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Plant cell growth responds rapidly to various stimuli, adapting architecture to environmental changes. Two major endogenous signals regulating growth are the phytohormone auxin and the secreted peptides rapid alkalinization factors (RALFs). Both trigger very rapid cellular responses and also exert long-term effects [Du et al., Annu. Rev. Plant Biol. 71, 379–402 (2020); Blackburn et al., Plant Physiol. 182, 1657–1666 (2020)]. However, the way, in which these distinct signaling pathways converge to regulate growth, remains unknown. Here, using vertical confocal microscopy combined with a microfluidic chip, we addressed the mechanism of RALF action on growth. We observed correlation between RALF1-induced rapid Arabidopsis thaliana root growth inhibition and apoplast alkalinization during the initial phase of the response, and revealed that RALF1 reversibly inhibits primary root growth through apoplast alkalinization faster than within 1 min. This rapid apoplast alkalinization was the result of RALF1-induced net H + influx and was mediated by the receptor FERONIA (FER). Furthermore, we investigated the cross-talk between RALF1 and the auxin signaling pathways during root growth regulation. The results showed that RALF-FER signaling triggered auxin signaling with a delay of approximately 1 h by up-regulating auxin biosynthesis, thus contributing to sustained RALF1-induced growth inhibition. This biphasic RALF1 action on growth allows plants to respond rapidly to environmental stimuli and also reprogram growth and development in the long term.

The two principal growth regulators cytokinins and ethylene are known to interact in the regulation of plant growth. However, information about underlying molecular mechanism and positional specificity of the cytokinin/ethylene crosstalk in root growth control is scarce. We have identified spatial specificity of cytokinin-regulated root elongation and root apical meristem (RAM) size, both of which we demonstrate to be ethylene biosynthesis-dependent. Upregulation of the cytokinin biosynthetic geneISOPENTENYLTRANSFERASE(IPT) in proximal and peripheral tissues leads to both root and RAM shortening. In contrast,IPTactivation in distal and inner tissues reduces RAM size while leaving the root length comparable to mock-treated controls. We show that cytokinins regulate two steps specific to ethylene biosynthesis, the production of ACC by ACC SYNTHASEs (ACSs), and its conversion to ethylene by ACC OXIDASEs (ACOs). We describe cytokinin- and ethylene-specific regulation controlling the activity ofACSsandACOsthat are spatially discrete along both proximo/distal and radial root axes. Using direct ethylene measurements, we identifyACO2, ACO3andACO4as being responsible for ethylene biosynthesis and the ethylene-regulated root and RAM shortening in cytokinin-treatedArabidopsis. Finally, we describe the tight cooperation between cytokinin and ethylene signaling in cytokinin-induced, ethylene-regulated control ofACO4due to the direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), a member of the multistep phosphorelay cascade and the C-terminal portion of ETHYLENE INSENSITIVE 2 (EIN2-C), a key regulator of canonical ethylene signaling.

Waterlogging leads to hypoxic conditions in the root zone that subsequently cause systemic adaptive responses in the shoot, including leaf epinasty. Waterlogging-induced epinasty in tomato has long been ascribed to the coordinated action of ethylene and auxins. However, other hormonal signals have largely been neglected, despite evidence of their importance in leaf posture control. To adequately cover a large group of growth regulators, we performed a tissue-specific and time-dependent hormonomics analysis. This analysis revealed that multiple hormones are differentially affected throughout a 48 h waterlogging treatment, and, more importantly, that leaf development defines a framework in which this hormonal control is regulated. In addition, we could distinguish early hormonal signals that might contribute to fast responses towards oxygen deprivation from those that potentially sustain the waterlogging response. For example, abscisic acid (ABA) levels peak in petioles within the first 12 h of the treatment, while its metabolites only rise much later, suggesting ABA transport is altered. At the same time, cytokinins (CK) and their derivatives drastically decline during waterlogging in leaves of all ages. This drop in CK possibly releases the inhibition of ethylene and auxin mediated cell elongation to establish epinastic bending. Auxins themselves rise substantially in the petiole of mature leaves, but mostly after 48 h of root hypoxia. Based on our hormone profiling, we propose that ethylene and ABA might act synergistically to dynamically fine-tune the balance of IAA and CK in the petiole, ultimately leading to differential growth and epinasty during waterlogging.

