. This sparked the use of the technique, leading to the accumulation of publications, from which gathering all relevant information can appear daunting. Here we present iNClusive (https://non-canonical-aas.biologie.uni-freiburg.de/), a manually curated, extensive repository using standardized nomenclature that provides organized information on ncAAs successfully incorporated into target proteins as verified by mass spectrometry. Since we focused on tRNA synthetase-based tRNA loading, we provide the sequence of the tRNA and aaRS used for the incorporation. Derived from more than 687 peer-reviewed publications, it currently contains 2432 entries about 466 ncAAs, 569 protein targets, 500 aaRSs and 144 tRNAs. We foresee iNClusive will encourage more researchers to experiment with ncAA incorporation thus contributing to the further development of this exciting technique.

ReverseDock: a web server for blind docking of a single ligand to multiple protein targets using AutoDock Vina.

Several platforms exist to perform molecular docking to computationally predict binders to a specific protein target from a library of ligands. The reverse, that is, docking a single ligand to various protein targets, can currently be done by very few web servers, which limits the search to a small set of pre-selected human proteins. However, the possibility to in silico predict which targets a compound identified in a high-throughput drug screen bind would help optimize and reduce the costs of the experimental workflow needed to reveal the molecular mechanism of action of a ligand. Here, we present ReverseDock, a blind docking web server based on AutoDock Vina specifically designed to allow users with no computational expertise to dock a ligand to 100 protein structures of their choice. ReverseDock increases the number and type of proteins a ligand can be docked to, making the task of in silico docking of a ligand to entire families of proteins straightforward. We envision ReverseDock will support researchers by providing the possibility to apply inverse docking computations using web browser. ReverseDock is available at: https://reversedock.biologie.uni-freiburg.de/.

Cellulose acetate membranes loaded with combinations of tetraphenylporphyrin, graphene oxide and Pluronic F-127 as responsive materials with antibacterial photodynamic activity.

The development of polymeric fabrics with photoinduced antibacterial activity is important for different emerging applications, ranging from materials for medical and clinical practices to disinfection of objects for public use. In this work we prepared a series of cellulose acetate membranes, by means of phase inversion technique, introducing different additives in the starting polymeric solution. The loading of 5,10,15,20-tetraphenylporphyrin (TPP), a known photosensitizer, was considered to impart antibacterial photodynamic properties to the produced membranes. Besides, the addition of a surfactant (Pluronic F-127) allowed to modify the morphology of the membranes whereas the use of graphene oxide (GO) enabled further photo-activated antibacterial activity. The three additives were tested in various concentrations and in different combinations in order to carefully explore the effects of their mixing on the final photophysical and photodynamic properties. A complete structural/morphologycal characterization of the produced membranes has been performed, together with a detailed photophysical study of the TPP-containing samples, including absorption and emission features, excited state lifetime, singlet oxygen production, and confocal analysis. Their antibacterial activity has been assessed in vitro against S. aureus and E. coli, and the results demonstrated excellent bacterial inactivation for the membranes containing a combination of the three additives, revealing also a non-innocent role of the membrane porous structure in the final antibacterial capacity.

A historical perspective of biomedical explainable AI research.

The black-box nature of most artificial intelligence (AI) models encourages the development of explainability methods to engender trust into the AI decision-making process. Such methods can be broadly categorized into two main types: post hoc explanations and inherently interpretable algorithms. We aimed at analyzing the possible associations between COVID-19 and the push of explainable AI (XAI) to the forefront of biomedical research. We automatically extracted from the PubMed database biomedical XAI studies related to concepts of causality or explainability and manually labeled 1,603 papers with respect to XAI categories. To compare the trends pre- and post-COVID-19, we fit a change point detection model and evaluated significant changes in publication rates. We show that the advent of COVID-19 in the beginning of 2020 could be the driving factor behind an increased focus concerning XAI, playing a crucial role in accelerating an already evolving trend. Finally, we present a discussion with future societal use and impact of XAI technologies and potential future directions for those who pursue fostering clinical trust with interpretable machine learning models.

