Mashanov et al. present a method to image and track individual fluorescently labeled molecules in live cardiac tissue slices using total internal reflection fluorescence (TIRF) microscopy. They show that the movement and interaction of G protein–coupled receptors can be observed in real time. Recent advances in light microscopy allow individual biological macromolecules to be visualized in the plasma membrane and cytosol of live cells with nanometer precision and ∼10-ms time resolution. This allows new discoveries to be made because the location and kinetics of molecular interactions can be directly observed in situ without the inherent averaging of bulk measurements. To date, the majority of single-molecule imaging studies have been performed in either unicellular organisms or cultured, and often chemically fixed, mammalian cell lines. However, primary cell cultures and cell lines derived from multi-cellular organisms might exhibit different properties from cells in their native tissue environment, in particular regarding the structure and organization of the plasma membrane. Here, we describe a simple approach to image, localize, and track single fluorescently tagged membrane proteins in freshly prepared live tissue slices and demonstrate how this method can give information about the movement and localization of a G protein–coupled receptor in cardiac tissue slices. In principle, this experimental approach can be used to image the dynamics of single molecules at the plasma membrane of many different soft tissue samples and may be combined with other experimental techniques. The physico-chemical properties of lipid membranes of unicellular organisms (prokaryotes and primitive eukaryotes) are critical to cell viability, cell maintenance, and cell signaling, while in multicellular organisms, cell–cell contacts and interactions also become critical to the development, viability and integrity of the organism. Direct observation of integral membrane proteins within the plasma membrane of living cells and of cells embedded within live tissue samples opens new avenues for research.Over the past 25 yr, light microscopy has undergone a radical transformation due to development of sensitive cameras, solid-state lasers, and fast computers. So, it is now relatively straightforward to visualize individual fluorophores in aqueous solution and in living cells. Early single-molecule imaging studies were made using surface-immobilized, biological molecules in vitro . Later, 8 biological molecules, many of which autofluoresce or produce Raman scattering. So, synthetic fluorophores (such as rhodamine) and fluorescent proteins (such as GFP) must be used as molecular probes. Their intense fluorescence at long wavelengths (λ > 450 nm) means they appear as bright spots of light above the diffuse cytosolic autofluorescence. A green fluorophore (λ = 550 nm) viewed in aqueous solution  appears as a circular spot, ∼250 nm in diameter, in the x-y plane  and ∼600 nm along the focal axis (2λ/NA2). So, in order to visualize two fluorophores as distinct objects, they must be separated by ∼250 nm . To achieve the requisite fluorophore separation, either labeled molecules must be naturally present at low concentration , or only a fraction (<0.1%) of the molecules should be tagged, or fluorophores that blink on and off, with a low duty-cycle ratio, can be used such that they spend perhaps 0.1% of the time in a bright fluorescent on state and 99.9% of the time in a dark off state.To measure the kinetics of a molecular process, data must be sampled faster than the relaxation rate of the underlying biochemical or biophysical process. Kinetics of protein–lipid interactions vary over more than seven orders of magnitude from microseconds to tens of seconds. So, while some processes can be observed via video imaging methods , others require more sophisticated optical correlation techniques. In this paper, we focus on direct observation of single molecules by video microscopy, which is currently limited to a temporal range from around 10 ms out to several tens of seconds (bandwidth of ∼0.01–50 Hz). This is sufficient to fully characterize the lateral diffusion of proteins and their collisional interactions in lipid bilayers but, for instance, is insufficient to directly observe their rotational diffusion. To visualize single fluorophores within a live biological specimen, fluorophores must be bright, sparse, and tolerant to photobleaching. The microscope must have diffraction-limited performance with good mechanical stability and be equipped with a camera system that is sensitive, fast, and low-noise. To put some approximate numbers on things, a cubic micron of cytosol contains >102 area). Given the planar nature of the plasma membrane, TIRFM is ideally suited to studies of membrane proteins and protein–lipid interactions. Other approaches to reduce background fluorescence include confocal microscopy, light-sheet microscopy, and laser-based, oblique-angle, illumination microscopy (TIRF microscopy (TIRFM) offers a great improvement over standard episcopic fluorescence excitation because the illuminating light beam no longer penetrates the entire specimen, and background autofluorescence is greatly reduced. To produce TIRF excitation, a focused laser beam enters the extreme edge of the objective lens (NA must be >1.4) so it is then incident to the specimen plane at greater than the critical angle (>63°), leading to total internal reflection at the interface between the glass coverslip and the (lower refractive index) aqueous medium of the biological specimen. This illumination method gives rise to an evanescent field that penetrates the specimen to a narrow depth  and the duration of the observation (longest times), called the “observation time window,” which is equivalent to bandwidth in the frequency domain. From a practical standpoint, excitation intensity  is adjusted to give an emission rate and fluorophore bleaching rate that optimize the observation time window for a specific measurement , allowing large datasets to be accumulated. Then, knowing the lateral diffusion coefficient (Dlat) of a typical protein  in cytoplasm is ≈20 µm2 ⋅ s−1 and in lipid membrane is ≈0.2 µm2 ⋅ s−1 (lat/fps)0.5 ≤ 300 nm, giving ≥500 fps for molecules in cytoplasm and ≥5 fps for molecules in membranes. With this knowledge, laser power and camera frame rate can be adjusted to suit the task, and in fact, they can be systematically varied while viewing the same specimen in order to sample different observation time windows.To make video recordings of individual fluorophores, the camera system should have high signal-to-noise ratio, i.e., high photon collection efficiency (product of detector quantum efficiency and pixel fill factor) compared with the sum of all noise sources . To track individual molecules in aqueous solution, at room temperature, the exposure time (shutter speed) must be long enough to collect sufficient light required to localize the fluorophore but brief enough that the image is not blurred by diffusive motion. Fluorophore brightness (emission rate) increases linearly with laser power until the emission is limited by the relaxation time of the excited state, called “saturation,” which approaches ∼10m2 ⋅ s−1 , we can 2 muscarinic receptors move fourfold faster in intact mouse hearts (Dlat ≈ 0.6 µm2 ⋅ s−1) compared with isolated primary cardiomyocytes  or cultured adherent mammalian cells, either from stable cell lines or primary cell culture . It is gchannels , signalichannels , and shoAll chemicals were obtained from Sigma-Aldrich unless stated otherwise.−1 heparin and 100 U ⋅ ml−1 penicillin-streptomycin for 5 min. After washing, the heart was moved into ice-cold “relaxing PBS”  for 5 min. The washed heart was then placed into the 8-mm-diameter cavity of a custom-made plastic matrix cutting block  solution supplemented with 10 U ⋅ mlng block  oriented2 muscarinic receptors with the tight-binding fluorescent ligand. In all cases, heart slices were washed several times in fresh relaxing PBS solution to remove any unbound Cy3B-telenzepine. Cy3B-telenzepine bound to M2 receptors is not removed by washing because of its extremely slow dissociation rate. The prepared slice was moved to a fibronectin-coated circular coverslip  inserted into a custom-made imaging chamber (2) stretched in a stainless steel ring. The chamber was sealed and filled with Hanks’ balanced salt solution supplemented with 20 mM HEPES (pH 7.4). The imaging was performed at 23°C or 37°C.The prepared tissue slices were placed in relaxing PBS solution supplemented with 10 nM of Cy3B-telenzepine  at 4°C f chamber . The tisConfocal microscopy was performed at 23°C using a Leica SP5-II confocal microscope (Leica Microsystems) equipped with a GaAsP hybrid photomultiplier detector, 561 nm laser excitation source, and 20×, NA 1.0, water-dipping objective lens, giving, 200 nm/pixel in the x-y plane and using 1-µm z-steps to acquire specimen volume sections.Slices were fixed in 0.1 M phosphate buffer  for 1 h at room temperature, then washed in 0.1 M PB (three times for 5 min) and incubated in 1% reduced osmium (1% osmium tetroxide and 1.5% potassium ferricyanide) for 1 h at 4°C. Slices were then washed in 0.1 M PB (three times for 5 min) followed by double-distilled water (three times for 5 min) and dehydrated in a graded ethanol series  and dried using a critical-point dryer . Slices were mounted onto scanning electron microscope stubs  using silver paint  and coated in 2 nm of platinum . Images were acquired using an Everhardt-Thornley secondary electron detector in a scanning electron microscope  at 6 keV, 2 nm spot size, 5 µs dwell time, and 15 mm working distance.Like all other physical sectioning methods, cell damage occurs at the cut interface. We visualized the tissue surface by critical-point drying and scanning EM and by scanning confocal microscopy . Muscle −2, and the incident laser beam angle was adjusted to ∼63° to create the evanescent field at the glass–aqueous medium interface. A second small mirror was placed at the opposite edge of the objective lens back aperture to remove the returning  laser beam from the microscope, and a narrow band-pass emission filter FF01-585/29 (Semrock) was used to block the scattered 556 nm laser light and other unwanted light. An electron multiplying charge-coupled device camera  captured full-frame (512 × 512 pixels) video sequences at 20 fps, and using the Andor camera “cropped-mode”  at 100 fps. Data were stored on a computer hard drive for later analysis.A custom-built TIRFM microscope was used, as described previously . Brieflylat = 0.13 µm2 ⋅ s−1  that was similar to Dlat = 0.12 µm2 ⋅ s−1  measured at the basal surface  and apical cell membranes by labeling an atrial cardiomyocyte line, HL-1, with Cy3B-telenzepine . MSD versus dT plots were then generated to examine the particle motion; a linear relationship is expected for simple Brownian motion, but curvature indicates anomalous diffusion. We analyzed trajectories where single fluorescent spots were tracked for at least eight consecutive frames with a maximum displacement of ≤0.7 µm between frames  in order to reveal the details of cellular structures. In live-cell imaging, time-lapse video microscopy is often used to reveal intra-cellular movements. The approach used here is to apply high-speed “movie-mode” imaging of sparsely labeled cells where the motion of individual molecules can be studied. Data can be visualized as a “z-projection” (where the z-dimension is time) whereby each pixel value represents a statistical descriptor of pixel intensity over time. Commonly used z-projections are median, average, maximum, or SD in pixel value measured over the duration of the video time series. The SD in pixel intensity should scale with the square root of mean intensity (shot-noise limit). However, for sparsely labeled live-cell video recordings’ fluctuations in pixel intensity are dominated by fluorophore motion and/or appearance and disappearance of individual fluorophores at a given pixel location. In this situation, the normalized SD projection,movement .