Microalgae play an essential role in agriculture as bio-fertilizers and biostimulants, but some also produce various plant hormones, such as cytokinins (CK). CK are a class of natural plant hormones and there is evidence that exogenous or bacterial CK can modulate plant defense. However, there is a lack of knowledge about the effect of specific microalgae or microalgae-produced cytokinins on this response. In our study, we have evaluated the cytokinins-producing microalgae Chlamydomonas reinhardtii (Cri) on its ability to prime Nicotiana tabacum plant defenses against its natural pathogen Pseudomonas syringae pv. tabaci. Our work functionally verifies that Cri primes plant defense response, with cytokinins being a crucial component. We have used the CRISPR/Cas9 system to generate CriLOG and IPT gene knockouts, which are major points of the CK biosynthesis pathways. While Cri shows strong protection potential, the CK-deficient mutants have a reduced ability to affect plant defense, where the degree of protection correlates with the CK levels - the IPT mutants show less protection than the LOG mutants. Additionally, by measuring plant defense gene expression by RTq-PCR we have shown that Cri treatment stimulates tobacco defense response by priming.

Fluctuating environmental conditions trigger adaptive responses in plants, which are regulated by phytohormones. During photoperiod stress caused by a prolongation of the light period, cytokinin (CK) has a protective function. Auxin often acts as an antagonist of CK in developmental processes and stress responses. Here, we investigated the regulation of the photoperiod stress response in Arabidopsis thaliana by auxin and its interaction with CK. Transcriptome analysis revealed an altered transcript abundance of numerous auxin metabolism and signaling genes after photoperiod stress treatment. The changes appeared earlier and were stronger in the photoperiod-stress-sensitive CK receptor mutant arabidopsis histidine kinase 2 (ahk2),3 compared to wild-type plants. The concentrations of indole-3-acetic acid (IAA), IAA-Glc and IAA-Asp increased in both genotypes, but the increases were more pronounced in ahk2,3. Genetic analysis revealed that the gain-of-function YUCCA 1 (YUC1) mutant, yuc1D, displayed an increased photoperiod stress sensitivity. In contrast, a loss of the auxin receptors TRANSPORT-INHIBITOR-RESISTANT 1 (TIR1), AUXIN SIGNALING F-BOX 2 (AFB2) and AFB3 in wild-type and ahk2,3 background caused a reduced photoperiod stress response. Overall, this study revealed that auxin promotes response to photoperiod stress antagonizing the protective CK.

Significance Mitochondrial adrenodoxins (ADXs) are small iron–sulfur proteins that function as mobile shuttles transferring electrons. Their function has been largely known in animals, as they transfer electrons between an adrenodoxin reductase (ADXR) and mitochondrial P450s, which is a crucial step that leads to steroidogenesis. Here we show that a functional mitochondrial ADX–ADXR–P450 pathway is essential for steroid biosynthesis and that its function is required for plant sexual reproduction.

Significance Mitochondrial adrenodoxins (ADXs) are small iron–sulfur proteins that function as mobile shuttles transferring electrons. Their function has been largely known in animals, as they transfer electrons between an adrenodoxin reductase (ADXR) and mitochondrial P450s, which is a crucial step that leads to steroidogenesis. Here we show that a functional mitochondrial ADX–ADXR–P450 pathway is essential for steroid biosynthesis and that its function is required for plant sexual reproduction., Mitochondrial adrenodoxins (ADXs) are small iron–sulfur proteins with electron transfer properties. In animals, ADXs transfer electrons between an adrenodoxin reductase (ADXR) and mitochondrial P450s, which is crucial for steroidogenesis. Here we show that a plant mitochondrial steroidogenic pathway, dependent on an ADXR–ADX–P450 shuttle, is essential for female gametogenesis and early embryogenesis through a maternal effect. The steroid profile of maternal and gametophytic tissues of wild-type (WT) and adxr ovules revealed that homocastasterone is the main steroid present in WT gametophytes and that its levels are reduced in the mutant ovules. The application of exogenous homocastasterone partially rescued adxr and P450 mutant phenotypes, indicating that gametophytic homocastasterone biosynthesis is affected in the mutants and that a deficiency of this hormone causes the phenotypic alterations observed. These findings also suggest not only a remarkable similarity between steroid biosynthetic pathways in plants and animals but also a common function during sexual reproduction.

