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Purpose: In-stent restenosis (ISR) following internal carotid artery (ICA) stenting is relatively common with an estimated incidence of 5%. Treatment options include repeat angioplasty with conventional or drug-eluting balloons (DEB), repeat stent angioplasty and surgical intervention. Application of DEB in ISR of the coronary and peripheral arteries is an established method; however, data on DEB treatment of ICA ISR are sparse. In this work, results from a retrospective cohort of 45 patients harboring 46 ICA ISR lesions treated with DEB angioplasty are presented. Methods: Clinical, procedural and imaging data from DEB angioplasty treatment of 46 high-grade ICA ISR lesions in 45 patients, performed between 2013 and 2021 were collected. A single type of DEB (Elutax, Aachen Resonance, Aachen, Germany) was used in all procedures. Imaging follow-up was performed by regular Doppler ultrasound (DUS), verified by computed tomography angiography (CTA) in cases suspicious for a recurrent ISR. Results: Technical success was 100%. Intraprocedural and postprocedural complications were not encountered. Clinical follow-up was obtained in all patients. Recurrent stroke in the affected territory was not encountered. A recurrent ISR following DEB treatment was confirmed by DUS and CTA in 4/46 (8.7%) of the lesions and were retreated with DEB. A third recurrent ISR occurred in a single case (2%) and following a second DEB retreatment there were no signs of a fourth recurrence after 36 months follow-up. Conclusion: The use of DEB angioplasty is a safe and effective treatment of ICA ISR lesions, yielding significantly better results compared to other modalities. Randomized multicenter studies are warranted. [ABSTRACT FROM AUTHOR]

The restoration of invasion-resistant plant communities is an important strategy to combat the negative impacts of alien invasions. Based on a systematic review and meta-analysis of seed-based ecological restoration experiments, here we demonstrate the potential of functional similarity, seeding density and priority effect in increasing invasion resistance. Our results indicate that native priority is the most promising mechanism to control invasion that can reduce the performance of invasive alien species by more than 50%. High-density seeding is effective in controlling invasive species, but threshold seeding rates may exist. Overall seeding functionally similar species do not have a significant effect. Generally, the impacts are more pronounced on perennial and grassy invaders and on the short-term. Our results suggest that biotic resistance can be best enhanced by the early introduction of native plant species during restoration. Seeding of a single species with high functional similarity to invasive alien species is unpromising, and instead, preference should be given to high-density multifunctional seed mixtures, possibly including native species favored by the priority effect. We highlight the need to integrate research across geographical regions, global invasive species and potential resistance mechanisms. A systematic review and meta-analyses of seed-based ecological restoration experiments identify native priority as a promising mechanism for controlling invasion of alien plant species, which can reduce the performance of invasive species by more than 50%. [ABSTRACT FROM AUTHOR]

Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses. Piezo1 ion channels are directly gated by mechanical forces and play an essential role in cellular mechanotransduction. However, readouts of Piezo1 activity are mainly examined by invasive or indirect techniques, such as electrophysiological analyses and cytosolic calcium imaging. Here, we introduce GenEPi, a genetically-encoded fluorescent reporter for non-invasive optical monitoring of Piezo1-dependent activity. We demonstrate that GenEPi has high spatiotemporal resolution for Piezo1-dependent stimuli from the single-cell level to that of the entire organism. GenEPi reveals transient, local mechanical stimuli in the plasma membrane of single cells, resolves repetitive contraction-triggered stimulation of beating cardiomyocytes within microtissues, and allows for robust and reliable monitoring of Piezo1-dependent activity in vivo. GenEPi will enable non-invasive optical monitoring of Piezo1 activity in mechanochemical feedback loops during development, homeostatic regulation, and disease. Stretch-activated ion channel Piezo1 contribures to mechanotransduction in many tissues, but its output is mostly measured indirectly. Here, the authors introduce GenEPi, a fluorescent reporter for directly visualizing Piezo1 activation-dependent calcium influx. [ABSTRACT FROM AUTHOR]

Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. From ultrastructural analysis, it is known that presynaptic boutons along single axons differ in the number of vesicles docked at the active zone. It is not clear whether the probability of these vesicles to get released (pves) is homogenous or also varies between individual boutons. Here, we optically measure evoked transmitter release at individual Schaffer collateral synapses at different calcium concentrations, using the genetically encoded glutamate sensor iGluSnFR. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. We find that Schaffer collateral boutons typically release single vesicles under low pves conditions and switch to multivesicular release in high calcium saline. The potency of individual boutons is highly correlated with their vesicular release probability while the number of releasable vesicles affects synaptic output only under high pves conditions. It is not fully understood if the probability of synaptic vesicle release is homogenous or varies between individual boutons. Here the authors perform optical quantal analyses of individual Schaffer collateral synapses, showing that multivesicular release enables a tenfold increase in glutamate output and that the vesicular release probability is not uniform between synapses. [ABSTRACT FROM AUTHOR]

To help upscale ecological restoration of degraded lands, landscape factors and longer time scales should be considered when assessing restoration efforts. We evaluated the impact of initial restoration intervention, landscape composition, and elapsed time since the restoration began on the long-term progress of Pannonic sand grassland restoration. Treatments (seeding, mowing, and carbon amendment) were implemented for 6–7 years and monitoring lasted up to 23 years after the first treatment applications in eight experimental blocks belonging to three field experiments. The abundance of target/neophyte species, and distance from primary grasslands and plantations (as major source of target/neophyte species) were estimated in 500 m landscape buffers around each block to characterize landscape composition. Restoration progress was calculated as the difference between the relative cover of target/neophyte species in treatment and control plots. Restoration intervention and neophyte abundance in the landscape had a significant effect on the restoration progress, but time did not. Seeding had the highest positive effect on target species and also prevented invasion by neophyte species. Higher abundance of neophytes in the landscape and the proximity to plantations increased the cover of neophytes in treatment plots. We conclude that restoration interventions may have a greater impact on restoration progress in the longer term than landscape factors or elapsed time. Seeding proved to be the best method in restoring sand grasslands by both favoring target species and controlling invasion. From the landscape factors, the abundance of neophyte species and distance to plantations should be considered when prioritizing areas and efforts for restoration. [ABSTRACT FROM AUTHOR]

In response to vascular damage, P-selectin molecules are secreted onto the surface of cells that line our blood vessels. They then serve as mechanical anchors to capture leucocytes from the blood stream. Here, we track individual P-selectin molecules released at the surface of live endothelial cells following stimulated secretion. We find P-selectin initially shows fast, unrestricted diffusion but within a few minutes, movement becomes increasingly restricted and ~50% of the molecules become completely immobile; a process similar to a sol-gel transition. We find removal of the extracellular C-type lectin domain (ΔCTLD) and/or intracellular cytoplasmic tail domain (ΔCT) has additive effects on diffusive motion while disruption of the adapter complex, AP2, or removal of cell-surface heparan sulphate restores mobility of full-length P-selectin close to that of ΔCT and ΔCTLD respectively. We have found P-selectin spreads rapidly from sites of exocytosis and evenly decorates the cell surface, but then becomes less mobile and better-suited to its mechanical anchoring function. P-selectin recruits leucocytes to regions of blood vessel damage. Using single molecule imaging, we find newly secreted P-selectin spreads rapidly across the plasma membrane and then becomes immobilized and better-suited to leucocyte capture. [ABSTRACT FROM AUTHOR]