−1). After drift correction, each fluorescent spot was centroided, and its floating-point, x-y coordinate pair (resolved with ∼10 nm precision), was replotted onto a new, up-sampled (“zoomed”), image frame to produce a super-resolution map of the trajectories. In these representations, if the original camera image was 512 × 512 pixels, with 100 nm per pixel calibration, the resulting super-resolution image might be an image of 5,120 × 5,120 pixels at 10 nm per pixel. This type of stop-motion analysis allows us to map cellular ultrastructures such as the cytoskeleton , and immobile spots were manually selected and used as fiducial markers so video data could be drift-corrected  of fluorescent molecules on the surface of a cubic cell (3 × 3 × 3 µm3), which has cylindrical protrusions .2-expressing cardiomyocytes in a tissue slice do not correctly report the shape of the cell because the single molecules are relatively sparse. However, because the receptors are highly mobile, fluorophores soon explore the entire plasma membrane. When the normalized SD z-projection of the video frames is computed, cell shape then becomes apparent . The average Dlat can be measured by least-squares linear regression to the initial slope of the MSD versus dT plot compiled from all molecular trajectories , and individual fluorescent spots arising from Cy3B-telenzepine–labeled receptors showed unitary amplitudes  compared with cardiomyocytes, and receptor mobility maps indicated lateral diffusion was slower in the axons  allowed nerve architecture to be visualized at unprecedented resolution (∼20 nm) in a live tissue slice . There wWe have shown that single molecules can be observed at the plasma membrane of living cells from freshly explanted tissue slices. We used cardiac muscle as a model system and two different model organisms, mouse and zebrafish, to demonstrate how the technique can reveal information about protein–membrane interactions. We developed a number of novel single-molecule image-processing methods to explore our datasets, and this allowed us to investigate the molecular dynamics of a model G protein–coupled receptor (muscarinic acetylcholine receptors) within the plasma membrane of freshly extracted cardiac tissue slices.Drosophila melanogaster), for which it is sometimes difficult to establish primary cell cultures due to poor cell survival rates or instances where the biological question demands the use of intact tissue.Serial tissue sections were made using a custom-built matrix-sectioning device, and the cutting block cavity was readily modified to accommodate different-sized organ samples (mouse versus zebrafish hearts). In principle, the matrix block design could be adapted to section other organs, for example, skeletal muscle, liver, kidney, blood vessels, or brain. The imaging methods presented in this paper may be of particular value for studies of small model organisms (like zebrafish and perhaps 2 receptors in mouse heart slices from zebrafish was slightly faster than mouse (Dlat = 0.7 µm2 ⋅ s−1 versus 0.6 µm2 ⋅ s−1). However, imaging was conducted at 23°C for both preparations, which is 14°C lower than the body temperature of mouse and 5°C lower than for zebrafish . We have shown here that M2 receptor mobility increases twofold for a 10°C increase in temperature in live tissue samples. This implies the Dlat of M2 receptors at physiological temperature for zebrafish and mouse might be more similar (Dlat ∼1.3 µm2 ⋅ s−1). In the future, it would be interesting to compare the mobility of membrane proteins in zebrafish acclimatized to different temperatures. There is evidence showing membrane viscosity is subject to homeostatic acclimation . However, this difference is most readily explained by the change in dimensionality of the diffusive motion. Our super-resolution stop-frame reconstruction revealed the diameter of the nerve fibers was ∼100 nm , and the model geometry consisted of a “cell” (3 × 3 × 3 µm3) with narrow, closed, tubular protrusions of various diameters in order to mimic nerve fibers (We found an apparent difference in membrane viscosity between cardiac and neuronal tissue (Dr of two  as the dle video . Fluorese fibers . The sime fibers  using thIn previous studies, a combination of light microscopy and EM has shown the pattern of nerve fibers across the vertebrate heart . FluoresThe results of our experiments show that the single-molecule imaging in ex vivo tissue slices provides a new level of understanding in membrane–protein interactions. The methodological approaches we have described will hopefully enable other studies of individual proteins in the lipid bilayer of live cells within their native multicellular tissue context.


This study aimed to determine the effects of caffeine supplementation on CrossFit® performance. Twenty CrossFit®-trained men  were randomized in a double-blind, crossover design. Participants completed two sessions separated by a seven-day washout period, 60 min after consuming 5 mg/kg body mass of caffeine or a placebo. In each session, participants completed as many rounds as possible in 20 min of five pull-ups, 10 push-ups, and 15 air squats. CrossFit® performance was the total number of repetitions completed in 20 min. Paired-samples t-tests were used to compare CrossFit® performance between caffeine and placebo conditions and to test for a potential learning effect between the first and second sessions. CrossFit® performance was not significantly different during the caffeine condition compared to the placebo . A significant learning effect was identified between the first and second sessions , with no significant effect of treatment order (p = 0.438). Caffeine’s ergogenic effect were not present during the CrossFit® workout “Cindy”; however, future research should include familiarization sessions and examine other CrossFit® workouts in novice and women participants.Caffeine’s ergogenic effects persist during various exercise modalities; however, information establishing its efficacy during CrossFit Caffeine supplementation is pervasive in sporting disciplines, with up to 74% of Spanish elite athletes consuming caffeine prior to competition for its ergogenic effects . Support−1 body mass) [2 availability are reported to increase muscle function by enhancing the metabolic steady-state of muscle tissue during both upper and lower-extremity exercise performed until failure [The mechanisms of caffeine’s physiological effects have spanned decades of research, which has offered multiple theories for caffeine’s ergogenic properties . Caffeindy mass) . Caffeindy mass) . To datedy mass) . These mdy mass) ,12. Emerdy mass) ,14. Incr failure ,16.In a recent meta-analysis of the effects of caffeine supplementation on muscle function, the authors reported significant improvements (+6–7%) in muscular endurance after caffeine supplementation . A major®, which has an annual competition called the CrossFit® Games [®, repetitions can approach up to 700 in a 20-min training session [® athletes likely possess high levels of health- and skill-related aspects of physical fitness, and research has significantly correlated CrossFit® performance with aerobic capacity, muscular strength (upper- and lower-body), and power [® performance [The recent dramatic increase in high-intensity functional training—which aims to enhance multiple domains of physical fitness by temporally exposing athletes to varying modes of exercise  within and between each session, for varying durations , at a relatively high-intensity —has beent® Games ,24,25,26 session . Similarnd power ,28. Unfoformance .® performance is limited, with only one investigation examining the effect of acute caffeine supplementation on CrossFit® performance [® performance; however, their investigation may be limited by the small sample size (n = 9), CrossFit® performance that included power-based exercises (snatches and double-unders), and short duration of the CrossFit® workout (10 min). These limitations may be resolved with larger sample sizes, using exercises that tax other aspects of muscular fitness, and greater workout durations, the latter of which may reveal caffeine’s ergogenic properties [® performance for a 20-min muscular endurance workout (“Cindy”) were examined. Similar to previous investigations documenting caffeine’s ergogenic effects for other types of training programs [® performance during a high-volume muscular endurance workout that taxed multiple muscle groups with limited recovery, as well as a decrease in perceptual responses to exercise . The current evidence examining the effects of dietary supplementation on CrossFitformance . The recoperties ,32. Osteoperties ,31,33. Hprograms ,31,33, i® performance. Inclusion criteria were having ≥6 months of CrossFit® experience; previously completing “Cindy” or a workout with similar repetition volumes for the included movements; being of male sex; and being 18–45 years of age. Participants were excluded if they had any known health-problem , answered “Yes” to any physical activity readiness questionnaire items, reported allergies or negative side-effects with caffeine use, were unable to perform “Cindy” as prescribed, or were taking medication for seizures. An a priori power analysis was conducted using a large effect size, powered at 80%, and an α of 0.05 [This study used a randomized, double-blind, crossover design to determine the effects of caffeine on CrossFit2, percent body fat = 19.3% ± 3.4%, experience = 3.7 ± 2.9 years, daily caffeine intake = 288.3 ± 287.5 mg/day) were randomized to consume either caffeine or the placebo. To determine the treatment order (placebo then caffeine vs. caffeine then placebo), participants were randomized using a random number generator (0–99), with odd numbers being placed into the placebo condition and even numbers being placed into the caffeine condition for the first visit . The caffeine pill(s) (Prolab) was/were provided at 5 mg·kg−1 body mass [® workout—“Cindy”—60 min after the consumption of the pill(s). Sixty min was selected based on caffeine’s pharmacokinetics and on investigations evaluating the effects of acute caffeine supplementation [® workout, and it has been previously described in the literature [® movement standards were followed; kipping was allowed for the pull-ups; push-ups were performed on the toes, with the subject lowering himself with a straight body until his chest touched the floor; and air squats required subjects to reach full knee and hip extension at the top of each repetition and have their hip crease below their knee at the bottom of each repetition [® Level 1 or 2 Certificates, verbally counted repetitions. Repetitions that did not meet movement standards were not counted, and participants were provided with feedback to meet the movement standards. CrossFit® performance was the total number of repetitions completed in 20 min. Participants were given a post-exercise survey to determine their ratings of perceived exertion (RPE) achieved during the workout on a scale of 1–10, and if they perceived an effect from the supplement given (yes/no) [Participants were asked to refrain from caffeine, alcohol, vigorous exercise, and nicotine for 24 h, maintain their normal diet, adequately hydrate, refrain from eating 3 h prior to testing, and otherwise maintain