Arabidopsis thaliana) reporter lines that express a chimeric polypeptide with an SNF1/SnRK1/AMPK-specific phosphorylation site. We investigated responses during an equinoctial diel cycle and after perturbing this cycle. As expected, in vivo SnRK1 activity rose toward the end of the night and rose even further when the night was extended. Unexpectedly, although sugars rose after dawn, SnRK1 activity did not decline until about 12 h into the light period. The sucrose signal metabolite, trehalose 6-phosphate (Tre6P), has been shown to inhibit SnRK1 in vitro. We introduced the SnRK1 reporter into lines that harbored an inducible trehalose-6-phosphate synthase construct. Elevated Tre6P decreased in vivo SnRK1 activity in the light period, but not at the end of the night. Reporter polypeptide phosphorylation was sometimes negatively correlated with Tre6P, but a stronger and more widespread negative correlation was observed with glucose-6-phosphate. We propose that SnRK1 operates within a network that controls carbon utilization and maintains diel sugar homeostasis, that SnRK1 activity is regulated in a context-dependent manner by Tre6P, probably interacting with further inputs including hexose phosphates and the circadian clock, and that SnRK1 signaling is modulated by factors that act downstream of SnRK1.Sucrose-nonfermenting 1 (SNF1)–related kinase 1 (SnRK1) is a central hub in carbon and energy signaling in plants, and is orthologous with SNF1 in yeast and the AMP-activated protein kinase (AMPK) in animals. Previous studies of SnRK1 relied on in vitro activity assays or monitoring of putative marker gene expression. Neither approach gives unambiguous information about in vivo SnRK1 activity. We have monitored in vivo SnRK1 activity using Arabidopsis ( SnRK1 is implicated in maintaining cellular homeostasis and energy balance alongside linking carbon (C) availability and growth, and has been extensively studied in recent years (Plant sucrose-nonfermenting 1–related protein kinase 1 (SnRK1) belongs to a eukaryotic family of Cant years . These fS.X.X.X.[LFIMV] or the less favored [MLVFI].[RKH].X.X.X.S.X.X.X.[LFIMV]  subunit and two regulatory (β and βγ) subunits that modify complex stability, activity, interactions and cellular localization . It is l.[LFIMV] . Such st.[LFIMV] . In mamm.[LFIMV] . In cont.[LFIMV] . Inhibit.[LFIMV] . These i.[LFIMV] . Subsequ.[LFIMV] . HoweverGlc6P and Glc1P are intermediates in glycolysis, gluconeogenesis, and in the synthesis and degradation of major carbohydrate reserves like starch and sucrose. Tre6P is an intermediate in the pathway of trehalose biosynthesis. It is synthesized from UDP-Glc and Glc6P by trehalose-6-phosphate synthase (TPS) and is dephosphorylated to trehalose by trehalose-6-phosphate phosphatase (TPP) . Trehaloin planta has not been addressed in detail. Interactions between Tre6P and SnRK1 have been more extensively studied, mainly by investigating changes in SnRK1 marker transcript abundance (DARK INDUCIBLE 6 (DIN6), DIN1, BRANCHED CHAIN AMINO ACID TRANSFERASE 2 (BCAT2), and EXPANSIN 10 (EXP10), whose expression is regulated by SnRK1-mediated phosphorylation of upstream transcription factors, such as BASIC LEUCINE-ZIPPER 11  led to changes in transcript abundance for a subset of these SnRK1 downstream target genes that were consistent with Tre6P acting to inhibit SnRK1  kernels , which was used to normalize for the level of expression of the reporter polypeptide see .Two transgenic lines were used: (i) expressing a nuclear reporter (NUC), that contains the SV40 nuclear location signal (NLS) and (ii) expressing a general reporter (GEN), in which GFP is observed in both the nucleus and the cytoplasm . They also showed that there is less phosphorylation in protoplasts prepared from inducible snrk1α knockdown plants than in protoplasts prepared from wild-type plants.The specificity of the ACC1 reporter peptide was previously tested in yeast by incubating WT and activity . The pepK48M line expressing a catalytically inactive mutant protein . Compared to the NUC control, reporter phosphorylation in NUC × 35S:SnRK1K48M was significantly lower at ZT0, 8, 24, and 28  when Tre6P levels decreased and phosphorylation of both NUC and GEN polypeptides increased. In addition, no reciprocal changes were observed between NUC or GEN phosphorylation and sugar levels, except for sucrose in the last part of the night. However, Glc6P and Glc1P did show a reciprocal relationship with SnRK1 activity through the night; Glc6P and Glc1P initially increased after dusk (from ZT12 to ZT16), and then decreased (from ZT16 to ZT24), while phosphorylation of NUC and GEN decreased during the first part of the night and then increased .To further explore how SnRK1 is regulated by changes in metabolites, by light and/or by endogenous (e.g. circadian clock) rhythms, we investigated NUC and GEN phosphorylation during perturbations of the light regime.−2 s−1 irradiance for 19 d, and then transferred to 60 µmol m−2 s−1 for one light period (treatment light period), darkened at ZT12 at the same time as in the initial growth conditions (treatment night), and then returned to 160 µmol m−2 s−1 on the next day .−2 s−1 irradiance for 19 d and then transferred to continuous lower irradiance. The original study of The second treatment was based on an experiment reported by −2 s−1, then transferred them, at dawn on Day 20 after sowing, to continuous light with an irradiance of 90 µmol m−2 s−1, and sampled them at 4-h intervals for 32 h . As previously seen (We grew plants in a 14-h light/14-h dark (T28) or a standard 12-h light/12-h dark (T24) cycle for 19 d with 160 µmol mectively . This exsly seen , in a T2sly seen . Correspsly seen . After iEscherichia coli TPS under the control of the Aspergillus nidulans ethanol-inducible ALCOHOL DEHYDROGENASE REGULATOR  and alcA promoter to investigate the impact of a short-term induced increase in Tre6P on starch, organic acid and nitrogen metabolism (TPS (NUC × iTPS #6.3 or GEN × iTPS #2.4), and two control lines with only the alcR gene .Tre6P is a signal of sucrose levels  and is wtabolism . We now tabolism  and into−2 s−1 irradiance). On Day 21 after sowing, plants were sprayed at ZT1 with either water (control) or 2% (v/v) ethanol to induce the expression of the bacterial TPS  ethanol toward the end of the night (ZT20) and harvested at ZT20, ZT22, ZT24, ZT25, and ZT27  for 21 DAS with 160 µmol mand ZT27 . We incland ZT27 . Tre6P land ZT27 , with Trand ZT27 . This reenolpyruvate (PEP) levels (The increase in Tre6P levels after ethanol spraying in the night was accompanied by a slight slowing of starch breakdown and lower glucose levels . This co) levels  may alsoOverall, these results show that Tre6P is not the sole regulator of SnRK1, and its inhibitory effect appears to be context dependent. Indeed, in some conditions, secondary changes caused by Tre6P induction may lead, indirectly, to a stronger positive effect on SnRK1 activity than the direct inhibitory effect of elevated Tre6P itself.Taken together, our experiments point to complex and somewhat flexible regulation of SnRK1 activity in conditions where C status is changing in a range above that prevailing during C starvation. We therefore investigated more systematically the relationship between diel changes of metabolite levels and NUC or GEN phosphorylation.R2 = 0.007, 0.004, and 0.004) or GEN phosphorylation (R2 = 0.001 and 0.046). Glc6P levels were not consistently related to NUC phosphorylation with R2 values of 0.048, 0.21 , and 0.02 but were negatively correlated with GEN phosphorylation with R2 values of 0.22 (P = 0.03) and 0.58 (P = 2.24 × 10−5). Similar relationships were observed for Glc1P which, due to the near-equilibrium reaction catalyzed by phosphoglucomutase, can be expected to change in parallel with Glc6P. Starch was not correlated with either NUC or GEN phosphorylation. Sucrose was not correlated with NUC and was negatively correlated with GEN phosphorylation in one but not the other experiment. Glucose was not correlated with NUC, and positively correlated with GEN phosphorylation in one but not the other experiment.We did this first for the three control NUC  and the two control GEN (Controls A and B) data