Genome-editing tools from Oligonucleotide-Directed Mutagenesis (ODM) to CRISPR system use synthetic oligonucleotides for targeted exchange of nucleotides. Presently, majority of genome-editing protocols are dependent on the in vitro cell or tissue culture systems with somaclonal variation, and limitations in plant regeneration. Therefore, here, we report an alternative in planta cellular test system for optimization of the ODM, based on the injection of oligonucleotide solution into the apical meristematic region of haploid maize seedlings. Using 5′-fluorescein-labeled oligonucleotides, we detected accumulation of synthetic DNA molecules in cells of the shoot apical meristem and of the vascular bundles of leaf primordia. For silencing or knocking down of the phytoene desaturase gene in somatic cells, 41-mer long single-stranded oligonucleotides with TAG stop codon were injected into maize seedlings. We detected out-growing M1 plantlets that developed leaves with white stripes or pale-green color. Confocal microscopy of white stripes showed that in addition to the chlorophyll fluorescence-deficient tissue region, chlorophyll containing cells are present in white stripes. The Ion Torrent sequencing of DNA samples from the white stripes indicated 0.13–1.50% read frequency for the TAG stop codon in the phytoene desaturase gene. Appearance of chlorotic abnormalities supports the mutagenic nature of oligonucleotide molecules after injection into the shoot apical meristem region of maize seedling. The described protocol provides basis for early seedling stage characterization of functionality of a mutagenic oligonucleotide with different chemistry and testing efficiency of various treatment combinations at plant level. [ABSTRACT FROM AUTHOR]

Assessing global variation in phenotypic traits and linking that variation to colonization and range expansion is notably rare in invasion biology. Here, we studied variation in seed size in Centaurea solstitialis, a weed with worldwide distribution. Additionally, we explored how seed size variation affects seedling survival of C. solstitialis under favorable precipitation conditions in Anatolia, an ancestral region, and unfavorable precipitation conditions in Argentina, a non-native region. To that end, we conducted seed collections following dispersal pathways of C. solstitialis in ancestral, expanded, and non-native ranges. Locally, collections followed elevation gradients. Also, we performed a greenhouse experiment with C. solstitialis populations varying in seed size and water additions simulating precipitation patterns in Anatolia and Argentina. Seeds from ancestral populations at low elevation were smaller than those from the rest of study populations. Also, seed size in populations at high elevation in the expanded range, the main source of non-native populations, was similar to that in all, but one non-native population, where seeds exhibited further increase. Increments in seed size thus track range expansion in C. solstitialis. Locally, seed size increased with elevation in all three ranges, suggesting convergent responses to that gradient. Seedlings from larger seeds displayed greater survival than those from smaller seeds only under Argentinean conditions. Consequently, populations with large seeds may have been instrumental for colonizing that non-native region. Our findings suggest that ancient and recent dispersal of large-seeded populations contribute to explain the reported global pattern of seed size divergence and worldwide distribution of C. solstitialis. [ABSTRACT FROM AUTHOR]

Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK.'. The original article has been corrected. Correction to: I Nature Communications i https://doi.org/10.1038/s41467-023-40134-y, published online 19 July 2023 In the original version of this article, the given and family names of Armando Del Rio Hernandez were incorrectly structured. [Extracted from the article]

Improving efficiency of oligonucleotide-directed mutagenesis (ODM) is a prerequisite for wide application of this gene-editing approach in plant science and breeding. Here we have tested histone deacetylase inhibitor treatments for induction of relaxed chromatin and for increasing the efficiency of ODM in cultured maize cells. For phenotypic assay we produced transgenic maize cell lines expressing the non-functional Green Fluorescent Protein ( mGFP) gene carrying a TAG stop codon. These transgenic cells were bombarded with corrective oligonucleotide as editing reagent to recover GFP expression. Repair of green fluorescent protein function was monitored by confocal fluorescence microscopy and flow cytometry was used for quantification of correction events. Sequencing PCR fragments of the GFP gene from corrected cells indicated a nucleotide exchange in the stop codon (TAG) from T to G nucleotide that resulted in the restoration of GFP function. We show that pretreatment of maize cells with sodium butyrate (5-10 mM) and nicotinamide (1-5 mM) as known inhibitors of histone deacetylases can cause elevated chromatin sensitivity to DNase I that was visualized in agarose gels and confirmed by the reduced presence of intact PCR template for the inserted exogenous mGFP gene. Maize cells with more relaxed chromatin could serve as an improved recipient for targeted nucleotide exchange as indicated by an average of 2.67- to 3.62-fold increase in GFP-positive cells. Our results stimulate further studies on the role of the condition of the recipient cells in ODM and testing the application of chromatin modifying agents in other, programmable nuclease-based genome-editing techniques in higher plants. [ABSTRACT FROM AUTHOR]