Elucidating the Excited State Behavior of Pyridyl Pyridinium Systems via Computational and Transient Absorption Studies of Tetrahedral Multichromophoric Arrays and their Model Compounds.

The tetrahedral shape-persistent molecule 14+ , containing four identical pyridyl pyridinium units connected via a sp3 hybridized carbon atom, has been investigated in detail by means of steady-state and time resolved spectroscopy. Remarkable photophysical properties are observed, particularly in comparison with protonated and methylated analogues (1H4 8+ , 1Me4 8+ ), which exhibit substantially shorter excited state lifetimes and lower emission quantum yields. Theoretical studies have rationalized the behavior of the tetrameric molecules relative to the monomers, with DFT and TD-DFT calculations corroborating steady-state (absorption and emission) and transient absorption spectra. The behavior of the monomeric compounds (each consisting in one of the four identical subunits of the tetramers, i. e., 2+ , 2H2+ and 2Me2+ ) considerably differs from that of the tetramers, indicating a strong electronic interaction between the subunits in the tetrameric species, likely promoted by the homoconjugation through the connecting sp3  C atom. 2+ is characterized by a peculiar S1 -S2 excited state inversion, whereas the short-lived emitting S1 state of 2H2+ and 2Me2+ exhibits a partial charge-transfer character, as substantiated by spectro-electrochemical studies. Among the six investigated systems, only 14+ is a sizeable luminophore (Φem =0.15), which is related to the peculiar features of its singlet state.

Non-Phenomenological Description of the Time-Resolved Emission in Solution with Quantum-Classical Vibronic Approaches-Application to Coumarin C153 in Methanol.

We report a joint experimental and theoretical work on the steady-state spectroscopy and time-resolved emission of the coumarin C153 dye in methanol. The lowest energy excited state of this molecule is characterized by an intramolecular charge transfer thus leading to remarkable shifts of the time-resolved emission spectra, dictated by the methanol reorganization dynamics. We selected this system as a prototypical test case for the first application of a novel computational protocol aimed at the prediction of transient emission spectral shapes, including both vibronic and solvent effects, without applying any phenomenological broadening. It combines a recently developed quantum-classical approach, the adiabatic molecular dynamics generalized vertical Hessian method (Ad-MD|gVH), with nonequilibrium molecular dynamics simulations. For the steady-state spectra we show that the Ad-MD|gVH approach is able to reproduce quite accurately the spectral shapes and the Stokes shift, while a ∼0.15 eV error is found on the prediction of the solvent shift going from gas phase to methanol. The spectral shape of the time-resolved emission signals is, overall, well reproduced, although the simulated spectra are slightly too broad and asymmetric at low energies with respect to experiments. As far as the spectral shift is concerned, the calculated spectra from 4 ps to 100 ps are in excellent agreement with experiments, correctly predicting the end of the solvent reorganization after about 20 ps. On the other hand, before 4 ps solvent dynamics is predicted to be too fast in the simulations and, in the sub-ps timescale, the uncertainty due to the experimental time resolution (300 fs) makes the comparison less straightforward. Finally, analysis of the reorganization of the first solvation shell surrounding the excited solute, based on atomic radial distribution functions and orientational correlations, indicates a fast solvent response (≈100 fs) characterized by the strengthening of the carbonyl-methanol hydrogen bond interactions, followed by the solvent reorientation, occurring on the ps timescale, to maximize local dipolar interactions.

Multi-Responsive Eight-State Bis(acridinium-Zn(II) porphyrin) Receptor.

A multi-responsive receptor consisting of two (acridinium-Zn(II) porphyrin) conjugates has been designed. The binding constant between this receptor and a ditopic guest has been modulated (i) upon addition of nucleophiles converting acridinium moieties into the non-aromatic acridane derivatives and (ii) upon oxidation of the porphyrin units. A total of eight states has been probed for this receptor resulting from the cascade of the recognition and responsive events. Moreover, the acridinium/acridane conversion leads to a significant change of the photophysical properties, switching from electron to energy transfer processes. Interestingly, for the bis(acridinium-Zn(II) porphyrin) receptor, charge-transfer luminescence in the near-infrared has been observed.

Requirements for mammalian promoters to decode transcription factor dynamics.