their usual training regimen throughout the study. Anthropometric measures were taken on the first laboratory visit. Height was measured using a stadiometer. Body mass and percent body fat were determined using bioelectrical impedance analysis in standard mode . Daily caffeine consumption was determined using a 7-day caffeine recall . Followiody mass . (A) 300entation ,36. The terature ,26,37. B(yes/no) . Partici(yes/no) ,40. The ® performance variables were normally distributed and were described as mean and standard deviation. RPE was not normally distributed and was analyzed with a Wilcoxon Signed Rank test and described as median and standard deviation. Paired samples t-tests were used to determine differences between the caffeine and placebo conditions in the total numbers of repetitions. An additional paired samples t-test was used to determine if a learning effect was present between the first and second sessions. An analysis of covariance was used to determine differences in the total number of repetitions performed during the caffeine condition between the different treatment order groups (placebo–caffeine vs. caffeine–placebo), while controlling for the total number of repetitions performed during the placebo condition. Descriptive data are provided as mean ± standard deviation. The degrees of freedom (df), critical value (cv), level of significance (p), and effect size (ES) were reported for each paired samples t-test (t). The magnitude to treatment effects (ES) were estimated with Cohen’s D and classified as “trivial” (<0.19), “small” (0.20–0.49), “moderate” (0.50–0.79), and “large” (>0.80) [p < 0.05.Data were entered into SPSS 25  for analysis. Dependent variables were tested for normality using the Kolmogorov-Smirnov test. The CrossFit (>0.80) . A signi® performance  between the caffeine (468.6 ± 114.7 repetitions) and placebo conditions  = −0.178, p = 0.861)  (t(19) = −3.791, p = 0.001, ES = 0.28 (0.09 0.50). After controlling for the total number of repetitions performed during the placebo condition, no significant treatment order effect was observed for the total number of repetitions performed during the caffeine condition between the different treatment order groups; F = 0.632, p = 0.438. No significant differences were found for CrossFit= 0.861) . No sign= 0.637) . A signiTen of the 20 subjects (50%) perceived an effect of the supplement during the caffeine condition, while seven subjects (35%) perceived an effect of the supplement during the placebo condition. After the caffeine condition, participants reported nervousness (n = 1), gastrointestinal problems (n = 1), headache (n = 1), and increased activeness (n = 6). After the placebo condition, some participants reported nervousness (n = 2), gastrointestinal problems (n = 3), headaches (n = 1), and increased activeness (n = 2).® performance and perceptual responses to exercise in CrossFit®-trained men. Because of the well-documented ergogenic effects of caffeine that are likely due to central mechanisms [® performance. The hypothesis was not supported, as no significant improvement in CrossFit® performance or perceptual responses was found after caffeine supplementation. However, a significant learning effect between the first and second sessions of “Cindy” was found. Lastly, this study aimed to provide a novel contribution to the literature regarding caffeine supplementation and muscular endurance by providing a unique high-volume muscular endurance challenge. On average, participants in the current investigation performed over 400 repetitions for the 20 min workout during the caffeine and placebo conditions, which adequately addressed this challenge. To the best of the authors’ knowledge, this is the first study to determine the effects of caffeine supplementation on performance during the CrossFit® workout “Cindy”, which taxes multiple muscle groups for an extended period of time with minimal rest.The purpose of this investigation was to determine the effects of acute caffeine supplementation on CrossFitchanisms ,3,5,42, −1 body mass) did not significantly increase the mean CrossFit® performance. These results echo the findings of Fogaca and colleagues, who found no effect of a 6 mg·kg−1 body mass dose of caffeine on CrossFit® performance prior to a CrossFit® bout including as many rounds as possible of double-unders and power snatches in 10 min [In the current study, caffeine supplementation  participants, seems to follow those trends regarding the numbers of repetitions performed. The significant learning effect observed appears to echo findings by Crawford and associates [® workout and reported a ~16% increase in work capacity after 6 weeks of a high-intensity functional training intervention that followed a CrossFit® template for novice healthy adult participants. The current investigation assessed CrossFit to date ,26,37. Bectively ,27. The sociates , who mea® performance would likely pose a meaningful advantage during competition. Collectively, these findings may be a concern for researchers and practitioners, as the learning effect during CrossFit®-based workouts may outweigh the effects of ergogenic aids, even among trained CrossFit® participants, where the learning effect is thought to be low. While the intervention by Crawford did elicit improvements in physical fitness (range 3.3–8.8%), it is possible that the increase in work capacity reported could be due to a learning effect, as the investigators did not familiarize subjects with the work capacity test . Moreove®-trained men who were able to complete “Cindy” as prescribed as a high-volume muscular endurance workout, and the fact that it is the first study to document a learning effect during a CrossFit® workout. However, the current investigation does not escape limitations. The significant learning effect documented may have been absolved with the inclusion of a familiarization session; however, efforts were made to reduce the participant burden with just two laboratory visits. Although 20 CrossFit®-trained men completed the study, the sample size is reasonably small. However, the average sample size reported in a recently published systematic review on the effects of caffeine in trained soccer players was 15 participants; therefore, the current investigation reflects similar sample sizes compared to other studies regarding caffeine’s ergogenic effect in trained populations [The strengths of the current investigation include a robust study design, with subjects serving as their own controls, the recruitment of CrossFitulations . Participant training volume was not reported leading up to or during the investigation. Although participants were instructed not to exercise vigorously for 24 h prior to each testing session or change their training regimen during the study period, fatigue and/or delayed onset muscle soreness from other training sessions could impact the results. However, fatigue and/or delayed onset muscle soreness was unlikely given the significant increase in performance during the second session. Additionally, invasive measures to determine blood caffeine concentration and caffeine metabolism were not evaluated. As recently identified, caffeine supplementation may have a “responder” vs. “non-responder” nature, which limits the translation to “non-responder” populations . The cur® participants. Caffeine supplementation in women is complicated by the effects of estrogen and oral contraceptive steroids on caffeine metabolism, both of which appear to prolong the effects of caffeine in the body [® protocols. These results also reflect the findings of Fogaca and colleagues who reported no difference in post-workout RPE between the caffeine and placebo conditions using the CR10 Borg Scale [® athletes [® performance.Additionally, the effects of caffeine supplementation were evaluated in men and may not be generalizable to women CrossFitthe body ,51. To ithe body ,53,54. Trg Scale . Althougathletes ,55. Lastathletes . However−1 body mass ingested 60 min prior to the CrossFit® workout “Cindy” did not change the number of repetitions performed in CrossFit®-trained men. Additionally, despite the central effects of caffeine, no significant changes in perceptual responses during the exercise bout were found. Caffeine’s ergogenic effects are well documented in the literature but were not present during the CrossFit® workout “Cindy”. Practitioners and athletes should be aware that a learning effect with CrossFit® workouts seems likely and that some caffeine “non-responders” may exist; thus, evaluating caffeine’s effectiveness prior to competition for a specific athlete is encouraged.In conclusion, caffeine supplementation at 5 mg·kg


Azotobacter vinelandii mutant strains in shake flasks under microaerophilic conditions. In cultures developed at oxygen transfer rate (OTR) values close to 5 mmol L−1 h−1, the highest viscosifying power (1.75 L g−1) and weight-average molecular weight (3112 ± 150 kDa) were achieved in cultures performed with the AT9 strain. These values were higher than those obtained for the alginates produced by the parental strain ATCC 9046 grown under similar OTR conditions. In contrast, the alginate produced by the GG9 and OPAlgU + exhibited a very low weight-average molecular weight and therefore a poor viscosifying power. Our results have shown that by the cultivation of AT9 strain under microaerophilic conditions it is possible to obtain a polymer having a high weight-average molecular weight and excellent viscosifying capacity. Therefore, it could be a viable strategy for producing alginates for industrial applications.Alginates are polysaccharides that are of interest in various industrial applications. This is due to the viscosifying properties of alginates, which depends on the weight-average molecular weight. The aim of the present study was to evaluate the changes in alginate quality, in terms of the viscosifying power and weight-average molecular weight of the polymer produced by  These polymers have gel-forming and viscosifying properties, besides, they are characterized by their non-antigenicity, biocompatibility and biodegradability ). Th et al.,  and Díaz et al., . It was  et al.,  have sho culture . In this culture  found thA. vinelandii have proven to be a very useful tool for alginate production with a high yield and specific chemical characteristics. Mutants as OPAlgU+ and GG9, among others, have shown to be potential sources for the production of alginate with a high molecular mass in shake flasks and bioreactor [A. vinelandii mutant derivative of strain AEIV , which has the mucG gene inactivated , has shA. vinelandii complemented strain OPAlgU+, in which alginate production was restored, showed a specific oxygen consumption rate (O2q) and alginate production very similar to that of strain ATCC9046. It is important to point out that in those studies, the strains were grown under diazotrophic and high aeration conditions (OTRmax = 30 mmol L−1 h−1), where alginate synthesis is not best.In a more recent study, Castillo et al.  found thA. vinelandii mutant strains under microaerophilic conditions and no-nitrogen fixation.In spite of the potential for the production of alginates having high weight-average molecular weight reported for these strains; the studies mentioned were carried out under culture conditions (nitrogen fixation and high oxygen transfer) that are not the best to promote the synthesis of high molecular weight alginate. Therefore, the aim of this study was to evaluate the viscosifying power and weight-average molecular weight of alginates synthesized by 22.1A. vinelandii strains used in this work were the wild type ATCC 9046, and its derivative mutant AT9 , the GG9 strain (mucG::mTn5) derived from the AEIV strain [algU−) strain [mucG::miniTn5 insertion, this strain was transformed with 5 μg of chromosomal DNA from mutant GG9, according to previous reported [mucG locus, to carry the mucG::miniTn5 mutation (data not shown). The resulting strain was named AT9.The V strain  and stra) strain . In ordereported . Transfo2.2−1 of spectinomycin was used. In the case of OPAlgU + mutant strain, 2 μg mL−1 of kanamycin were added.All strains were grown in modified Burk’s medium as previously described Gómez-Pazarín et al. . The ini2.3A. vinelandii strains were conducted in shake flasks of 250 mL with 50 mL of filling volume. Cultivations were maintained at 29 °C with a shaking frequency of 200 rpm in an orbital incubator with a shaking diameter of 25 mm. Before inoculation, protein was quantified in the inoculum by the Lowry´s method in order to adjust the inoculum volume to get an initial concentration of protein in the range of 0.06–0.08 g L−1, equivalent to 0.15–0.2 g L−1 of dry weight biomass, considering a protein content of 40 %. Evaluation of the OTR and the respiratory quotient (RQ) were determined by a respiration activity monitoring system (RAMOS), described by Anderlei et al. [Cultures of the i et al. . In addi2.42.4.1Microbial growth was evaluated through protein measurements using the Lowry method, as described by García et al. . Sucrose2.4.2P3HB content was quantified by HPLC after its conversion into crotonic acid, as it was previously described by Castillo et al. . Alginat2.4.3−1. The detector signal was processed with a PC compatible software . Calibration of the columns were performed via a standard calibration method using as molecular weight standards, the standard pullulans within a range of 58–2350 kDa. For this purpose, the standard kit P-82 (58–1600 kDa) and P2500 (2350 kDa) from Shodex.Co (Japan) were used. Alginate solutions were filtered through 0.22 μm Millipore membranes to remove microgels [Viscosity of culture broths was measured using a cone/plate rheometer as described by Peña et al. . The algicrogels .2.4.40 (g L−1) was the initial protein concentration, O2q (mmol g-1  h−1) the specific oxygen uptake rate, RQ (-) is the respiratory quotient, CTR (mmol L-1  h−1) is the carbon dioxide transfer rate and OTR (mmol L-1  h−1) is the oxygen transfer rate.Equations used for the determination of specific oxygen uptake rates and respiratory quotient were the following:The growth rate (μ) was calculated as described by Eq. 333.1A. vinelandii. For this purpose, we constructed, as described in Materials and methods, strain AT9, an ATCC 9046 derivative carrying a mucG::miniTn5 insertion. In addition to the MucG deficient strains AT9 and GG9, the OPAlgU + strain was also included in this study.Considering the high mean molecular mass that the absence of MucG conferred to the alginate produced by mutant GG9, we wanted to explore this effect in the background of strain ATCC 9046, which is a natural alginate over-producing strain of max) value was reached after 5 h of cultivation and this remained practically constant until 30 h for the all strains evaluated. Specifically, the OTRmax reached values of 4.8 ± 0.28, 5.23 ± 0.18, 4.84 ± 0.33 and 5.24 ± 0.29 mmol L−1 h−1 for ATCC 9046, OPAlgU+, GG9 and AT9, respectively. After 30 h of cultivation, a straight increase in the OTR was observed in the cultures with the OPAlgU+, GG9 and AT9 strains. This increase could be related with changes in the metabolism of bacteria, from a condition of no-nitrogen fixation to a condition of nitrogen-fixation. It is known that when A. vinelandii is grown under diazotrophic conditions, a high respiration rate is induced by the respiratory protection of the nitrogenase [−1 of yeast extract) were conducted. Analyzing the OTR profiles (max =1.15 ± 0.04 g L−1) and specific growth rate (μ = 0.10 ± 0.021 h−1) were achieved using the GG9 strain (The oxygen transfer rate (OTR) evolution of the cultivations of strains ATCC 9046, AT9, GG9 and OPAlgU + are shown in 9 strain . These v3.2O2q), is of great importance in order to understand the metabolism in each strain. The highest value of O2q was achieved in cultures with the AT9 strain   . For exa−1 h−1, the RQ was 8–10 % higher in the cultures with the ATCC 9046 and GG9 strains, with respect to the values for the cultures with the AT9 and OPAlgU + strains. These results suggest that the AT9 and OPAlgU + strains are channeling a higher amount of acetyl-CoA through the TCA cycle. This hypothesis is supported by the increase in 25 % of the O2q observed in cultures conducted with the AT9 strain, as well as in its RQ value of 1  for AT9 strain  is a very useful parameter for analyzing alginate and poly-3-hydroxybutyrate (P3HB) biosynthesis . It is ilue of 1 , Table 19 strain . These dO2q increased, the carbon fluxes through the alginate biosynthetic pathway also increased [A. vinelandii mutant strains, low values of RQ (close to 1.05) were associated with a higher conversion of the carbon source into alginate; whereas high RQ values (close to 1.13) are related with the use of the carbon source for the P3HB biosynthesis.Previous studies with the ATCC 9046 strain have shown that when the OTR, and consequently, the ncreased ,20,21. Increased ,11. On t3.3−1) reached by AT9 strain was higher (27 ± 1.8 mPas) with respect to that obtained with the wild type strain ATCC 9046 and than those determined for the GG9 and OPAlgU + strains . This value of weight-average molecular weight was higher than that previously reported for this strain grown under similar OTRmax conditions. For example, Gómez-Pazarín et al., [−1 h−1, respectively. To our knowledge, the weight-average molecular weight of the alginate produced by AT9 strain is one of the highest reported so far, and also we confirmed that MucG inactivation results in the production of alginates with a high weight-average molecular weight.As shown in  et al.,  reportedIn a previous work, it was reported that the MucG protein had a negative effect on the alginate molecular mass . The culA. vinelandii and the molecular mechanism by which MucG controls this trait.4Our results have shown that the viscosifying power, as well as the weight-average molecular weight of the alginates produced are affected by the strains under study. It was possible to obtain alginates with a high weight-average molecular weight and cultures broths of high viscosity using the mutant AT9 under non-diazothrophic conditions. This finding will open up new possibilities for the implementation of alternative molecular strategies for the scaling-up of alginates with improved viscosifying.The authors declare that they have no competing interests.Andres García: Investigation, Writing - original draft. Tania Castillo: Investigation, Conceptualization. Diego Ramos: Investigation. Carlos L. Ahumada-Manuel: Writing - review & editing. Cinthia Núñez: Writing - review & editing. Enrique Galindo: Writing - review & editing. Jochen Büchs: Writing - review & editing. Carlos Peña: Writing - review & editing, Supervision, Project administration, Funding acquisition.


The identification of drought-tolerant olive tree genotypes has become an urgent requirement to develop sustainable agriculture in dry lands. However, physiological markers linking drought tolerance with mechanistic effects operating at the cellular level are still lacking, in particular under severe stress, despite the urgent need to develop these tools in the current frame of global change. In this context, 1-year-old olive plants growing in the greenhouse and with a high intra-specific variability (using various genotypes obtained either from cuttings or seeds) were evaluated for drought tolerance under severe stress. Growth, plant water status, net photosynthesis rates, chlorophyll contents and the extent of photo- and antioxidant defenses  were evaluated under well-watered conditions and severe stress (by withholding water for 60 days). Plants were able to continue photosynthesizing under severe stress, even at very low leaf water potential of −4 to −6 MPa. This ability was achieved, at least in part, by the activation of photo- and antioxidant mechanisms, including not only increased xanthophyll cycle de-epoxidation, but also enhanced α-tocopherol contents. “Zarrazi” (obtained from seeds) and “Chemlali” (obtained from cuttings) showed better performance under severe water stress compared to the other genotypes, which was associated to their ability to trigger a higher antioxidant protection. It is concluded that (i) drought tolerance among the various genotypes tested is associated with antioxidant protection in olive trees, (ii) the extent of xanthophyll cycle de-epoxidation is strongly inversely related to photosynthetic rates, and (iii) vitamin E accumulation is sharply induced upon severe chlorophyll degradation. Olea europaea L.) is one of the most widespread crops in Mediterranean-type agroecosystems, with high economic and social importance. In semi-arid areas of the Mediterranean basin, the scarcity of precipitation combined with extreme climatic conditions such as high temperatures increase water stress in olive trees. It is particularly in this context that serious concerns have been raised about the impact of climate change on olive agricultural productivity. According to Olive tree  and in the case of tocopherols additionally preventing lipid peroxidation in thylakoid membranes . By taking advantage of the variability associated to the genotypic diversity of various young olive trees, we aimed here to unravel the link, if any, between leaf water potential, photosynthesis rates and chlorophyll loss with mechanisms of photo- and antioxidant protection in juvenile olive trees subjected to severe drought. Furthermore, it is discussed how the development of new photo- and antioxidant markers may be useful in order to select and develop new drought-tolerant olive tree cultivars for oil production in semi-arid regions. Due to the difficulty of performing studies in mature olive trees in field trials, we used here trees at an early stage of development. The juvenile phase of trees usually represents the most abiotic stress sensitive stage in woody plants ; therefoOlea europaea L.) were grown under natural daylight conditions with a mean maximum daily photosynthetically active radiation (PAR) of 1017 μmol m–2 s–1 in a plastic greenhouse at the Olive Tree Institute of Sfax. Plants were obtained from cuttings and/or from germination of seeds. Rooted cuttings were obtained from the two main Tunisian olive cultivars “Chemlali” and “Chetoui,” while seedlings were regenerated from the seeds of wild olive tree “Oleaster” and from three olive cultivars “Chetoui,” “Chemlali” and “Zarrazi” which are cultivated, respectively, in the north, the center and the south of Tunisia. All seeds were collected from the experimental station “Taous” of the Olive Tree Institute , except for the wild olive tree “Oleaster,” whose seeds were collected from the National Park of Ichkeul .One-year-old olive trees  and then irrigated twice a week with half-strength Hoagland”s solution , immediately frozen in liquid nitrogen and stored at −20°C until analyses.Hi, Li), at 30 days  and at 60 days  of drought. Stem growth rate (SGR) and leaf production rate (LPR) were estimated from these parameters according to the equations described by Stem elongation (cm) and the number of leaves per plant were measured for each plant at the beginning of stress (L) was measured using a Scholander pressure chamber model SKPM 1400  between 9:00 and 11:00 local time. Net photosynthesis rate (An), stomatal conductance (gs), transpiration (E), water use efficiency (A/E) and intercellular to ambient carbon dioxide