sets in undisturbed equinoctial diel cycles see . Tre6P lWe also performed regression analysis separately on the light period (ZT4–ZT12) and night (ZT16–ZT24) data sets . There wPairwise correlation analyses between metabolites  revealedR2 and lowest P-values).The diel changes in SnRK1 activity and metabolite levels included abrupt changes after illumination or darkening. These may not be well represented in correlations based on changes across the entire cycle. To place more weight on rapid transient changes, we calculated the difference between the average absolute levels at consecutive time points in the diel cycle and divided this by the time difference to obtain the direction and rate of change of a given trait in a given time interval. Compared with absolute values, derivative values provide more information about changes in the balance in metabolism. The derivative values were used for linear regression analyses as described above, except that analyses were only performed for the entire diel cycle . ChangesR2 = 0.46, 0.41, and 0.23; P = 2.6 × 10−4, 0.001, and 0.018, respectively) or GEN phosphorylation  after transfer to continuous low light. There were significant correlations between Glc6P and NUC  phosphorylation activity in a T28 cycle. In experiments in which Tre6P was induced in the light period, NUC phosphorylation correlated negatively with Tre6P in the control water-sprayed plants  but not when ethanol was sprayed to increase Tre6P  to a minimum at ZT16 and then rose until the end of the night . ActivitA second unexpected finding was that SnRK1 activity did not increase in response to a decrease in overall C availability in the light. Illumination from dawn onwards at a lower irradiance than growth irradiance did not lead to an increase in the phosphorylation status of either the NUC or GEN reporter polypeptides, compared with that at growth irradiance  and 3. AThese observations point to SnRK1 being regulated in a more flexible manner in the light than in the dark. This flexibility is also revealed by the response when plants are transferred to low irradiance for one light period and then returned to growth irradiance. This perturbation led to higher levels of sugars and faster accumulation of starch when plants were returned to growth irradiance, compared with control plants that had been left at growth irradiance . These oThere may also be more ways for the plant to cope with a lower rate of photosynthesis in the light than there are to adjust to a shortfall of starch at night. For example, the impact of decreased irradiance on Glc6P levels in the light period was less marked than the impact on sugar levels . This maA third finding was that an endogenous circadian rhythm may contribute to the diel changes in SnRK1 activity, at least in the middle of the 24-h cycle, when SnRK1 activity decreased between about ZT12 and ZT16. The decrease after ZT12 was partly retained in low continuous light  and was The unexpected diel response pointed to SnRK1 operating within a network that controls C utilization in the daytime and at night to maintain C homeostasis. SnRK1 activity integrates various inputs including multiple signals from metabolism . We askeThe role of Tre6P in regulation of SnRK1 has attracted wide attention . To direThe possible role of hexose phosphates like Glc6P and Glc1P in the in vivo regulation of SnRK1 activity has attracted less attention, despite evidence from in vitro assays that they can inhibit SnRK1 . SeveralThe question also arises what endogenous factor might be responsible for the decline in SnRK1 activity between ZT12 and ZT16, which occurred even when plants were not darkened at ZT12 (see above). One possible explanation is that this decline is driven by diel changes in starch turnover. As shown in Taken together, our results point to both Tre6P and hexose phosphates contributing to the regulation of SnRK1 in vivo. These metabolites provide complementary information about C status. Tre6P provides information about the amount of sucrose , which iAn additional regulatory mechanism that might contribute to observed diel changes in SnRK1 activity would be changes in subunit composition, including expression or stability of its subunits, such as by SUMOylation and ubiquitination of SnRK1α1, SnRK1β1, SnRK1β2 SnRK1β3, and by myristoylation of SnRK1β1 and SnRK1β2 . These wAXP and EXP10). This pattern contrasts with the response of the NUC or GEN reporter polypeptide phosphorylation, which was relatively high in the light period and fell in the first part of the night  genotypes were in the Columbia-0 (Col-0) background. Lines expressing the NUC or GEN SnRK1-reporters were generated as described in Rattus norvegicus) acetyl CoA carboxylase (rACC1), containing the AMPK recognition motif and Ser79 phosphorylation site, fused at the C-terminus to eGFP and a double hemagglutinin tag . Pollen from the parental parent was transferred by rubbing anthers from mature flowers against exposed stigmas of emasculated maternal plants under a binocular microscope, and the inflorescence meristems and unopened flower buds were removed. After crossing, the maternal plants were grown on, with regular watering, for 2–3 wk until siliques had fully developed and started to dry. Siliques were collected, left to dry for a further 3–4 d at room temperature and then kept at 10 °C until seeds were germinated on selective media. After surface sterilization by treatment with 2% (w/v) sodium hypochlorite for 5 min at room temperature and washing in five changes of water, seeds were placed on 0.5 × Murashige and Skoog medium  containihttps://www.stender.de) and vermiculite in 6-cm diameter pots, covered and kept at 4 °C in the dark for 48 h then transferred to a controlled environment chamber  with a 16-h photoperiod (160 µmol m−2 s−1 irradiance provided by white LEDs) and day/night temperatures of 21 °C/19 °C. After germination, seedlings were transferred to 10-cm diameter pots (four or five seedlings per pot), and grown as above or as described for individual experiments. Whole rosettes were harvested in situ and immediately quenched in liquid nitrogen, with four or five plants from the same pot being pooled for each biological replicate. Frozen plant material was ground to a fine powder and stored at −80 °C until use.Seeds were sown on a 1:1 mixture of compost  and control alcR lines  were gro×g (4 °C) for 3 min. Aliquots of the supernatant containing approx. 12 µg of soluble protein were electrophoresed on a 12% (w/v) polyacrylamide-SDS gel. Proteins were electro-blotted onto nitrocellulose membrane, visualized by staining with 0.2% (w/v) Ponceau Red in 1% (v/v) acetic acid and imaged for use as a protein-loading reference. After blocking in Tris-buffered saline containing 5% (w/v) milk powder, the membrane was incubated overnight at 4 °C with rabbit anti-phospho-acetyl CoA carboxylase (Ser79) antibody  at a dilution of 1:1,500. After washing, NUC/GEN phosphorylation was detected by incubation with IRDye 800CW goat anti-rabbit IgG (H + L) secondary antibody  at a dilution of 1:9,000 and scanning with an Odyssey Imaging Scanner (Li-COR), and quantified using Image Studio Live 5.2 software (Li-COR). Total NUC/GEN polypeptide abundance was detected by probing duplicate blots with a mouse anti-GFP antibody , Darmstadt, Germany; www.sigmaaldrich.com) at a dilution of 1:1,500, followed by incubation with IRDye 680RD goat anti-mouse IgG (H + L) secondary antibody (Li-COR) at a dilution of 1:9,000, and quantified as above.Proteins were extracted from aliquots (10 mg FW) of frozen, finely powdered plant material by suspension in 100 µL of pre-warmed (90 °C) sample buffer  quickly vortex mixed, incubated at 90 °C for 5 min, then chilled on ice and centrifuged at 13,500To minimize the impact of gel-to-gel variation, aliquots from an extended night sample were loaded onto each gel and the signal from this sample was used to normalize the signals from the other samples on the corresponding immunoblot. Two aliquots of the protein extract from each biological replicate were analyzed on separate gels and, after normalization as described above, the average of the two technical replicates was calculated as the reported value for that