In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode dynamics. Here, we use optogenetics to directly manipulate the nuclear localization of a synthetic TF in mammalian cells without affecting other processes. We generate pulsatile or sustained TF dynamics and employ live cell microscopy and mathematical modelling to analyse the behaviour of a library of reporter constructs. We find decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Using the knowledge acquired, we build a synthetic circuit that allows obtaining two gene expression programs depending solely on TF dynamics. Finally, we show that some of the promoter features identified in our study can be used to distinguish natural promoters that have previously been experimentally characterized as responsive to either sustained or pulsatile p53 and NF-κB signals. These results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build complex synthetic circuits steered by TF dynamics.

Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells.

The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.

Antibacterial Photodynamic Therapy in the Near-Infrared Region with a Targeting Antimicrobial Peptide Connected to a π-Extended Porphyrin.

The increase of antimicrobial resistance to conventional antibiotics is worldwide a major health problem that requires the development of new bactericidal strategies. Antimicrobial photodynamic therapy (a-PDT) that generates reactive oxygen species acting on multiple cellular targets is unlikely to induce bacterial resistance. This localized treatment requires, for safe and efficient treatment of nonsuperficial infections, a targeting photosensitizer excited in the near IR. To this end, a new conjugate consisting of an antimicrobial peptide linked to a π-extended porphyrin photosensitizer was designed for a-PDT. Upon irradiation at 720 nm, the conjugate has shown at micromolar concentration strong bactericidal action on both Gram-positive and Gram-negative bacteria. Moreover, this conjugate allows one to reach a low minimum bactericidal concentration with near IR excitation without inducing toxicity to skin cells.

Novel Cu(I)-5-nitropyridine-2-thiol Cluster with NIR Emission: Structural and Photophysical Characterization.

A novel Cu(I) cluster compound has been synthesized by reacting CuI with the 2,2'-dithiobis(5-nitropyridine) ligand under solvothermal conditions. During the reaction, the original ligand breaks into the 5-nitropyridine-2-thiolate moiety, which acts as the coordinating ligand with both N- and S-sites, leading to a distorted octahedral Cu6S6 cluster. The structure has been determined by single-crystal X-ray diffraction and FT-IR analysis, and the photophysical properties have been determined in the solid state by means of steady-state and time-resolved optical techniques. The cluster presents a near-infrared emission showing an unusual temperature dependence: when passing from 77 to 298 K, a blue-shift of the emission band is observed, associated with a decrease in its intensity. Time-dependent-density functional theory calculations suggest that the observed behavior can be ascribed to a complex interplay of excited states, basically in the triplet manifold.

Synthetic microbiology applications powered by light.

Synthetic biology is a field of research in which molecular parts (mostly nucleic acids and proteins) are de novo created or modified and then used either alone or in combination to achieve new functions that can help solve the problems of our modern society. In synthetic microbiology, microbes are employed rather than other organisms or cell-free systems. Optogenetics, a relatively recently established technology that relies on the use of genetically encoded photosensitive proteins to control biological processes with high spatiotemporal precision, offers the possibility to empower synthetic (micro)biology applications due to the many positive features that light has as an external trigger. In this review, we describe recent synthetic microbiology applications that made use of optogenetics after briefly introducing the molecular mechanism behind some of the most employed optogenetic tools. We highlight the power and versatility of this technique, which opens up new horizons for both research and industry.

Experimental Characterization of In Silico Red-Shift-Predicted iLOVL470T/Q489K and iLOVV392K/F410V/A426S Mutants.

iLOV is a flavin mononucleotide-binding fluorescent protein used for in vivo cellular imaging similar to the green fluorescent protein. To expand the range of applications of iLOV, spectrally tuned red-shifted variants are desirable to reduce phototoxicity and allow for better tissue penetration. In this report, we experimentally tested two iLOV mutants, iLOVL470T/Q489K and iLOVV392K/F410V/A426S, which were previously computationally proposed by (KhrenovaJ. Phys. Chem. B2017, 121 ( (43), ), pp 10018-10025) to have red-shifted excitation and emission spectra. While iLOVL470T/Q489K is about 20% brighter compared to the WT in vitro, it exhibits a blue shift in contrast to quantum mechanics/molecular mechanics (QM/MM) predictions. Additional optical characterization of an iLOVV392K mutant revealed that V392 is essential for cofactor binding and, accordingly, variants with V392K mutation are unable to bind to FMN. iLOVL470T/Q489K and iLOVV392K/F410V/A426S are expressed at low levels and have no detectable fluorescence in living cells, preventing their utilization in imaging applications.