The ability to optically image cellular transmembrane voltage at millisecond-timescale resolution can offer unprecedented insight into the function of living brains in behaving animals. The chemigenetic voltage indicator Voltron is bright and photostable, making it a favorable choice for long in vivo imaging of neuronal populations at cellular resolution. Improving the voltage sensitivity of Voltron would allow better detection of spiking and subthreshold voltage signals. We performed site saturation mutagenesis at 40 positions in Voltron and screened for increased ΔF/F0 in response to action potentials (APs) in neurons. Using a fully automated patch-clamp system, we discovered a Voltron variant (Voltron.A122D) that increased the sensitivity to a single AP by 65% compared to Voltron. This variant (named Voltron2) also exhibited approximately 3-fold higher sensitivity in response to sub-threshold membrane potential changes. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, with lower baseline fluorescence. Introducing the same A122D substitution to other Ace2 opsin-based voltage sensors similarly increased their sensitivity. We show that Voltron2 enables improved sensitivity voltage imaging in mice, zebrafish and fruit flies. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Test pattern compression techniques reduce the amount of transferred data while decompressed pattern flexibility remains high. The flexibility can be measured by the guaranteed number of specified test pattern bits, equal to or greater than the number of care bits in the pattern. This paper addresses the problem of test pattern compression and decompression using nonlinear codes. Test patterns can be encoded using a linear code that leads to an XOR network that decompresses the compressed patterns transferred from a tester. This XOR network can be completed or replaced by a standard (nonlinear) combinational network with substantially improved compression efficiency. The problem of finding the code was formulated as a clique cover problem. This approach’s tight throat lies in computational complexity and the needed memory capacity to temporarily retain intermediate results of clique coverage search attempts. The paper proposes a method that substantially reduces the computational time to get a robust nonlinear code with decompression ability. It shows that random search algorithms cannot reach the code parameters obtained by a restricted iterative search proposed in the paper. Additional knowledge concerning the check-bit truth table characteristics was used to avoid unsuccessful investigations. The proposed decompressor schemes have higher efficiency than till now known decompressor constructions. On the other hand, finding other heuristics that provide efficient codes for a broader class of code parameters is challenging than the proposed solution.

Visualizing neuronal activity in vivo Imaging the changes in fluorescence of voltage-sensitive reagents would enable monitoring of the activity of neurons in vivo. Abdelfattah et al. created such a voltage indicator by designing a protein that combines the voltage sensor domain from microbial rhodopsin with a domain that captures a dye molecule with exceptional brightness and photostability. When the protein was expressed in mice, flies, or zebrafish, they could monitor single action potentials in dozens of neurons simultaneously for many minutes. Science , this issue p. 699

Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits its speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and computationally recovers high-resolution images, attaining voxel rates of over 1 billion Hz in structured samples. Using a static image as a prior for recording neural activity, we imaged visually evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 µm and frame rates over 1 kHz. Dendritic glutamate transients in anesthetized mice are synchronized within spatially contiguous domains spanning tens of micrometers at frequencies ranging from 1–100 Hz. We demonstrate millisecond-resolved recordings of acetylcholine and voltage indicators, three-dimensional single-particle tracking and imaging in densely labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime. A two-photon computed tomography approach, called scanned line angular projection microscopy, enables high-speed imaging at over 1 kHz frame rates, as demonstrated for glutamate imaging in the in vivo mouse brain.

Protective coatings deposited on traditional Zr-based fuel cladding materials potentially enhance accidental tolerance of Light Water Reactors due to reduced interaction between water and Zr alloys during accidental conditions. Enhancement of cladding performance and reduction of negative effects, such as heat generation, hydrogen production, oxidation, and embrittlement can be achieved by deposition of various coatings that are widely studied around the world. Even though thermal, mechanical, and chemical responses during accidental conditions are the main concern for these materials, the actual application of coated materials will be linked to standard operating conditions including economic consequences and transient response. Reference VVER-1200 fuel system with five coatings (Cr, CrN, ZrSi2, Cr2AlC, Fe(Cr)(Al)) is investigated from the neutron-physical point of view using the calculation package SCALE 6.2.2. In addition to the multiplication factor, specific fuel safety criteria are also examined for both the fresh fuel assemblies and fuel assemblies with burn-up up-to 70 MWd/ t HM . All studied coatings entail certain negative effects in comparison with the reference case. The closest behavior to the reference VVER-1200 fuel assembly is found for ZrSi2 coating followed by Cr2AlC and Fe(Cr)(Al) coatings. Both Cr and CrN materials exhibit significant differences compared to the reference case.