biological replicate. The signal ratio pACC/GFP was used to normalize for any differences in reporter protein abundance in the tissue and differences in gel loading. See https://www.leica-microsystems.com) using a 20× objective. To visualize nuclei, NUC samples were stained with 10 μg/mL propidium iodide (PI) solution for 5–10 min. A 488 nm laser (40% intensity) was used to excite GFP and fluorescence emission was detected in the 524–555 nm range with a gain setting of 860. A 561 nm laser (10% intensity) was used to excite PI and fluorescence emission was detected in the 571–640 nm range with a gain setting of 550. Confocal Z sections were acquired every 0.5 μm.To determine the subcellular localization of the NUC and GEN reporter polypeptides, GFP was visualized by laser confocal microscopy of 6-d-old seedlings. Confocal imaging was performed on a Leica TCS SP5 microscope . Significance of changes in metabolite and SnRK1 activity was tested by one-way ANOVA using a pairwise multiple comparison procedure, with post-hoc testing by the Holm–Sidak method (P < 0.05), as described in each figure legend.Technical replicates were always averaged to generate a single value for each biological replicate, statistical analysis was restricted to biological replicates and performed using Sigma-Plot 14.5 software (Systat Software GmbH, Düsseldorf, Germany; kiad066_Supplementary_DataClick here for additional data file.


Articular screw penetration is one of the most common hardware-related problems after scaphoid fracture fixation, occurring in up to two-thirds of patients, in particular into the scaphotrapezotrapezoidal (STT) joint. The aim of this study was to investigate whether this clinically important issue could be detected using standard anteroposterior (AP) and lateral, as well as additional nonstandard fluoroscopic views using direct open visualization with magnifying loupes as reference standard.Ten fresh cadaver wrists were used for this imaging study. A 2.2 mm cannulated compression screws with a length of 24 mm was placed in the scaphoid and incrementally left to protrude at the STT joint up to 2 mm. Eight fluoroscopic views of the wrist were then obtained by rotating the forearm using goniometric measurements, keeping the image beam parallel to the floor: (1) anteroposterior with the wrist in neutral rotation, (2) anteroposterior with the wrist in ulnar deviation, (3) supinated oblique 60° from neutral (60° supinated oblique), (4) supinated oblique 45° from neutral (45° supinated oblique), (5) a true lateral, (6) a true lateral with the wrist in radial deviation, (7) pronated oblique 45° from neutral (45° pronated oblique), and (8) a pronated oblique 60° from neutral (60° pronated oblique).p < 0.0001).Standard anteroposterior and lateral fluoroscopy views  of a percutaneous cannulated screw fixation of a scaphoid fracture were insufficient to detect distal articular penetration, missing half the amount of screw penetrations in the current study. The 45° pronated oblique view was found as the most sensitive in detecting STT penetration (Standard anteroposterior and lateral fluoroscopy views of a percutaneous cannulated screw fixation of a scaphoid waist fracture are insufficient to detect STT screw penetration. According to the current study, standard views would have missed half the amount of screw penetrations, which seems to reflect the high incidence of this problem in current practice. The most sensitive view was the 45° pronated oblique view, which detected STT screw penetration in all cases.Level of Evidence Not applicable. Problems related to hardware are likely the most common complication of scaphoid fracture fixation and remain either underreported or underemphasized, with highly heterogeneous rates across the literature –3. LikelA previous cadaveric study focused only on screw penetration of the proximal scaphoid articular surface and found that the semi-pronated 60°) oblique view was the most important fluoroscopic image to obtain . However0° obliquThe aim of this study was to investigate whether this overly common and clinically important issue could be detected using standard anteroposterior (AP) and lateral, as well as additional nonstandard fluoroscopic views using direct open visualization with magnifying loupes as reference standard, to prevent distal articular screw penetration, delayed union, and symptomatic STT arthritis. As a secondary aim, the addition role of the 45° semi-pronation view (versus a previously demonstrated 60° semi-pronation view) is studied.Ten fresh cadaver wrists were used for this imaging study. There were no visible wrist scars to be noted on any of the cadavers and no radiographic signs of STT arthritis. A mini-open incision with capsulotomy were performed at the STT joint to expose the entry point of the screw at the volar scaphoid tubercle. A 2.2 mm cannulated compression screws (Medartis SpeedTip®) with a length of 24 mm was advanced over a pre-drilled K-wire in the scaphoid along its longitudinal axis. The screw was placed under direct vision until it was flush with the subchondral bone and its position confirmed fluoroscopically with an image intensifier . Eight fluoroscopic views of the wrist were then obtained by rotating the forearm using goniometric measurements, keeping the image beam parallel to the floor: (1) anteroposterior with the wrist in neutral rotation, (2) anteroposterior with the wrist in ulnar deviation, (3) supinated oblique 60° from neutral (60° supinated oblique), (4) supinated oblique 45° from neutral (45° supinated oblique), (5) a true lateral, (6) a true lateral with the wrist in radial deviation, (7) pronated oblique 45° from neutral (45° pronated oblique), and (8) a pronated oblique 60° from neutral (60° pronated oblique).In the first part of the study—according to a published study protocol —the screIn the second part of the study, all screws were reversed so as to allow visual protrusion distally of 2 mm, measured with a caliper from the radial side of the screw visualized with magnifying loupes, and all views were repeated  was run to determine whether the direction of the radiograph (using the eight different views) had an effect on the determination of articular penetration. A Kruskal–Wallis test for nonparametric continuous variables was used for comparison of the amount of screw penetration between different fluoroscopic views. The level of statistical significance was set at the 0.05 level (two sided).A Cochran’s p < 0.0001), identifying screws penetrating 0.5 mm in 8 out of 10 wrists, and in all wrists following the protocol with increments up to 1.5 mm  followed by the true lateral view with the wrist held in radial deviation (1.9 mm) and the 60° pronated oblique view (1.6 mm).In the second part of the study, screws that were visually measured to protrude distally by 2 mm were radiographically measurable on all but both semi-supinated views Table . The 45°Screw protrusion is likely the most common surgical complication after scaphoid fracture fixation . In the As opposed to volar (mini-)open approaches, percutaneous retrograde scaphoid screw fixation not only presents potential problems at the proximal pole  but moreAnother study on screw fixation of scaphoid fractures showed that in four of eight patients with scaphotrapezial joint space narrowing, osteoarthritic changes were related to attrition from prominence of the screw . Howeverp = 0.049) after nonoperative treatment and suggested this difference resulted from possible iatrogenic STT injury [At 12 years follow-up, Saedén et al. reported an incidence of STT osteoarthritis of 61% (14 of 23) following scaphoid fracture fixation, and only 25% .This importance of the semi-pronated scaphoid view is consistent with the findings of Kim et al. that proximal screw penetration was best visualized on a 60° pronated oblique view . HoweverConsistent with their findings, small amounts of screw penetration (0.5 mm) were not always detected on fluoroscopic views as in two of out ten cases it remained unidentified on all views, and with 1 mm of penetration in one out of ten cases. Larger amounts of distal articular screw penetration 1.5–2 mm were detected in all cases with at least the 45° pronated