concentration ratio (Ci/Ca) were determined between 9:00 and 11:00 using a portable gas exchange system (Cl-340 Handheld Photosynthesis System). During leaf gas exchange measurements, photosynthetically-active radiation was set at 1250 μmol⋅m–2⋅s–1, leaf temperature at 35°C, CO2 concentration at 430 ppm and vapor pressure deficit at 5 KPa. All measurements were carried out on fully-expanded young leaves taken from the median part of the shoot. Leaf water potential determination was performed using the whole leaf with the petiole, taking care the latter remained intact during measurements.Leaf water potential  on a Dupont non-end capped Zorbax ODS-5-μm column  at 30°C at a flow rate of 1 ml⋅min–1 for 38 min. The solvent mixture for the gradient, detection at 445 nm  and calculation of pmol/are ratios for quantification of chlorophyll a, chlorophyll b, neoxanthin, violaxanthin, antheraxanthin, zeaxanthin, lutein, and β-carotene were performed as described by The extraction and HPLC analysis of chlorophylls and carotenoids were performed as described by The same methanolic extract was used for the determination of tocopherols, which were separated isocratically on a normal-phase HPLC system  and quantified using a fluorescent detector as described by p ≤ 0.05. Correlation between variables was determined according to Pearson tests.Statistical analyses were carried out with the SPSS Base 20.0 software . A two-way analysis of variance (ANOVA) was used to examine cultivar and treatment effects. Duncan”s multiple range tests were used for mean comparison at p < 0.001) (Drought and cultivar effects on growth were observed . Leaf pr< 0.001) .L showed the lowest values (−1.4 MPa) in both “Chetoui” rooted cuttings and seedlings, which significantly differ to other genotypes except “Chemlali” (L decreased significantly (p < 0.001) in all olive trees compared to control plants (p < 0.001). The reduction of ΨL ranged from 71 to 88%.In irrigated conditions, Ψhemlali” . The higl plants . The resn decreased significantly after 60 days of drought stress in all genotypes, irrespective of whether the plants were obtained from cuttings or seeds , no significant difference was observed in gs and E between irrigated and stressed plants  was recorded on leaf chlorophyll (Chl a + b) content after 60 days of drought stress (A significant interaction between cultivar and water regime (t stress . Comparet stress .p < 0.001). The increase in VZA/Chl ranged in all genotypes between 1.3 and 1.9-fold in stressed plants compared to their respective controls. The highest VZA/Chl ratio was recorded in “Zarrazi S,” followed by “Chemlali” rooted cuttings, whereas the lowest one was obtained for “Oleaster S” (p < 0.001). The stress-related increase in DPS ranged between 1.2 and 1.5-fold relative to controls, respectively, in “Chetoui” seedlings (“Chetoui S”) and “Zarrazi S.” In contrast, the β-carotene per unit of chlorophyll (β-car/Chl) decreased significantly under stress conditions in all olive trees with “Zarrazi S” showing the lowest decrease (27.2%). However, the lutein to Chl ratio (Lut/Chl) increased significantly with stress in “Chemlali S “and remained unchanged in the rest of the olive trees compared to control plants (Drought stress increased significantly the xanthophyll cycle pool expressed per unit of chlorophyll (VZA/Chl) and the de-epoxidation state of the xanthophyll cycle (DPS) in olive trees compared to control plants  increased significantly in stressed olive trees compared to control plants, with the exception of “Chemlali S,” “Oleaster S” and “Chetoui” rooted cuttings, in which no significant changes were observed . This inL , An , Chl a + b . It appears that a linear and significant increase of DPS and VZA levels occurred when the ΨL decreased below −4 MPa and An was reduced below 5 μmol CO2⋅m–2⋅ s–1. Moreover, a significant correlation was observed between ΨL and α-Toc/Chl, and between Chl a + b and α-Toc/Chl  of ΨL. This difference is probably associated with the different mechanisms of osmotic adjustment adopted by the olive trees under water stress conditions, as previously reported by n was recorded in “Zarrazi S” and, in a second degree, in “Chemlali” rooted cuttings, which allows them to withstand water stress more effectively than the other tested olive trees. These results are in accordance with those found by n in “Chemlali” classified as a drought-tolerant cultivar. However, “Oleaster S” can be considered the most sensitive to water stress as they showed the highest depressive effect of water stress on An (92.8% reduction compared to control). In susceptibility to water stress, “Oleaster S” was followed by “Chemlali S,” “Chetoui S” and “Chetoui” rooted cuttings with a mean of 88.4% reduction on An compared to its respective control. Moreover, the increase in water use efficiency  could be considered as a drought tolerance index  and a high vapor pressure deficit (5 KPa), which explains the low stomatal conductance and photosynthesis rates together with the high Ci/Ca values observed in well-watered plants. Similar stomatal conductance, net photosynthesis rates and Ci/Ca ratios were observed for anti-sense Rubisco transgenic tobacco plants . The lowest VZA/Chl level (302 mmol⋅mol–1) was recorded in “Oleaster S.” Indeed, water stress is known to induce the activation of the xanthophyll cycle, in which violaxanthin is de-epoxidized to antheraxanthin and then zeaxanthin, enabling excess light energy to be dissipated as heat (L and An (L decreased below −4 MPa and An turned less than 5 μmol CO2⋅m–2⋅s–1. Indeed, loss of leaf turgor and photosynthetic limitation might be involved in triggering the activation of photoprotective mechanisms in young olive trees under severe water stress. β-carotene/Chl ratio was significantly decreased under drought conditions. A similar response was reported by L decreases paralleled an accumulation of α-Toc/Chl in leaves in all studied cultivars, although significantly only for “Chemlali” rooted cuttings, “Chetoui S” and “Zarrazi S.” Differences observed in α-Toc/Chl level could be related to genotypic differences in antioxidant capacity among olive trees. α-Tocopherol is involved in the elimination of 1O2 (It is interesting to note that despite the very low values of −6 MPa achieved, some photosynthetic activity was maintained in some cultivars of olive trees. At leaf water potentials below −4 MPa, net photosynthesis rate ranged between 0.45 and 3.57 μmol COistachio . Indeed,o plants . Leaf gao plants . Hence, o plants . Our res as heat . The incL and An , 4. It an of 1O2  and amonn of 1O2 . α-Toc/Cn of 1O2 . The accn of 1O2 .A summary of the differences in the response to severe water stress among olive trees are shown in In conclusion, withholding irrigation for 60 days induced a considerable decrease in growth, leaf water potential and net photosynthesis rates in 1-year-old olive trees. This might lead to the activation of photoprotective mechanisms, including, increases in xanthophyll cycle de-epoxidation, and accumulation of α-tocopherol as chlorophyll loss progressed in severely stressed plants. These increases may contribute to the survival of young olive trees under severe water stress conditions. The study also revealed that “Zarrazi” seedlings may serve as drought-tolerant rootstock which exhibited a lower decrease in growth and net photosynthesis rate, as well as a higher antioxidant activity. “Oleaster,” “Chemlali” and “Chetoui” seedlings could be comparatively considered in this order as more drought-sensitive rootstocks. “Chemlali” rooted cuttings showed the tolerance behavior closer to that of “Zarrazi” seedlings, thus, “Chemlali” rooted cuttings are interesting to create vegetatively propagated drought-tolerant rootstocks and/or cultivars used for breeding programs.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SB, AC-R, and SM-B conceived and designed the study. SB, EF, and MM performed the experiments. SB, OE, and SM-B analyzed the data and wrote the manuscript, while other authors performed a critical revision of the manuscript. AC-R, FBA, LF, and SM-B supervised the work. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


APOE, sex, and longitudinal cognitive decline.The abnormal deposition of tau begins before the onset of clinical symptoms and seems to target specific brain networks. The aim of this study is to identify the spatial patterns of tau deposition in cognitively normal older adults and assess whether they are related to amyloid-β (Aβ), APOE genotype, and sex. Linear mixed effects models were built to test whether tau in each component predicted cognitive decline. Finally, we ordered the spatial components based on the frequency of high tau deposition to model tau spread.We included 114 older adults with cross-sectional flortaucipir (FTP) and Pittsburgh Compound-B PET in addition to longitudinal cognitive testing. A voxel-wise independent component analysis was applied to FTP images to identify the spatial patterns of tau deposition. We then assessed whether tau within these patterns differed by Aβ status, We found 10 biologically plausible tau patterns in the whole sample. There was greater tau in medial temporal, occipital, and orbitofrontal components in Aβ-positive compared with Aβ-negative individuals; in the parahippocampal component in ε3ε3 compared with ε2ε3 carriers; and in temporo-parietal and anterior frontal components in women compared with men. Higher tau in temporal and frontal components predicted longitudinal cognitive decline in memory and executive functions, respectively. Tau deposition was most frequently observed in medial temporal and ventral cortical areas, followed by lateral and primary areas.APOE ε2ε3, sex and cognitive decline. These patterns could be used to predict the regional spread of tau and perform in vivo tau staging in older adults.These findings suggest that the spatial patterns of tau in asymptomatic individuals are clinically meaningful and are associated with Aβ, The online version of this article (10.1007/s00259-019-04669-x) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) is characterized by a long-lasting preclinical phase, in which the presence of progressively increasing pathology is not yet accompanied by clinical symptoms . In thisAPOE, sex, and cognition on these patterns have not yet been assessed. In addition, no studies have examined tau PET differences between APOE ε2ε3 and ε3ε3 carriers [APOE, sex, or longitudinal cognitive decline. We also sorted the spatial patterns based on the frequency of tau positivity across subjects to determine a proxy for regional tau spread.The relationship between the spatial patterns of tau and biomarkers associated with risk of developing AD remain unexplored in older adults. In particular, the effects of Aβ-positivity, carriers , 10. In One hundred and fourteen cognitively normal individuals ≥ 60 years of age were included from the Berkeley Aging Cohort Study. All individuals underwent structural MRI, Pittsburgh Compound-B PET (PiB), and Flortaucipir (FTP) PET imaging in addition to neuropsychological testing. Inclusion criteria included a baseline Mini-Mental State Examination (MMSE) score ≥ 25; normal performance on cognitive tests; no neurological, psychiatric, or major medical illness; no medications affecting cognition; and no imaging contraindications, and that all participants were community-dwelling.Z scores for memory and executive function domains were created, as previously described [Z scores were calculated using the means and standard deviations from the first cognitive session data of a larger sample of 150 healthy participants from the Berkeley Aging Cohort Study that also included the 86 older