COVID-19, classified as SARS-CoV-2, is causing an ongoing global pandemic. The pandemic has resulted in the loss of lives and has caused economic hardships. Most of the devices used to protect against the transmission of the novel COVID-19 disease are related to textile structures. Hence, the challenge for textile professionals is to design and develop suitable textile structures with multiple functionalities for capturing viruses, passivating them, and, at the same time, having no adverse effects on humans during the complete period of use. In addition to manufacturing efficient, biocompatible, and cost-effective protective face masks, it is also necessary to inform the public about the benefits and risks of protective face mask materials. The purpose of this article is to address the concerns of efficiency and efficacy of face masks by primarily reviewing the literature of research conducted at the Technical University of Liberec. The main focus is on the presentation of problems related to the specification of aims of face mask applications, mechanisms of capture, durability, and modes of sterilization. The recommendations, instead of conclusions, are addressed to the whole textile society because they should be leading players in the design, creation, and proper treatment of face masks due to their familiarity with the complex behavior of textile structures and targeted changes of structural hierarchy starting from polymeric chains (nano-level) and ending in planar textile structures (millimeter level) due to action by mechanical, physical and chemical fields. This becomes extremely critical to saving hundreds of thousands of lives from COVID-19.

The paper deals with design of new SCRAM experimental device at Training reactor VR-1. Crucial works cowers the detector development, transient calculations and principal design of the device. Conceptual design will be prepared in 2019–2020. This paper presents current work status.

The seemingly random and unpredictable nature of seizures is a major debilitating factor for people with epilepsy. An increasing body of evidence demonstrates that the epileptic brain exhibits long-term fluctuations in seizure susceptibility, and seizure emergence seems to be a consequence of processes operating over multiple temporal scales. A deeper insight into the mechanisms responsible for long-term seizure fluctuations may provide important information for understanding the complex nature of seizure genesis. In this study, we explored the long-term dynamics of seizures in the tetanus toxin model of temporal lobe epilepsy. The results demonstrate the existence of long-term fluctuations in seizure probability, where seizures form clusters in time and are then followed by seizure-free periods. Within each cluster, seizure distribution is non-Poissonian, as demonstrated by the progressively increasing inter-seizure interval (ISI), which marks the approaching cluster termination. The lengthening of ISIs is paralleled by: increasing behavioral seizure severity, the occurrence of convulsive seizures, recruitment of extra-hippocampal structures and the spread of electrographic epileptiform activity outside of the limbic system. The results suggest that repeated non-convulsive seizures obey the ‘seizures-beget-seizures’ principle, leading to the occurrence of convulsive seizures, which decrease the probability of a subsequent seizure and, thus, increase the following ISI. The cumulative effect of repeated convulsive seizures leads to cluster termination, followed by a long inter-cluster period. We propose that seizures themselves are an endogenous factor that contributes to long-term fluctuations in seizure susceptibility and their mutual interaction determines the future evolution of disease activity.

The training reactor VR-1 is a nuclear facility operated by Czech Technical University in Prague (CTU in Prague). It is a zero power reactor utilised mainly for the education and training and more than 25 experiments at multiple levels of difficulty are offered. The reactor is firmly connected in the international organizations and has the highest reputation for the training performance. In December 2020, the reactor will achieve 30 years of its successful operation. The anniversary is a good opportunity for a thorough retrospective of such a unique facility. History of the VR-1 reactor operation can be used as an inspiration for other operators of similar nuclear facilities.

The VR-1 reactor core safety characteristics calculations are performed by standardized MCNP code, however due to license requirements, it is desired to replace it by the Serpent2 code. In order to do that, Serpent2 calculation must be standardized by State office of nuclear safety. Comparison of Serpent2 and MCNP multiplication factors for several reactor cores is presented to demonstrate the capability and accuracy of Serpent2 results. In addition to this, Serpent2 calculation setup is tested and the impact of different nuclear data libraries is examined. The comparison of results from Serpent2 and standardized MCNP calculation proved that results are similar and therefore the Serpent2 code can be used for the VR-1 reactor core calculations, especially for criticality predictions.