The absence of a good interface between scientific and other knowledge holders and decision-makers in the area of biodiversity and ecosystem services has been recognised for a long time. Despite recent advancements, e.g. with the Intergovernmental Platform on Biodiversity and Ecosystem Services (IPBES), challenges remain, particularly concerning the timely provision of consolidated views from different knowledge domains. To address this challenge, a strong and flexible networking approach is needed across knowledge domains and institutions. Here, we report on a broad consultation process across Europe to develop a Network of Knowledge on biodiversity and ecosystem services (NoK), an approach aiming at (1) organising institutions and knowledge holders in an adaptable and responsive framework and (2) informing decision-makers with timely and accurate biodiversity knowledge. The consultation provided a critical analysis of the needs that should be addressed by a NoK and how it could complement existing European initiatives and institutions at the interface between policy and science. Among other functions, the NoK provides consolidated scientific views on contested topics, identification of research gaps to support relevant policies, and horizon scanning activities to anticipate emerging issues. The NoK includes a capacity building component on interfacing activities and contains mechanisms to ensure its credibility, relevance and legitimacy. Such a network would need to ensure credibility, relevance and legitimacy of its work by maximizing transparency and flexibility of processes, quality of outputs, the link to data and knowledge provision, the motivation of experts for getting involved and sound communication and capacity building. [ABSTRACT FROM AUTHOR]

The accumulation of toxic compounds generated by the interaction between reactive oxygen species and polyunsaturated fatty acids of membrane lipids can significantly damage plant cells. A plethora of enzymes act on these reactive carbonyls, reducing their toxicity. Based on the chromosomal localization and on their homology with other stress-induced aldo-keto reductases (AKRs) we have selected three rice AKR genes. The transcription level of OsAKR1 was greatly induced by abscisic acid and various stress treatments; the other two AKR genes tested were moderately stress-inducible. The OsAKR1 recombinant protein exhibited a high nicotinamide adenine dinucleotide phosphate-dependent catalytic activity to reduce toxic aldehydes including glycolysis-derived methylglyoxal (MG) and lipid peroxidation-originated malondialdehyde (MDA). The function of this enzyme in MG detoxification was demonstrated in vivo in E. coli and in transgenic plants overproducing the OsAKR1 protein. Heterologous synthesis of the OsAKR1 enzyme in transgenic tobacco plants resulted in increased tolerance against oxidative stress generated by methylviologen (MV) and improved resistance to high temperature. In these plants lower levels of MDA were detected both following MV and heat treatment due to the activity of the OsAKR1 enzyme. The transgenic tobaccos also exhibited higher AKR activity and accumulated less MG in their leaves than the wild type plants; both in the presence and absence of heat stress. These results support the positive role of OsAKR1 in abiotic stress-related reactive aldehyde detoxification pathways and its use for improvement of stress tolerance in plants. [ABSTRACT FROM AUTHOR]

We demonstrate using Ca2+-dependent calmodulin (CaM)-affinity chromatography and overlay with biotinylated CaM that the adaptor proteins growth factor receptor bound (Grb)7 and Grb7V (a naturally occurring variant lacking the Src homology 2 (SH2) domain) are CaM-binding proteins. Deletion of an amphiphilic basic amino-acid sequence (residues 243–256) predicted to form an α-helix located in the proximal region of its pleckstrin homology (PH) domain demonstrates the location of the CaM-binding domain. This site is identical in human and rodents Grb7, and shares great homology with similar regions of Grb10 and Grb14, and the Mig10 protein from Caenorhabditis elegans. We show that Grb7 and Grb7V are present in the cytosol and bound to membranes, while the deletion mutants (Grb7Δ and Grb7VΔ) have less capacity to be associated to membranes. Grb7Δ maintains in part the capacity to bind phosphoinositides, and CaM competes for phosphoinositide binding. Activation of ErbB2 by heregulin β1 decreases the pool of Grb7 associated to membranes. The cell-permeable CaM antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), but not the CaM-dependent protein kinase II inhibitor KN93, prevents this effect. Highly specific cell-permeable CaM inhibitory peptides decrease the association of Grb7 to membranes. This suggests that CaM regulates the intracellular mobilization of Grb7 in living cells. Direct interaction between enhanced yellow fluorescent protein (EYFP)-Grb7 and enhanced cyan fluorescent protein (ECFP)-CaM chimeras at the plasma membrane of living cells was demonstrated by fluorescence resonance energy transfer (FRET). The FRET signal dramatically decreased in cells loaded with a cell-permeable Ca2+ chelator, and was significantly attenuated when enhanced yellow fluorescent protein-Grb7 chimera (EYFP-Grb7)Δ instead of EYFP-Grb7 was used. Finally, we show that conditioned media from cells transiently transfected with Grb7Δ and Grb7VΔ lost its angiogenic activity, in contrast to those from cells transiently transfected with their wild-type counterparts.Oncogene (2005) 24, 4206–4219. doi:10.1038/sj.onc.1208591 Published online 4 April 2005 [ABSTRACT FROM AUTHOR]