patcHwork: a user-friendly pH sensitivity analysis web server for protein sequences and structures.

pH regulates protein function and interactions by altering the charge of individual residues causing loss or gain of intramolecular noncovalent bonds, which may lead to structural rearrangements. While tools to analyze residue-specific charge distribution of proteins at a given pH exist, currently no tool is available to investigate noncovalent bond changes at two different pH values. To make protein pH sensitivity analysis more accessible, we developed patcHwork, a web server that combines the identification of amino acids undergoing a charge shift with the determination of affected noncovalent bonds at two user-defined pH values. At the sequence-only level, patcHwork applies the Henderson-Hasselbalch equation to determine pH-sensitive residues. When the 3D protein structure is available, patcHwork can be employed to gain mechanistic understanding of the effect of pH. This is achieved using the PDB2PQR and PROPKA tools and noncovalent bond determination algorithms. A user-friendly interface allows visualizing pH-sensitive residues, affected salt bridges, hydrogen bonds and aromatic (pi-pi and cation-pi) interactions. patcHwork can be used to identify patches, a new concept we propose of pH-sensitive residues in close proximity on the protein, which may have a major impact on function. We demonstrate the attractiveness of patcHwork studying experimentally investigated pH-sensitive proteins (https://patchwork.biologie.uni-freiburg.de/).

Photophysical and Computational Insights into Ag(I) Complexation of Porphyrinic Covalent Cages Equipped with Triazoles-Incorporating Linkers.

The photophysical characterization of four supramolecular complexes based on covalent cages 2H-S-2H, 2H-L-2H, Zn-S-2H, and Zn-L-2H, consisting in either two free-base porphyrins or one Zn(II) porphyrin and one free-base porphyrin connected by four flexible linkers of different lengths incorporating triazole binding sites, and their Ag(I) complexation are reported. The complexation processes have been followed by means of absorption and emission spectroscopies, and a comprehensive computational study explains the behavior of the free-base porphyrin-containing cages. Absorption and emission features have been interpreted on the bases of conformational changes, metalation processes, and modification of energy transfer efficiencies occurring in the different cases. In all cages, except 2H-L-2H, the coordination of four Ag(I) ions to the lateral triazole groups of the linkers leads to the enlargement of their cavity. Only for 2H-L-2H is a different behavior observed, where the process of silver metalation of the porphyrins' core prevails.

Temporal control of the integrated stress response by a stochastic molecular switch.

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response by integrating quantitative experiments with mathematical modeling and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2α, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 messenger RNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

Deterministic Lateral Displacement Microfluidic Chip for Minicell Purification.

Deterministic lateral displacement (DLD) is a well-known microfluidic technique for particle separation with high potential for integration into bioreactors for therapeutic applications. Separation is based on the interaction of suspended particles in a liquid flowing through an array of microposts under low Reynolds conditions. This technique has been used previously to separate living cells of different sizes but similar shapes. Here, we present a DLD microchip to separate rod-shaped bacterial cells up to 10 µm from submicron spherical minicells. We designed two microchips with 50 and 25 µm cylindrical posts and spacing of 15 and 2.5 µm, respectively. Soft lithography was used to fabricate polydimethylsiloxane (PDMS) chips, which were assessed at different flow rates for their separation potential. The results showed negligible shear effect on the separation efficiency for both designs. However, the higher flow rates resulted in faster separation. We optimized the geometrical parameters including the shape, size, angle and critical radii of the posts and the width and depth of the channel as well as the number of arrays to achieve separation efficiency as high as 75.5% on a single-stage separation. These results pave the way for high-throughput separation and purification modules with the potential of direct integration into bioreactors.