oblique view. In contrast to their study assessing proximal articular screw penetration, no Steinmann pins were used in the present study. The choice for a standardized 24 mm cannulated screw was based on the aim to simulate a real surgical screw fixation as closely as possible.One way to improve distal screw visualization on a true lateral wrist view is to radially deviation the wrist thereby flexing the scaphoid that displaces the screw head volarly and away from the trapezium. The 45° pronated oblique view was the most sensitive view for this purpose, detecting all of the up to 1.5 mm penetrating screws as well as providing the longest radiographically measurable screw penetration.Another potential solution has been advocated by Patel et al. to use shorter screws as in their biomechanical study in 18 cadaveric scaphoids, in which maximizing screw length did not provide superior fixation . They foThe findings of the present study should be interpreted in light of its limitations inherent to a cadaveric setting, which may not perfectly reflect a clinical setting in the operating room, although in our experience it was an optimal simulation of reality. First, we focused only on STT screw penetration as it seems to be the most common contemporary problem related to hardware, and the fact that data on proximal screw penetration have not yet been published. Second, radiographic penetration past the outer cortical line likely overestimates true articular penetration as scaphoid cartilage thickness is estimated to be up to 1 mm in cadaveric wrists on the distal tubercle at the STT joint. This could in part explain the fact that on the 45° pronated oblique view the average protrusion was larger by 0.1 mm than measured by caliper, although the inherent imprecision to the latter methodology as well as differentiating increments of 0.5 mm should be taken in account. Alternative advanced imaging techniques would also have had inherent limitations such as the difficulty of discerning cartilage on CT and metal-induced artifacts on magnetic resonance imaging (MRI). Third, the combination of pronation and radial or ulnar deviation is difficult in specimens, but this is a variable that would be interesting to study for clinical application.Even in case of minimal and clinically nonvisible screw penetration Fig. B of lessIn conclusion, standard anteroposterior and lateral fluoroscopy views of a percutaneous cannulated screw fixation of a scaphoid waist fracture are insufficient to detect STT screw penetration. According to the current study, standard views would have missed half the amount of screw penetrations, which seems to reflect the high incidence of this problem in current practice. The most sensitive view was the 45° pronated oblique view, which detected STT screw penetration in all cases.


This study aimed at demonstrating the intentional modulation of bimanual coordination dynamics at transition frequency and determining whether it is associated with perceptual and/or motor inhibition capacities. Healthy adults (N = 29) performed in a random order: i) bimanual anti-phase (AP) movements at the maximal individual transition frequency, with the instruction to either let go, or intentionally maintain the initial movement pattern and oppose to the spontaneous transition to in-phase (IP) movements, and ii) The Motor and Perceptual Inhibition Test, giving separate scores for perceptual and motor inhibition. Results showed that in the intentional condition participants were able to delay (more movement cycles before the transition) and suppress  the spontaneous transition from AP to IP. A statistically significant, though weak, correlation was found between motor performance and perceptual inhibition scores. We interpreted our findings as an indicator of the presence of an inhibitory mechanism underlying intentional dynamics that is partially associated to perceptual inhibition in healthy adults. This could have implications in populations with compromised inhibitory capacities, which might entail motor repercussions, and suggests the possibility of using bimanual coordination as means to stimulate both cognitive and motor capacities. •Intentional modulation of spontaneous dynamics is possible at transition frequency.•Healthy adults can intentionally delay and suppress the spontaneous transition.•Intentional motor performance positively correlates with perceptual inhibition score. Their stability and flexibility result from a coalition of multiple constraints, that are of musculoskeletal, perceptual, cognitive, and neural origins . In thisCyclical bimanual coordination has been a widely used paradigm for studying cognitive-motor interactions underlying complex movements . IndeedTo meet task demands, the intrinsic dynamics can be modulated by cognition . SpecifiInhibition is unarguably a multifaceted process that can be studied through different paradigms, and has been accounted for from different theoretical perspectives. For instance, the Stroop task  has beenOne way to evaluate perceptual and motor inhibition capacities through separate scores is to use the Motor and Perceptual Inhibition Test (MAPIT) ,32. The Overall, in this study, we were interested in demonstrating the intentional modulation of bimanual dynamics at transition frequency and determining whether it is associated with perceptual and/or motor inhibition capacities. Participants performed, in a conter-balanced order, the MAPIT, and bimanual AP movements at transition frequency with the instruction to either let go or intentionally maintain AP and oppose to the spontaneous transition to IP. To analyze motor performance, we compared the number of trials without transition and the number of movement cycles before the transition between the two instruction conditions. We were expecting to observe more trials without transition and more movement cycles before the transition in the intentional maintenance condition. Regression analyses were then conducted between inhibition scores and motor performance change from the “let go” to the “maintain and resist” condition. We were expecting both perceptual and motor inhibition capacities to correlate with AP pattern intentional maintenance, with potentially a stronger correlation with perceptual inhibition.22.1Twenty-nine healthy volunteers  participated in this study. None of the participants self-declared having previous or current neurological or psychiatric disorders, recent or current musculoskeletal upper limb problems, an ongoing psychopharmacological medication, an uncorrected vision, an addiction to illegal substances or alcoholism. Following self-report of their hand use, 26 of our participants were right-handed, and only three were left-handed.Prior to their enrolment, all participants were given detailed written information about the study, without stating its precise objective or the underlying hypotheses. They all gave their written informed consent to the experimental procedure that agreed with the Declaration of Helsinki and was approved by the Ethics Committee for Research in Science and Techniques of Physical and Sports Activities (CER STAPS n° IRB00012476-2020-15-12-78).2.2Participants performed the bimanual and inhibition tasks presented below in a randomized counter-balanced order. During all the experimental session, they were seated in a height adjustable chair without armrests, with their back straight leaning against the seatback.2.2.1The experimental setup was the same as the one used in Temprado et al.  ,11. It cThe task began with a familiarization period, during which participants performed 50 cycles of IP and AP bimanual movements at 1 Hz and at 1.5 Hz. Then, participants performed a block of 6 trials to identify their transition frequency, that is the frequency for which a spontaneous transition from AP to IP occurred. During these 6 trials, the metronome frequency was increased stepwise by 0.5 Hz every 10 s, starting from 1 Hz until the occurrence of the transition. Participants were instructed to adopt the AP pattern, and not to intervene (let go) if they felt the need at some point to switch to the IP pattern. The transition frequency was defined as the higher frequency of the 6 trials at which the participants switched from AP to IP. The individual transition frequency was used to perform the experimental task under two conditions presented in a randomized order: the “let go” condition ,8 to obsFor each trial, we calculated the RP (in degrees) between the oscillations of the right and left hands, with the right hand as reference. The RP represented the difference in phase angles between the two hands:2.2.2https://trello.com/b/EtNCNrZH/ice). The MAPIT is a reaction time task that requires responding by pressing as fast as possible a left or right key according to the direction (left or right) or the location  of a black arrow presented on a white background computer screen . In this study, the participant responded using a modified AZERTY keyboard on which only two letter keys were kept “Q” (corresponding to the answer “left”) and “M” (corresponding to the answer “right”). Participants were seated comfortably in front of the setup holding lightly without pressing their right index on the “M” key and their left index on the “Q” key. As soon as the stimuli appeared on the screen, they were requested to press as fast as possible the adequate key according to the given instruction before each set of the test. In between sets, a central fixation cross was presented on the screen, and disappeared with the onset of the stimulus. The presented stimulus remained on the screen until the participant responded.Motor and perceptual inhibition capacities were assessed using the Motor and Perceptual Inhibition Test (MAPIT) , that waThe test included three blocks of 80 trials each presented in a fixed order : a preliThe preliminary block included two sets. The first was a familiarization set. It consisted of two conditions, one with 20 arrows pointing left and one with 20 arrows pointing right. The left and right pointing arrows were presented in a random order at the center of the screen. The participant was required to press the key corresponding to the direction pointed by the arrow . The second set was used to elicit and reinforce the prepotent spatial responses. It consisted of two conditions, one with 20 black squares presented on left side of the screen (8.5 cm from the center), and one with 20 black squares presented on the right side of the screen (8.5 cm from the center). The resulting 40 trials were presented randomly to the participant who needed to press the key according to the position of the square .The perceptual inhibition block included two combined sets . This resulted in 80 randomly presented trials. In the perceptual congruent set, the spatial location of the arrow corresponded to its pointing direction , while in the perceptual incongruent set the spatial location of the arrow conflicted with its pointing direction . Participants were required to press the key corresponding to the arrow direction regardless of its location on the screen. In the incongruent set, the participant had to inhibit the natural tendency of processing the arrow’s location, and instead to focus on its pointing direction.The motor inhibition block included two separated sets  wherein the arrows were always located at the center of the screen. In the motor congruent set, the participant had to respond in accordance with the arrows' direction. In the incongruent set, the participant had to respond the opposite to where the arrow pointed , inhibiting thereby the over-learned response that is spatially compatible with the presented stimulus.For each condition of each set of every participant, the median response time in milliseconds (RT) of correct trials was calculated. We chose medians instead of means to minimize the influence of possible outliers. The perceptual inhibition (PI) and motor inhibition (MI) scores are derived from the median RTs as follows:Lower MI and PI scores were respectively associated to higher motor inhibition and perceptual inhibition capacities ,32.2.3M ± CI), with CI representing the 95% confidence interval normalized to account for the within-subjects design [Data were processed with MATLAB R2018b  and Microsoft Excel 2010 . For each trial of the bimanual coordination task, we removed the first 3 s to ensure the stabilization of the participant’s behavior. Then raw data were filtered with a dual-path Butterworth filter , with application of the correction factor . An ampls design ,37.For the MAPIT task, we ensured that all participants had more than 75% of correct answers and that none of the analyzed RT values exceeded 3 s or were below 200 ms. MAPIT data are reported as means with standard deviation (M ± SD).All analyzed variable were checked for outliers outside the range of Mean ± 3 × SD. No outlier values were found.2.4p < 0.05). Size effects are reported as partial eta square (η2). To determine whether and which inhibition processes could be associated with the ability to intentionally stabilize the AP bimanual coordination pattern and resist the spontaneous transition, simple linear regression analyses were conducted. In this analysis, the dependent variable was the participants change values from the “maintain and resist” condition to the “let go” condition  for which a statistically significant effect of condition was found. The predictor was the MAPIT inhibition scores  analyzed separately. The coefficient of determination is reported as R2. For the sake of brevity, only values relative to statistically significant effects are reported in the results.The studied dependent variables were statistically analyzed using JASP software . To verify the compliance with task instruction in the bimanual coordination task, we compared both conditions using a one-way repeated measure analysis of variance (ANOVA) with the factor condition . The level of significance was set to 5%  and NC  = 60.27, p < 0.001, η2 = 0.683), with more trials without transition . The ANOVA also revealed a significant effect of condition for NT  = 46.59, ansition A and moransition B in the 3.2p = 0.042, R2 = 0.144)  (The MAPIT results showed PI and MI scores of respectively 36.41 ± 33.69 ms and 46.68 ± 39.68 ms . As the = 0.144) A, with d = 0.09) B. Partic4First of all, our results showed that healthy adults were able to intentionally delay and suppress the spontaneous transition from AP to IP when performing the task at their highest individual transition frequency. Also, we found that perceptual inhibition capacities, as measured by the MAPIT’s PI score, were to some extent associated with the observed intentional stabilization performance.2 = 0.62 for NT, and η2 = 0.7 for NC). They corroborate and extend the previous findings reported by Lee, Blandin and Proteau [The capacity to intentionally maintain the AP pattern and delay or even suppress the spontaneous transition to IP at transition frequency was reflected by an increase in the number of movement cycles before transition, and an increase in the number of trials without transition in the “maintain and resist” condition, compared to the “let go” condition. These results were statistically robust with very large effect size .Nevertheless, as expected, the correlation analysis showed that higher perceptual inhibition capacities (lower PI scores) were to some extent associated with lower delta NT and tendentially with lower delta NC, that is with more trials without transition and more movement cycles before transition in the “maintain and resist” condition than in the “let go” condition. The effect sizes were however small, as attested by the regression’s slope and ROverall, the observed bimanual behavior under the two task instructions together with the shown link between inhibitory and motor performances suggest the presence of an inhibitory mechanism contributing to the intentional maintenance of the AP pattern at transition frequency. Thus, it can be concluded that better perceptual inhibition may contribute to better intentional stabilization of the AP pattern at high movement frequency, through suppressing and delaying the spontaneous transition. The role that seems to be played by perceptual inhibition relative to motor inhibition gives support to the importance of perceptual constraints in bimanual coordination dynamics ,26,38. HMotor constraints might also play a role, although a statistically significant link between MI and bimanual coordination performance was not found in this study. To be noted that this result remains unchanged even when removing the two extreme values noted in MI results , andIn future studies, inducing perceptual or motor conflicts could help better elucidate the contribution of these constraints of different nature that are long known to contribute in different ways to bimanual coordination ,26,28. OThe observed results could have implications in special populations suffering from compromised inhibitory capacities, which might entail motor repercussions. It suggests the possibility of using the tested bimanual paradigm as means to stimulate both cognitive and motor capacities.Rita Sleimen-Malkoun, PhD, PT: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Louise Devillers-Réolon, Msc: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Jean-Jacques Temprado, Prof: Conceived and designed the experiments; Wrote the paper.Data will be made available on request.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper


Although real-time 3D echocardiography (RT3DE) has only been introduced in the last decades, its use still needs to be improved since it is a time-consuming and operator-dependent technique and acquiring a good quality data can be difficult. Moreover, the additive value of this important diagnostic tool still needs to be wholly appreciated in clinical practice. This review aims at explaining how, why, and when performing RT3DE is useful in clinical practice. Real-time three-dimensional echocardiography RT3DE) is a new valuable imaging tool for studying the mitral valve (MV) anatomy in each plane orientation ,2. MitraDE is a nRecent developments in this technique allow to assess the detailed anatomical characterization of the MV morphology, the precise quantification of the regurgitation and the mechanisms of MV disease (MVD). The most appropriate treatment and the optimal timing can be determined and this technique has assumed a crucial role in MR management ,3,4,5,6.Tridimensional transthoracic echocardiography (3D-TTE) can be performed either from the parasternal or apical approach ,9; it is(1)Live RT3DE mode: Characterized by an optimal temporal resolution despite the low spatial resolution. This modality is useful to test the gain optimization.(2)Zoom mode: If zoom RT3DE is activated, two orthogonal 2D preview images show the ‘‘truncated’’ pyramid sector that the operator can move over the region of interest, regulating width and length and preventing respiratory or rhythm artifacts. However, it is limited by a low temporal resolution that can be improved by minimizing the sector’s width and length. B.(3)“Wide angle single beat”: This option has the same advantages of the zoom mode but a relatively lower temporal and spatial resolution.(4)“Full volume multiple beat” with or without color Doppler. This modality implies an electrocardiogram (ECG) synchronization permitting the analysis of an extensive volumetric data set by acquiring narrow subvolumes . High spatial and temporal resolutions and a global MV view with excellent image quality represent the greatest strength of this modality C.By simultaneous multiplanar mode (X-plane) two-dimensional (2D) imaging, two orthogonal planes can be simultaneously visualized. The X-plane 2D imaging allows both an excellent spatial and temporal resolution, resulting in an optimal study of challenging findings such as ruptured chordae or vegetations A. The X-Among these three modalities, only the “Full Volume” allows the RT3DE representation of the color Doppler D to be aThe full volume modality is the gold standard for anatomical and functional characterization of MV by RT3DE. However, breathing, patient movement, and arrhythmias limit this modality. We recommend applying some practical tricks to avoid these artifacts.The full volume modality is not provided with the color acquisition. The volume data set is acquired during a single heartbeat. The multi-beat acquisition (full volume) during a single breath-hold allows to post-process volume data set and RT3DE rendering. RT3DE is particularly useful in identifying the main regurgitant jets in the pre-operative planning of MV transcatheter edge-to-edge repair (MV-TEER). Notably, on one hand RT3DE is limited by lower frame rates, but on the other hand full volume modality is affected by stitching artifacts.The method allows the MV apparatus to be rendered and quantified in more than 50 measures around the annulus, leaflets, coaptation points, and tendinous chordae can be challenging.Mitral valve quantification and mitral valve navigation (MVQ/MVN) analysis can be performed only by 3D-TTE using dedicated software. We suggest using a checklist for an optimal acquisition:Step 1. The full volume or zoom RT3DE image is obtained, including the entire mitral annulus, the papillary muscle tips, and the left ventricle outflow tract (LVOT) with aortic root.Step 2. The dataset acquired is uploaded on the workstation, and the best RT3DE images are selected to run in the MV navigation tool.Step 3. The RT3DE image is split into three orthogonal spatial planes like multiplanar reconstruction . A fourth box shows a navigation guide .Step 4. The end-systolic frame position is automatically detected by the software or manually selected by the operator.Step 5. The planes are aligned following the schematic illustration of the tutorial box.Step 6. The operator selects six landmarks: four annular points , the nadir of the leaflets, and the aortic point to allow an automatic RT3DE reconstruction of the mitral annulus.Step 7. Annulus editing is useful for checking the correct positioning of the other annular points by sliding all the longitudinal slices. If no editing is necessary, proceed to the next step. At the end of this step, you can see an RT3DE annulus model overlapped on the RT3DE image.Step 8. Commissure editing: the correct commissural position is confirmed by comparing it with an RT3DE image box.Step 9. Leaflet editing: leaflets are automatically detected and drawn in their extension. Manual repositioning of the leaflet points is possible. The coaptation point is manually selected by the operator.Step 10. Border editing: scallop and commissure border points can be manually adjusted.Step 11. The papillary muscle tips must be marked to determine chord length if visible.Based on the reconstructed MV model, a lot of normal and pathological anatomical parameters are automatically calculated for the components of the MV apparatus, such as annular diameters, height, leaflet area, surface area, leaflet billowing height and volume, tenting height and volume, and leaflet closing angles, etc., .For obtaining an optimal RT3DE image quality, three main rules should be followed:(1) “Suboptimal 2D images result in suboptimal RT3DE data sets”. Therefore, optimizing pre-acquisition 2D gains is crucial;(2) Respiratory and rhythm artifacts must be avoided or reduced;(3) Acquisition modality choice should vary according to the information that is needed.We suggest verifying the RT3DE imaging quality by activating the RT3DE live modality. Volumetric RT3DE image quality strictly depends on pre-acquisition 2D gain, so we usually set the gain in a mid-range (no greater than 60) and the compression/dynamic range in a mid-range .To reduce respiratory and cardiac artifacts, the patient should be aware that the procedure is not dangerous and collaborate during the exam . Pharmacological sedation is performed with an intravenous benzodiazepine such as midazolam or with propofol (if it is not contraindicated).We could use a low B-blocker dose (metoprolol 1-2 mg i.v.) to reduce heart rate (HR) and minimize HR disturbance. An HR ranging from 70 to 85 bpm allows an increased number of scan lines per second, optimizing the zoom RT3DE and full volume acquisition modalities.We use the full volume modality providing that it is suitable. A broader sector acquisition is obtained by maintaining a higher frame rate and, consequently, a good quality RT3DE image.We switch to the RT3DE zoom modality if the patient is not compliant and if the MVD does not require a large sector or a high frame rate. Highly moving vegetation are