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Abstract The aim of the present study was to investigate the aggregation of cardiovascular risk factors (hyperinsulinaemia, impaired glucose tolerance, dyslipidaemia, and hypertension) in 180 (77 female, 103 male) obese and 239 control children. Blood glucose, serum insulin and lipid levels were determined from blood samples taken after an overnight fast. Oral glucose tolerance tests were performed and blood glucose concentrations were monitored. The body mass index, body fat (on the basis of skinfold measurements), lean body mass and waist/hip ratio were calculated and blood pressure was measured five times in all subjects. It was shown that only 14.4% of obese children were free from any risk factors, in contrast to 79.1% of the control children. Four risk factors (metabolic cardiovascular syndrome) were found in 8.9% of the obese children (8.7% in males and 9.1% in females) while none could be detected in controls. Considerable differences were also detected in the prevalence of one, two or three risk factors between control and obese children. Patients with the metabolic cardiovascular syndrome could not be characterized by any of the investigated anthropometric characteristics, but the duration of obesity was significantly longer in these children. Conclusion Potential risk factors for cardiovascular diseases already tend to cluster in childhood and they are strongly associated with obesity. Our observations suggest that the development of the metabolic cardiovascular syndrome has its origin in childhood. [ABSTRACT FROM AUTHOR]

Transgenic tobacco plants that synthesize alfalfa ferritin in vegetative tissues -- either in its processed form in chloroplasts or in the cytoplasmic nonprocessed form -- retained photosynthetic function upon free radical toxicity generated by iron excess or paraquat treatment. Progeny of transgenic plants accumulating ferritin in their leaves exhibited tolerance to necrotic damage caused by viral (tobacco necrosis virus) and fungal (Alternaria alternata, Botrytis cinerea) infections. These transformants exhibited normal photosynthetic function and chlorophyll content under greenhouse conditions. We propose that by sequestering intracellular iron involved in generation of the very reactive hydroxyl radicals through a Fenton reaction, ferritin protects plant cells from oxidative damage induced by a wide range of stresses. [ABSTRACT FROM AUTHOR]

The effects of d-Met, Pro-enkephalinamide (EA) on pain tolerance and some cognitive functions have been examined in healthy male volunteers. Dihydrocodeine (DC) was used as reference substance. Applying the submaximum effort tourniquet technique EA (10 mg SC) was found to elevate the pain threshold similarly to DC (20 mg SC). Neither DC nor EA impaired the performance in the symbol cancellation test, which quantitates the intensity of attention. In this assay rather a slight improvement was detected. In addition the short-term memory performance (Wechsler test) was also improved by EA and DC. No alteration was seen in the word fluency test, an indicator of long-term (semantic) memory. The data show that EA not only improves pain tolerance but some of its mental effects are similar to those of a classical morphine congener DC. [ABSTRACT FROM AUTHOR]

Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators. [ABSTRACT FROM AUTHOR]

Faithful reporting of temporal patterns of intracellular Ca2+ dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca2+ signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca2+ affinities and accelerated kinetics by weakening the Ca2+-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca2+ decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca2+ dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca2+ at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca2+ kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes. [ABSTRACT FROM AUTHOR]