adults studied here. The memory composite score included the short- and long-delay free recall tests of the California Verbal Learning Test [Eighty-six of the 114 subjects underwent longitudinal neuropsychological testing before and up to 1 year after the date of the FTP scan. These subjects had between 2 and 11 cognitive testing sessions  over a period of 4.6 ± 2.9 years, with an average delay of 1.2 ± 0.4 years between sessions. For each testing session, composite ing Test  and the ing Test . The exeing Test .APOE alleles was performed using a TaqMan Allelic Discrimination Assay using a Real-Time PCR system , as previously described [APOE information.The determination of escribed . One sub3).All PET scans were acquired at the Lawrence Berkeley National Laboratory on a Siemens  Biograph 6 Truepoint PET/computed tomography (CT) scanner in 3D acquisition mode. FTP was synthesized using a TracerLab FXN-Pro  synthesis module with a modified protocol based on an Avid Radiopharmaceuticals  protocol. Participants were injected with 10 mCi of tracer and scanned in list mode from 75 to 105 min post-injection. Data was subsequently reconstructed as 4 × 5-min frames within 80 to 100 min post-injection. PiB was also synthesized according to a previously published protocol . ImmediaT1-weighted structural MRI images were acquired on a 1.5-T Siemens Magnetom.Avanto scanner using a magnetization-prepared rapid gradient echo sequences with the following parameters: repetition time = 2110 ms, echo time = 3.58 ms, flip angle = 15°, voxel size = 1mm isotropic.Both PiB and FTP PET frames were motion-corrected, time-averaged, and coregistered to their T1-weighted images. For PiB images, distribution volume ratios (DVRs) were calculated using Logan graphical analysis on PiB frames corresponding to 35 to 90 min post-injection using all the frontal, temporal, and parietal regions provided by the Desikan-Killiany atlas of FreeSurfer as well as a cerebellar gray matter reference region , 20. Subhttps://fsl.fmrib.ox.ac.uk/fsl/fslwiki) and smoothed using an 8-mm Gaussian filter. Warping to MNI was performed using the transformation parameters derived from warping the T1-weighted images to the MNI152 template. The average delay between the PiB and FTP scans was 24.8 ± 99.2 days.For FTP, standardized uptake value ratio (SUVR) images were created using the mean inferior cerebellar gray matter uptake as the reference region –25. ThesIn this study, we did not apply partial volume corrections (PVC) to the FTP data because methods suitable for voxel-wise analyses fail to account for tracer binding in the multiple brain compartments  that can affect FTP images.Z score > 2.0, which corresponds to a two-tailed significance value of p < 0.05.FTP PET images were submitted to an independent component analysis using the group ICA of functional MRI Toolbox (GIFT) . This daAPOE genotype and sex, we compared mean SUVRs within these components between Aβ− and Aβ+ groups, subjects with different APOE genotypes, and male and female participants. These analyses were carried out using permutation tests with 1000 replicates, while controlling for age, sex, education, and PiB DVR, when appropriate. To identify and exclude potential outliers in tau SUVRs, we applied the Tukey method (1.5 × interquartile range). Adjustment for multiple comparisons was performed using false discovery rate (FDR) corrections [q < 0.05. For all significant group differences, we calculated effect sizes using Cohen’s d [Differences in baseline characteristics between groups were analyzed using chi-squared tests for binary variables and analyses of variance for continuous variables. To assess whether tau burden within each spatial component differed by Aβ status, rections  at q < 0ohen’s d .To evaluate whether the spatial patterns of tau were associated with change in cognition over time, we fitted linear mixed effect models, implemented in R using “lme4.” In these models, we included memory or executive scores as dependent variables, and tau component SUVRs, time since first cognitive evaluation, age, sex, education, and PiB DVR as fixed effects. These models included all main effects, the interaction between tau SUVRs and time, and random effects for intercepts and slopes: cognitive scores ~ time × tau network SUVR + age + sex + education + PIB DVR + (time|subject). We built separate models for each tau component.Z scores for the mean SUVR in each tau component across all participants. For each participant, components with a Z score > 2.5 were considered to be positive for tau deposition. Using this approach, the frequency of positive tau deposition was assessed for each component. Components were then ordered from highest to lowest frequency since we assumed that components with a higher frequency of positive tau deposition comprised earlier accumulating regions. This approach allowed us to estimate spatial tau spread using cross-sectional data.Finally, to determine the order of tau spreading across the different components, we selected Aβ− individuals whose FTP SUVR values in the entorhinal cortex were < 1.2 to obtain regional mean and standard deviation values of SUVR, following a previously described procedure . Using tAPOE genotypes: ε2ε3 (n = 11), ε3ε3 (n = 71), or ε3ε4 (n = 28). In addition, 3 participants were ε2ε4 carriers, which were excluded from the analyses due to the small number of subjects in this group.Characteristics of the sample are summarized in Table d = 0.4, p = 0.031) and had a higher prevalence of the APOE ε3ε4 genotype  than their Aβ-negative peers. As expected, ε3ε4 carriers showed higher PiB DVRs compared with both ε2ε3  and ε3ε3  carriers, and ε2ε3 were less educated than ε3ε3 carriers . In our sample, men had lower executive function scores compared with women .In this study, Aβ-positive individuals were less educated . This component was characterized by high tau signal in a thin elongated cluster surrounding the meninges . The meninges have been previously described as an off-target binding area for FTP as well as other tau PET tracers. Importantly, their signal varies across subjects and can potentially bleed into cortical areas [The optimal number of components identified in FTP data was 15, which explained 85.2% of the total variance. Five of these components were excluded from the analyses since they had white matter voxels or consisted of thin clusters surrounding the meninges, which is an off-target binding region for al areas , 35. Ourn = 10) were included in further analyses. These components involved bilateral gray matter regions, except for 2, which were unilateral , parahippocampal , left and right inferior occipital , lateral temporo-parietal  and orbitofrontal  components compared with Aβ-negative subjects  in ε3ε3 carriers compared with those in ε2ε3 carriers after adjusting for PiB DVR and for multiple comparisons  and lateral temporo-parietal  components compared with men . However, at an uncorrected level, we identified an interaction between time and the parahippocampal , left inferior occipital , and lateral temporo-parietal  components (Supplementary Table 3), indicating that participants with higher tau within these patterns had worse longitudinal decline in memory. In addition, we also found an interaction between the superior frontal tau pattern and time  (Supplementary Table 3), indicating that participants with higher tau within this pattern had worse executive decline.Our mixed effects models did not show any significant interactions between time and the different tau patterns in cognitive decline after adjusting for multiple comparisons with FDR . These models showed lower AIC and BIC values than the models with a single interaction term between time and tau (Supplementary Table 4), indicating they were a worse fit to our data.n = 29) pattern (I). This was followed by regional involvement of ventral areas that included the left inferior occipital  (II), right inferior occipital , orbitofrontal  (III), and medial temporal  (IV) components. Once most of the ventral areas were affected by tau pathology, the more frequent set of regions with high tau deposition included lateral and medial cortical areas such as the lateral temporo-parietal  (V), parietal  (VI), and superior frontal  (VII) components. Finally, the least affected patterns with tau were the sensorimotor  (VIII) and anterior frontal  (IX) components.The progression of tau pathology across the spatial patterns is displayed in Fig. APOE ε3ε3 carriers, or women. In addition, the association we found between some of these patterns and cognitive decline suggest they could also be used to understand the variability in longitudinal cognitive trajectories among older adults.With upcoming therapeutic approaches based on tau pathology, new tau biomarkers are urgently needed for early AD . In thisAPOE ε2 allele against in vivo tau deposition in clinically normal adults. We found that the ε2ε3 genotype was associated with lower tau in parahippocampal areas compared with the more frequent ε3ε3 genotype, before and after controlling for Aβ. In contrast, we found that the increases of tau load in ε3ε4 carriers were mostly mediated by Aβ. Our results agree with previous findings of reduced neurofibrillary tangle pathology in ε2 carriers [APOE ε4 and amyloid [There is increasing evidence showing that, rather than appearing in unrelated brain regions, tau pathology occurs within the boundaries of distributed networks , 38. Howcarriers , and the amyloid , which aOne of the earliest cognitive changes in aging, and the one that has probably received most attention due to its association with AD, is the decline of memory. Here we found that higher tau load in the parahippocampal, left inferior occipital, and lateral temporo-parietal patterns predicted retrospective longitudinal decline in memory in older adults, although these associations did not remain significant after adjustment for multiple comparisons. These results potentially strengthen the hypothesis that tau pathology plays a key role in memory decline in aging , which iIn this study, to determine the “spread” of tau across the different spatial components, we calculated the frequency of high tau in each component in all participants. This approach showed that tau deposition was most frequent in the parahippocampus, followed by ventral areas such as the inferior temporal, inferior occipital, and orbitofrontal cortices. Then, from these ventral areas, tau spread into lateral temporo-parietal and medial parietal regions, and finally reached frontal areas and the sensorimotor cortex. This regional progression of tau largely mirrors the spread of neurofibrillary tangles previously reported by Braak et al. . InteresAPOE, and sex effects. In particular, the number of ε2ε3 carriers was very small (n = 11), in agreement with the low prevalence of this genotype in the population [Our study has some limitations. Although comparable with previous tau PET studies, our sample size was modest and might have limited our ability to detect additional spatial patterns or their value in characterizing Aβ, pulation . Thus, opulation –30, it ipulation . In factpulation , 35, 55,Our findings indicate that distinct spatial tau patterns are present in cognitively normal adults and Aβ-negativity, ε2ε3 carriership, and male sex are protective factors against tau deposition within these patterns. In light of the increasing recognition of AD as a network disorder that affects distributed brain systems , we hopeESM 1(DOCX 20675 kb)