better detected by full volume, whereas RT3DE zoom may successfully study a prolapsing scallop.Multiplanar reconstruction (MPR) is essential in RT3DE analysis. The manual rotation of the dataset around a center point allows proper visualization ,15.MPR allows a more accurate measurement of the MV area, annulus, vena contracta area, vegetations, and masses by choosing a better section plane. This phase of reconstruction depends on the final image quality.We prefer to use multiplanar reconstruction before the cropping. By moving the three planes  perpendicularly at the region of interest, a more accurately rendered RT3DE image is obtained A.Cropping describes the process of manually moving a cutting plane from outside the 3D volume towards its center, thus providing a view from the cutting plane into the 3D volume.Cropping is performed following the orthogonal acquisition planes  or using a free plane.We prefer a semi-automated method based on a crop box positioned in the region of interest. In our case, the MV apparatus includes mitral commissures and an entire excursion of the leaflets avoiding the dropout of echoes throughout the cardiac cycle B.After obtaining the RT3DE volumetric cropped image, we can freely rotate the image 360 degrees in the best perspective  C,D.Usually, in the atrial view perspective, the aortic valve (AV) is displayed in an upper position ,2.The 3D reconstruction of the regurgitant jet can also be performed using the cropping mode, improving the detection of MVR jets origin.The introduction of 3DE has substantially improved the knowledge of anatomical and functional mechanisms of MV disease.The RT3DE assessment of MV allows a better MV disease understanding than two-dimensional echocardiography (2DE). 3D-TEE has been shown to be more useful than 2D-TEE in assessing mitral valve prolapse (MVP) .In addition, RT3DE allows us to visualize the whole MV.Even though MV diseases can be easily studied by 2DE, morphological RT3DE imaging provides a precise characterization, thus facilitating diagnosis and accurately showing anatomical functional features. The possibility of choosing the best perspective for each cardiac structure results in a detailed anatomical characterization useful for planning a surgical or interventional strategy in MV diseases.Preload conditions, as well as LV size and function, make MV evaluation challenging, particularly when a secondary etiology occurs ,18.Effective regurgitant orifice area (EROA) and regurgitant volume (RVol), as measured by proximal isovelocity surface area (PISA), may both overestimate and underestimate the severity of MR . LV voluVisualizing the entire surface of the leaflets allows an easy identification and classification of the prolapsing, flail, and tethered segments . SystemaThe utility of the full volume RT3DE analysis in complex MVP has been well assessed ,35.The angle views  help to detect commissural prolapse and its complications. The leaflet image’s motion flow and the realistic and dynamic vision of RT3DE imaging allows a “segment-oriented approach” method of analysis with significant implications in surgical planning A–D. It hAccording to our experience, in patients with endocarditis RT3DE anatomical imaging accurately detects vegetation characteristics, such as the site, the extension, and the relationship with near structures A–F.2DE may permit diagnosis of a traumatic or ischemic papillary muscle rupture. At the same time, RT3DE may be useful in differentiating the papillary muscle from its tip anchored to the leaflet prolapsing in the LA A–C. FurtVisualizing the MV apparatus by RT3DE using specific software allows a deep understanding of the mechanisms of transvalvular regurgitation. Assessing valve morphology, geometry of the annulus, shape and surface of the leaflets, and the relationship between MV, the papillary muscle, and LV is useful for evaluating a surgical or percutaneous MV repair strategy. The morphological and semiautomatic assessment of MV apparatus improves the accuracy of diagnosis even when performed by an unskilled sonographer. Notably, identifying a closure line and the relative position of the papillary muscle tips provides a quantitative analysis of the MV anatomy before and after MV surgery and percutaneous interventions. LV measurements such as tethering distance and tenting volume are also helpful in decision making.MVN analysis is used for distinguishing BD from FED . It has Both semi-quantitative and quantitative methods for MR grading have been recommended ,43,44,45RT3DE is also helpful for assessing ERO with the VCA method ,51,52.Even though mitral calcifications  are not detected by RT3DE due to its lower spatial resolution, the MPR modality can be used to more precisely measure the non-planar MV orifice at the tips of the leaflets compared with 2DE  52]..52].RT3DE is useful in surgical or clinical MV decision making. The use of a checklist in order to improve RT3DE study should be encouraged ,53.According to Carpentier’s classification, based on leaflet mobility anatomy and etiology (primary and secondary M.R), three types of MR have been proposed:Type I : this type includes secondary M.R. with dilated annulus due to LV dysfunction and remodeling and primary M.R. caused by congenital (cleft) or acquired (endocarditis) leaflet perforation. Typically, in primary MR the annulus is normal.Even though secondary MR often results in a central regurgitation jet, eccentric and multiple regurgitation jets may occur. In these cases, RT3DE color Doppler is more helpful than 2DE in identifying and quantifying ERO. An RT3DE color cropped LV view may be more useful than the LA view in detecting the flow convergence regions of a regurgitant jet using a power color map . These dType II (excessive leaflet motion): this type includes both primary and secondary MR. In primary MR, MV prolapse has been described. MV is thought to be caused by BD and FED complications such as chordal rupture and flail. 2DE usually permits us to distinguish BD from FED. However, the 3D anatomical characterization of MV morphology overcomes the limits of 2DE in identifying commissural pericommissural scallop prolapse  or a BD with complex anatomy ( anatomy . CharactType IIIa (systolic and diastolic restricted motion): this type includes primary MR due to degenerative calcifications of the annulus extended to the leaflets and a rheumatic etiology. 2DE has a higher accuracy than RT3DE for detecting mitral calcifications due to the better spatial resolution. RT3DE color is useful in detecting non-planar ERO (anar ERO A–D.Type IIIb (systolic restricted/tethered motion): this type includes secondary MR caused by LV dilatation leading to papillary muscle displacement and symmetrical or asymmetrical leaflet tethering , and PISA for calculating functional MV area, have several limitations ,57 that Both longitudinal parasternal and apical views have been proposed to obtain the ventricular perspective of the MV apparatus. In our experience, the apical view allows a better ventricular visualization of the stenotic mitral orifice. Remarkably, a multiplanar reconstruction of MVA with a better alignment of the image plane at the mitral tips provides a more accurate and reproducible planimetric measurement ,59. ThreThe MV area obtained with RT3DE PISA has also been compared with RT3DE and 2DE planimetry. The semi-automated RT3DE quantification of the MV area might be more accurate than conventional 2D methods ,62.RT3DE is used, together with 2DE, to evaluate the potential PMBV feasibility and to predict the success. An RT3DE-based score for PMBV has been developed to improve the prediction of success of the 2DE-based Wilkins score .The severity of valvular calcification and its extension is the stronger predictor of adverse events after PMBV ,65,66,67RT3DE planimetry is more accurate than 2D planimetry in identifying the non-planar orientation of MV leaflets. Moreover, the true MV area at the tip of the leaflets can be measured A,B.Conversely, the low spatial resolution of the RT3DE image does not easily allow us to differentiate calcium from valvular tissue. Therefore, we suggest using RT3DE only when the 2DE study is not useful for evaluating the MS severity and before