The paper describes verification of the MCNP model of manganese bath used at the Czech Technical University for education purposes. The bath consists of a spherical tank made of plexiglass filled with an aqueous solution of MnSO 4 with the total volume of 163.3 litres. Neutron sources are introduced into the centre of the bath manually using a dry plexiglass channel. The MCNP model contains all important geometric and material details of the bath including optional neutron source. Its verification was performed based on experimentally determined activity; as well, neutron emission rates for used radionuclide sources were compared. The experiments were performed with two various radionuclide neutron sources (Am-Be and 252Cf). Comparison of calculation results with ENDF/B-VII.1 and JEFF-3.2 nuclear data libraries was performed.

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

Recombinant adeno-associated virus (rAAV) has been widely used as a viral vector across mammalian biology and has been shown to be safe and effective in human gene therapy. We demonstrate that neural progenitor cells (NPCs) and immature dentate granule cells (DGCs) within the adult murine hippocampus are particularly sensitive to rAAV-induced cell death. Cell loss is dose dependent and nearly complete at experimentally relevant viral titers. rAAV-induced cell death is rapid and persistent, with loss of BrdU-labeled cells within 18 hours post-injection and no evidence of recovery of adult neurogenesis at 3 months post-injection. The remaining mature DGCs appear hyperactive 4 weeks post-injection based on immediate early gene expression, consistent with previous studies investigating the effects of attenuating adult neurogenesis. In vitro application of AAV or electroporation of AAV2 inverted terminal repeats (ITRs) is sufficient to induce cell death. Efficient transduction of the dentate gyrus (DG)—without ablating adult neurogenesis—can be achieved by injection of rAAV2-retro serotyped virus into CA3. rAAV2-retro results in efficient retrograde labeling of mature DGCs and permits in vivo 2-photon calcium imaging of dentate activity while leaving adult neurogenesis intact. These findings expand on recent reports implicating rAAV-linked toxicity in stem cells and other cell types and suggest that future work using rAAV as an experimental tool in the DG and as a gene therapy for diseases of the central nervous system (CNS) should be carefully evaluated.

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all organisms.

The exceptionally high cellular uptake of gold nanorods (GNRs) bearing cationic surfactants makes them a promising tool for biomedical applications . Given the known specific toxic and stress effects of some preparations of cationic nanoparticles, the purpose of this study was to evaluate, in an in vitro and in vivo in mouse, the potential harmful effects of GNRs coated with (16-mercaptohexadecyl)trimethylammonium bromide (MTABGNRs). Interestingly, even after cellular accumulation of high amounts of MTAB GNRs sufficient for induction of photothermal effect, no genotoxicity (even after longer-term accumulation), induction of autophagy, destabilization of lysosomes (dominant organelles of their cellular destination), alterations of actin cytoskeleton , or in cell migration could be detected in vitro. In vivo , after intravenous administration , the majority of GNRs accumulated in mouse spleen followed by lungs and liver. Microscopic examination of the blood and spleen showed that GNRs interacted with white blood cells (mononuclear and polymorphonuclear leukocytes) and thrombocytes, and were delivered to the spleen red pulp mainly as GNR-thrombocyte complexes. Importantly, no acute toxic effects of MTABGNRs administered as 10 or 50 μg of gold per mice, as well as no pathological changes after their high accumulation in the spleen were observed, indicating good tolerance of MTABGNRs by living systems.

This paper presents results obtained by reproducing a VVER-1000 lattice physics benchmark using the Serpent2 code. The benchmark data are related to the initial cycles of the Khmelnitskiy nuclear power plant. This study compares results obtained with the Serpent2 code against those calculations published in the benchmark document. Detailed descriptions are provided for two fuel assemblies; one without gadolinium in the fuel pins, and the other with gadolinium. A sensitivity study is performed utilizing different nuclear data libraries, different depletion sequences (including pin by pin burnup) and different number of radial regions within the fuel pins containing gadolinium. The infinite multiplication factor behavior and isotopic concentrations of major isotopes are compared and discussed. It is concluded that Serpent2 provided reasonable results in comparison with the other codes used in the benchmark.