Growing concern over the hazardous effect of radionuclides on the environment is driving research on mitigation and deposition strategies for radioactive waste management. Currently, there are many techniques used for radionuclides separation from the environment such as ion exchange, solvent extraction, chemical precipitation and adsorption. Adsorbents are the leading area of research and many useful materials are being discovered in this category of radionuclide ion separation. The adsorption technologies lack the ability of selective removal of metal ions from solution. This drawback is eliminated by the use of ion-imprinted polymers, these materials having targeted binding sites for specific ions in the media. In this review article, we present recently published literature about the use of ion-imprinted polymers for the adsorption of 10 important hazardous radionuclides—U, Th, Cs, Sr, Ce, Tc, La, Cr, Ni, Co—found in the nuclear fuel cycle. Spent nuclear fuel (SNF) and high-level radioactive waste (HLRW) are generated during nuclear power reactor operation and decommissioning of nuclear facilities, respectively. There are 62 different radionuclides (RNs) formed from ²³⁵U fission, which due to their instability, are subject to radioactive transformation. About 200 different radioisotopes are observed during the nuclear reactor operation, mainly in the core. Nuclear power plants around the world are the major producers of SNF and HLRW. Situated on the territory of the Slovak republic are in Jaslovské Bohunice  and Mochovce .Slovak legislation on SNF and radioactive waste (RW) management fully corresponds to international legislation and regulations. Requirements for the management of SNF and RW have been regulated in the Act No. 541/2004 Collection on the peaceful use of nuclear energy (Atomic Act) and in its implementing regulation and the Act No. 308/2018 Collection on the National Nuclear Fund and amending the Atomic Act. The Atomic Act defines RW as any unusable material in gaseous, liquid, or solid form, which cannot be released into the environment due to the content of RNs or the level of RN contamination. Details on the requirements for the management of nuclear materials, RW, and SNF are also defined by the Decree of the Nuclear Regulatory Authority of the Slovak Republic No. 30/2012 Collection. It is assumed that the individual units of nuclear power plants in Slovakia will produce 2500 tons of SNF and 3700 tons of HLRW during their operation period. Produced SNF can be considered either as a usable source of energy and RNs and can be reprocessed or can be destined for final disposal if it is considered as RW.(1)Fission products (FPs): formed by the fission of fuel nuclei by neutrons. These are highly active RNs; whose half-life ranges from a few seconds to thousands of years:-,¹⁰⁶Ru, ¹³¹,¹³³,¹³⁵I, ¹³⁴,¹³⁵,¹³⁷Cs, …),volatile substances ,non-volatile substances , tritium (³H).noble gases Activation products (APs): formed by the interactions of neutrons with inactive nuclides of the coolant, moderator, or reactor construction materials, respectively. By activating the reactor construction materials, the following main activation corrosion and erosion products are formed:-,¹¹⁰ᵐAg, ¹²⁴,¹²⁵Sb, ¹⁵²Eu, ¹⁶⁶ᵐHo,steel: ⁵¹Cr, ⁵⁴Mn, ⁶⁰Co, ⁶³Ni, ⁵⁹Fe, ⁹³Mo, ⁹⁴Nb, ¹⁰⁸ᵐ-,¹⁵⁴,¹⁵⁵Eu, ¹⁶⁰Tb, ¹⁸¹Hf, ¹⁸²Ta.concrete: ³⁶Cl, ⁴¹Ca, ⁴⁶Sc, ⁵¹Cr, ⁵⁹Fe, ⁵⁹, ⁶³Ni, ⁶⁰Co, ⁶⁵Zn, ⁸⁵Sr, ¹²⁴Sb, ¹³¹Cs, ¹⁵²By ²³⁵U fission two main groups of RNs are created:,²³⁹Np, ²³⁸,²⁴²Pu, ²⁴¹,²⁴³Am, ²⁴²,²⁴⁴Cm. The RNs with the half-life longer than >200,000 years, such as: ⁷⁹Se, ⁹³Zr, ⁹⁹Tc, ¹⁰⁷Pd, ¹²⁶Sn, ¹²⁹I, ¹³⁵Cs which are difficult to measure, play significant roles in the separation processes. However, there are also other problematically detected RNs with shorter half-life < 200,000 are i.e., ⁵⁹,⁶³Ni, ⁷⁹Sc, ¹⁴⁶Sm, and actinide ²³⁶U.The gradual capture of neutrons by fuel nuclei and their subsequent radioactive transformations produce transuranic elements. The most commonly used source nuclides of transuranic elements are ²³⁸U, ²³⁷Uranium is the most significant metal for energy production and also the primary waste generated from nuclear reactor operation. Uranium is known for its toxicity, apart from the fact that ingestion of uranium can damage the liver and cause acute renal failure in humans [Thorium is a metallic element of the actinides. For energy generation, thorium is a better choice than uranium due to its abundance on Earth, and the relatively less radioactive waste generated from its operation in a nuclear reactor. More than 99.99% of natural thorium is ²³²Th, the rest being represented by ²³⁰Th and ²²⁸Th. Thorium metal occurs naturally in the environment. The increasing release of these metals has spiked due to mining activity, e.g., milling, and processing operation, coal production, and phosphate production for fertilizers. Besides its attractive properties for nuclear energy generation, it is toxic element for human health, as a potentially carcinogenic substance. Thorium exposure is primarily due to inhalation, intravenous injection, ingestion and absorption through the skin [Cesium-137 and Strontium-90 are isotopes produced during a nuclear power plant’s operation by ²³⁵U fission and are the most harmful RNs due to their strong beta emission properties and long half-life . The nuclear accidents in Chernobyl and the Fukushima Daiichi released large amounts of cesium isotopes  into the surroundings [Cerium-144  is formed by the fission of uranium and accumulates in the human body leading to acute myocardial infarction, leukemia, and imbalance in blood biochemistry. It also causes a toxic effect damaging the cell membranes in marine life, consequently damaging the nervous system and reproduction system in organisms [Technetium-99, which as a FP is highly toxic due to its high mobility in water and a half-life of 2.11 × 105 years [Lanthanum-140  occurs as a FP of uranium; it has no biological role and is considered an environmental threat due to its radioactivity [Chromium-51  is an AP and can be used as a radioactive tracer in isotope dilution analysis. Chromium is needed in trace amounts for the proper functioning of the human body, but its excess can cause carcinogenic effects [Nickel-59, 63 are isotopes generated in the nuclear reactor as APs [Cobalt-60 is generated in the nuclear power plant. ⁶⁰Co is the source of high gamma radiation and has a half-life of 5.27 years. The presence of cobalt in the environment, considering its radioactivity, can cause detrimental effects on ecology and human health [n humans . The uran humans ,3,4. Accn humans . Thoriumthe skin . The disthe skin ,8. Cesiuoundings ,11. Cerirganisms ,13. The activity . Chromiu effects . Nickel-r as APs . ⁵⁹Ni anr as APs . Nickel r as APs . Cobalt-n health .For the mitigation of radionuclides’ environmental impact, various methods have been used to date such as phytoremediation , ion-exc-as a component in a multibarrier system for the safe storage and final disposal of SNFs and RWs, respectively,-as a separation component for RN isolation from RN mixtures, mainly for further use, or in radiochemical analysis for target radionuclide separation and subsequent qualitative and quantitative determination.The Department of Nuclear Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, has long been dedicated to the development and research on various highly selective and radiation-stable sorbents, which are used:Ion-imprinted polymers (IIPs) can solve this issue and are widely used for in radionuclide adsorption or removal processes . IIPs arN-(o-carboxyphenyl) maleamic acid (CPMA)—for Th(IV) ion separation. This adsorbent was made of a silica gel surface coating with a thin IIP layer. MIP and IIP preparation differ in the type of interaction between the monomers or ligands (MIPs) and ions (IIPs), i.e., covalent or noncovalent interactions. The covalent molecular imprinting technique method developed by Wulff et al. , involveA few reviews can be found on IIP technologies for adsorption/removal of a larger group of metal ions such as a review on IIPs for removing inorganic pollutants by Mafu et al. . FurtherThe synthesis of IIPs is commonly carried out in the following way: First, monomers containing functional groups are mixed with template ions, whereupon the monomers self-assemble around the ions. Second, a crosslinker is used to polymerize the monomers by using a photo- or thermal polymerization technique. Third, template ions are removed from the polymers, consequently generating specific binding sites that can capture target ionic species . IIPs shOne of the most crucial criteria for the effective binding and selectivity of the metal ion to the imprinted material is the interaction of the functional groups of monomers with the template . The temThe template should be inert, it should not affect the cross-linking and polymerization of the monomer, e.g., the functional moieties shouldn’t interfere with the polymerization by acting as inhibitors or reacting with the monomer.The template should be cost-effective.The template should be stable at the polymerization or crosslinking temperature or exposure to UV radiation.The selection of templates is a crucial requirement for IIP development. The templates’ chemical and physical properties are analyzed for their selection. The basic requirements for choosing a template are as follows:In order to prepare recognition sites in a polymer matrix monomer should have functional groups that will bind with the analytes. Monomer selection is an important factor for the design of IIPs. To prepare imprinted polymers a free radical polymerization (FRP) method is used since it is easy to execute and offers a wide range of monomer selection. Generally, in radical polymerization (RP) of IIPs vinyl monomers are used, e.g., methacrylic acid , acrylamThe purpose of the solvent in the polymerization is to solubilize the components of the reaction mixture such as monomers, templates, and crosslinkers. It helps in pore formation in a material, which improves the material’s adsorption properties . The solThe initiator is responsible for the activation of free radical polymerization in monomers. The initiator selection depends on the template ion’s stability and electrostatic interactions with the monomers, mainly for complex formation. If initiators are thermally activated, thermal stability of the template is also required. If the monomers form a complex with the template ions by hydrogen bond formation, then it is not appropriate to use a thermal method. However, a photochemically activated initiator is more reliable for using such a low stability template and its interactions. The initiator concentration is directly related to the generation of radicals in the system, which increases the particle size of the IIPs . Some of2 and -OH groups which can be modified with other compounds, in order to form a dendritic structure or crosslinked networks. For the preparation of crosslinked structures chitosan can be crosslinked with various difunctional molecules such as glutaraldehyde [The preparation of IIPs can be performed in several ways using multiple preparation methods. In this section, the most commonly used type of polymerization or imprinting strategies are described. The simplest method for IIP synthesis is radical polymerization (RP). Radical polymerization can be divided into bulk polymerization , emulsioaldehyde , epichloaldehyde  used 3-galdehyde .removal and the other one is equilibrium adsorbate concentration. The % removal represents the approximate value of the adsorption performance and it is shown in Equation (1):oC (mg/L) is the initial adsorbate concentration and eC (mg/L) equilibrium concentration in the solution. The equation for the adsorption performance (Equation (2)) is derived from the expression of the equilibrium concentration of the adsorbate eQ (mg/g) at the amount of adsorbate adsorbed at the equilibrium [m (g) is the dry mass of the used adsorbent; V (L) is the volume of the adsorbate solution.IIPs are known for their selectivity towards the analytes, and IIPs’ adsorption performance can be studied with respect to a non-imprinted polymer (NIP) which is synthesized using the same procedure but without the template ion. Two different equations are used for calculation, one is the % ilibrium :Qe = V ×eQ against eC. The Data modeling is achieved using different models such as Langmuir, Freundlich, etc. [eq is the equilibrium of the amount of uranyl ions adsorbed on the adsorbent (μmol/g), maxq is the maximum adsorption capacity (μmol/g), eC is the equilibrium concentration of uranyl ions in the solution (μmol/L), b is the Langmuir constant (L/μmol). maxq and b are the Langmuir constants, which can be calculated from the intercept and slope of the linear plot based on e1/q versus e1/C. Adsorption isotherms can be defined by the relationship between the amount of adsorbate adsorbed on the adsorbent at equilibrium and the adsorbate’s concentration at equilibrium in the solution. This relationship is determined by plotting ch, etc. . To undech, etc. . In IIPsch, etc. . A lineafK denotes the Freundlich isotherm constant and 1/n is the heterogeneity factor.The Freundlich model can be applied on heterogeneous surfaces with the extension of multilayer adsorption . SimilarThe Langmuir and Freundlich isotherms are commonly used isotherms in order to describe sorption experiments. However, other different isotherms models are also studied instead of the Langmuir or Freundlich isotherms. To investigate the adsorption of Cs⁺ ion on two different RAFT IIPs, Meng et al.  applied Changes in the solution acidity or basicity affect the concentration of H⁺ ions in a solution. At lower pH values, the self-assembly process reduces the choice of metal ions over H⁺ ions. Thus, a higher pH value favors complex formation. On the other hand, the pH also determines the material’s surface properties and protonation level, which directly control the electrostatic interactions with the interacting species. In the study carried out by Sadeghi et al. , experimExcept for the pH, other essential factors for optimal adsorption are the adsorbent dosage and adsorbate concentration. Tawengna et al. showed an increase in extraction efficiency from 47 to 80% and 35 to 62% with an increase in the amount of adsorbent from 10 to 50 mg for magnetic IIPs and NIPs, respectively. This increased extraction efficiency is explained due to the rise in adsorption sites and surface area for analyte adsorption. The initial concentration of the uranyl ion solution was varied from 0.5 and 8 mg/L using 50 mg of IIP adsorbent, and the experiments showed that the saturation point was reached at 2 mg/L of initial concentration. Maximum adsorption capacities of 1.04 ± 0.03 and 0.95 ± 0.02 mg/g were observed for magnetic IIPs and NIPs, respectively. This adsorption capacity is increased due to the increased strength of the transfer force and the mass transfer . In anotUranium occurs in Nature mainly in the forms of 4+ and 6+ oxidation states. The most common uranium form is uranium oxide, the uranyl cation UO₂⁺, and it is the most generally used template for IIP production . Ahmadi 3O4 nanoparticles. In another study, Monier et al. [p-aminostyrene (Schiff base), mainly used for the complex formation with UO₂⁺ ion and later used as crosslinked with divinylbenzene (DVB) [Pakade et al.  preparedr et al.  preparedne (DVB) . Tavengwne (DVB)  preparedne (DVB)  synthesine (DVB)  preparedne (DVB)  developeSuaeda glauca was used for this purpose to hyperaccumulate uranium ion from aqueous solution, later the material was kept for carbonization, mainly after successful ion removal from the carbonized material. The material was then used for further investigations. In another study, Wang et al. [Zhu et al.  used an g et al.  preparedIn a recent study, Zhong et al.  used a so-phenylenediamine (BASPDA)—on the surface of Fe3O4-SiO2. Magnetic chitosan crosslinked with epichlorohydrin (ECH)-based Th-IIPs were prepared by Huang et al. [Being the first true actinide, thorium still has an empty 5f orbital, and therefore Th(IV) is the most stable and plentiful oxidation state . The IIPg et al.  where thg et al.  reportedTo capture the cesium from the environment, different materials based on carbon, titanate, tungstate, vanadate, hydroxyapatite, metal oxides were used ,116,117.Shamsipur et al.  preparedN-isopropylacrylamide (NIPAM) and N,N-methylenebisacrylamide (MBAA) were used, which were polymerized onto the surface of the prepared silica.⁹⁰Sr is produced in the nuclear fission process of ²³⁵U and from nuclear weapons testing. In the environment it occurs in the 2+ oxidation state . For Sr²Research on graphene and graphene oxide-based materials is currently at the forefront due to graphene’s properties such as 2D dimensional layered structure, mechanical, thermal, and chemical properties. Liu et al.  preparedDithiocarbomate (-S₂CNR₂) can form strong metal-carbon bonds. This aspect favors dithiocarbomate-grafted chitosan Sr-IIP preparation. To achieve dithiocarbomate functionalization on chitosan beads, Liu et al.  applied 3+-IIP crosslinked with the γ-3-glycidoxypropyltrimethoxysilane (KH-560) for Ce sorption. Zhang et al. [3+ ion-imprinted polymer preparation. Chen et al. [4+ and Gd3+ ions. The polymer was prepared using free radical polymerization. The magnetic nanoparticle -COCl was spin-coated on the surface of the screen-printed carbon paste electrode after which it was polymerized with multiwalled carbon nanotubes (MWCNT) in the presence of Ce4+ and Gd3+ template ions 3 as a template. Kecili et al. [N,N-methylenebisacrylamide (MBAAm), cryopolymerization, resulting in poly(HEMA-co-(MAAP)₂Ce(H₂O)₂. Liu et al. prepared SBA-15-supported chitosan crosslinked with KH-560 imprinted polymer for Ce3+ removal [In another study, Alizadeh et al.  preparedi et al.  synthesi removal . The ads−) is generated in large amounts from nuclear waste, and its mitigation strategy is a key concern [− depicted in N-vinylimidazole(VI) were polymerized on the surface of vinylated Fe3O4-SiO2. The adsorption capacity was studied using the Langmuir model. The adsorption capacity was found to be equal to 62.8 mg/g at pH = 6 and a temperature of 298.15 K.Technetium has several different oxidation states in Nature. The oxidation state 7+ is generally the most common in the environment. The pertechnetate ion , N,N-dimethylaminoethyl methacrylate (DMAEMA), polymerized by thermal radical polymerization and crosslinked using EGDMA. In another study, Mustapa et al. [Among rare earth metals, lanthanum is the most abducent one; interest in it has expanded because of its unique physical and chemical properties. Many conventional methods for removing radionuclides and lanthanides from aqueous solutions were already presented . Ion-impa et al.  prepareda et al.  develope3O4-SiO2 as a support medium. The polymerization has been done using 4-VP and HEMA or EGDMA as a crosslinker. A similar type of study was carried out by Zhou et al. [3O4-SiO2 was modified with MPS and after surface imprinting polymerization was accomplished by using 4-VP. Kumar et al. [3O4-NPs, in the second step it was modified with amine (Fe3O4-SiO2-NH2). Finally, in the third step, the NPs were coated with polymer using 4-VP and MAA, and EGDMA as the crosslinker. Recently, for effective binding of Cr ions, a new type of IIPs was developed. Trzonkowska et al.  preparedu et al.  where Fer et al.  used a t2+, a novel ion-imprinted polymer was developed. Zhou et al. [2+-imprinted polymer by bulk polymerization. Ligands such as diphenylcarbazide (DPC) and N,N-azobisisobutyronitrile (AIBN) were used for Ni2+ adsorption. The study of Ni2+ removal from aqueous solutions using surface-modified molecularly imprinted ferrite nanomaterials as adsorbent was carried out by Ahmad et al. [2+ imprinted polymer using N-(4-vinylbenzyl)-2-(aminomethyl)pyridine (Vbamp) ligand to form a complex between Ni2+ and the complex crosslinked with EGDMA in a 1:3 ratio. The prepared material showed excellent retention capacity over other competitive ions. The properties of the abovementioned Ni-IIPs are presented in For the selective detection and removal of Niu et al.  preparedd et al. . They usd et al. . Lenobled et al.  prepared60Co, 58Co, 57Co that are highly radioactive gamma emitters. Exposure to these isotopes causes hair loss, skin burns, anemia, and cancer [2+ ions due to their selectivity and recognition ability. The use of inorganic materials  grafted on chitosan or polyethyleneimine, which have been crosslinked with epoxy functionalized organic or silane coupling agents, for the Co-IIPs preparation, are explored in various studies [Cobalt is one of the heavy metals that can cause several adverse health effects on humans, e.g., pneumonia, goiter, and gastrological diseases ,148. Cobd cancer . Recentl studies ,152,153.2+ and Mn2+ ions. Kang et al. [3O4-SiO2 magnetite nanoparticles. The method is depicted in Nishad et al.  preparedg et al.  prepared2-H2BDC) modified with glycylglycine [2+ tracer determination, using Co2+-8-hydroxyquinoline linked to cast magnetic nanoparticles at a glass carbon electrode surface. The procedure is illustrated in The use of activated carbon modified with -COOH groups as a crosslinker with hydrazine hydrate for the preparation of Co-IIP was developed by Turan et al. . Metal-olglycine . Torkash2+-BSA-CO chelated complex. It was introduced as template in a nanocomposite designed from a multi-walled carbon nanotube, Cu nanoparticles and carbon quantum dots-based Co-IIP sensors. Lee et al. [In another study, Sebastian et al.  developee et al.  used mage et al.  developee et al.  prepared− and Ce³⁺. Ion-imprinted polymers are based on organic substances that have a huge variability and complex formation abilities with metallic ions whereas inorganic-based materials show excellent thermal and environmental stress-related stability. The combination of both organic- and inorganic-based ion-imprinted polymers allow good preservation of their properties. On the other hand, there are some issues related to ion-imprinted polymers, e.g., regarding their lower reusability and large-scale production. The understanding of the chelating mechanism is important for future research aimed at improving the binding performance of ion-imprinted polymers. Indeed, concerning ligands and functional monomers, this can be achieved by computational methods. Other ways to improve the selective adsorption are by utilizing novel selection methods such as surface imprinting, stimuli-responsive imprinting, and dual/multiple component imprinting. New preparation techniques should be explored for better selectivity, cost reduction, and large-scale production of ion-imprinted polymers.In this review article, ion-imprinted polymers for the adsorption of 10 important radionuclides—U, Th, Cs, Sr, Ce, Tc, La, Cr, Ni, Co—which are found in the nuclear fuel cycle, are presented. These radionuclides were chosen for this study due to their commercial interest and environmental concerns. Recent updates in the field of ion-imprinted polymers for the radionuclide separation discussed with preparation methods, properties, adsorption capacities, and characterization methods used for its determination were discussed here. In recent years, research interest has been focused on the development of ion-imprinted polymers for radionuclide separation due to their properties such as selectivity and recognition ability for targeted ions. Despite the number of studies in this area, more research should be done on TcO₄