PMBV.The 3D-TEE detection of echographic features is very useful in evaluating percutaneous or surgical repair and accurately assessing MV, LA, interatrial septum, and catheter moving segments.A complete understanding of the underlying degenerative etiology, anatomical lesions, and leaflet or annulus dysfunction is mandatory to predict the efficacy of repair and to choose the most appropriate repair technique.Planning leaflet resection, evaluating the type and sizing of prosthetic rings, and avoiding systolic anterior motion (SAM) are the three goals to achieve .On the other hand, only a few pieces of data underline the importance of 3DE during a surgical procedure. Conversely, MV repair failure repair may be detected early using RT3DE .2 MV area, sufficient leaflet tissue, flail gap ≤ 10 mm, flail width ≤ 15 mm, coaptation depth ≤ 11 mm, and coaptation length ≥ 2 mm. However, it has been shown that complex MV lesions can be successfully treated with MitraClip [An anatomical assessment of the MV apparatus is needed before percutaneous repair. The EVEREST ,70 echocitraClip ,72,73,74itraClip ,77,78,79The initial step is the guidance of the transseptal puncture. The ideal location for the puncture in most patients is the mid-fossa in the bicaval view, and the puncture site is directly visualized by the ‘tenting’ of the fossa ovalis. 3D-TEE with X-plane modality provides imaging of the entire interatrial septum (IAS). It allows the correct position and orientation of the Mullins Catheter, avoiding complications  and advancement of the clip delivery system (CDS) into the L.A.; RT3DE with X-plane modality permits us to avoid LA free-wall injury C.3.The third step is positioning the Mitraclip above the MV. RT3DE with zoom modality en face view allows determination of when the clip is adequately positioned above the middle segments of the MV and if the orientation is perpendicular to the line of coaptation D.4.The fourth step is the advancement of the Mitraclip in the LV. RT3DE with zoom modality in the L.A. or LV views represent direct visualization of the MitraClip concerning the MV and the line of coaptation, lowering the overall gain to get a clear impression of the clip in the LV views E.5.The fifth step is grasping the leaflets and assessing proper leaflet insertion and clip detachment.2DE is the preferred method for monitoring the procedure in this crucial step because of the low frame rate of RT3DE imaging . This isHowever, many clips may be grasped in a non-central position, so the classic bi-dimensional three-chamber view is not appropriate, and the RT3DE X-plane modality provides accurate localization of the grasping view by placing the interrogation line through the clip in the commissural view and visualizing tissue grasp in the orthogonal view.6.The sixth and final step is assessing the procedure results. RT3DE color may be used to evaluate the residual MV regurgitation, and 3D-TEE multiplanar reconstruction may be used to assess the post-implant mitral valve area. The clip can be repositioned or withdrawn in case of severe mitral stenosis. In case of residual significant MV regurgitation, another clip can be released.3D-TEE is advantageous in the majority of the steps for the MitraClip procedure , includiIt has been hypothesized that 3DE is useful not only in surgical or clinical decision making but also in the short-term management of MVD due to the fact that it detects most complications early, such as detachments of the prostheses due to endocarditis or pannus or prosthesis thrombosis. Moreover, a role for RT3DE in the long-term management of MVD has been proposed .The main limitations of RT3DE are related to physicians’ expertise, considering that certificated training is required.In this paper, we highlighted the “tip and tricks“ of the RT3DE acquisition modalities and post-processing to provide a practical guide for the echocardiographer to facilitate and accelerate the learning process.Several advantages of RT3DE, such as allowing simultaneous visualization of more than one cardiac structure, which enhances the procedure’s safety; lower procedural complications; and shorter interventional times, have been reported.However, incorporating RT3DE into the workflow may be cost effective and useful in clinical practice . CurrentImportant advances in RT3DE imaging have recently been reported. The introduction of RT3DE in clinical practice using both transthoracic and transesophageal modalities has improved spatial image resolution and anatomical characterization of the cardiac structures, allowing a real-time visualization of the valves and subvalvular apparatus, volume quantification, surgical timing, intraprocedural guidance, and post-procedural evaluation.


Microscopy of ice cream samples after freezing and hardening shows the ability of β-glucan to form a greater number of energy bonds due to specific interaction with milk proteins. Analysis of the microstructure of ice cream samples during 28 d of storage confirms the ability of oat β-glucan to suppress the growth of ice crystals more effectively than Cremodan® SI 320. Oat β-glucan gives ice cream a rich creamy taste, increases overrun and resistance to melting, which brings this type of frozen dessert closer to a full-fat analogue (10% fat).The work is devoted to the study of the functional and technological properties of oat β-glucan in low-fat milky ice cream (2% fat) in comparison with the stabilization system Cremodan Ice cream as a complex heterogeneous food object, which is at the same time foam, emulsion and suspension, is characterized by aggregative instability throughout the entire technological process of production and during storage . In the The average size and distribution of ice crystals significantly affect the consumer properties of ice cream ,14, alon®  functional complex systems for ice cream are widely known, which along with hydrocolloids contain high-melting mono- and diglycerides of fatty acids (E471). However, now the priority of consumers is food products that are made from exclusively natural ingredients that are not identified as food additives. One of the natural stabilizers is β-glucan, which, due to the active binding of free moisture, is able to increase the viscosity of mixes, increase overrun, resistance to melting, promote uniform distribution of the air phase, and influence the growth and distribution of ice crystals under production and storage time [Today, there are numerous modern stabilization integrated systems for ice cream that contain food emulsifiers and a stabilizer. For example, Cremodanage time . Burkus age time  explaineage time , which mTherefore, due to the functional and technological properties indicated above, β-glucan in the composition of low-fat and non-fat ice cream can significantly affect the nature of freezing of free water during low-temperature processing of mixes and storage of the finished product, which has not been investigated before.In addition, β-glucan is a good delivery system with pronounced functional properties that provide a positive effect on the cardiovascular system (effect of reduce blood lipid and blood glucose), the digestive process, and the maintenance of beneficial human intestinal microflora ,24. UndoMilk fat not only gives an original taste, but also contributes to the formation of a finely dispersed and stable structure of ice cream. Therefore, the ability of oat β-glucan to mimic the presence of milk fat  in non-fThe purpose of the research is to investigate the functional and technological properties of β-glucan from oats in the composition of low-fat ice cream.-to conduct a comparative analysis of the patterns of the crystallization process of free water in samples of low-fat ice cream with a classic stabilization system and with β-glucan during low-temperature processing;-to investigate the dynamics of changes in the size of ice crystals in ice cream with various stabilizers during its storage;-to reveal the influence of β-glucan on the main physicochemical and organoleptic indicators of mixtures and ice cream.To achieve the goal of the study, the following tasks were formulated:An increase in the coefficient of dynamic viscosity was observed, due to the high structuring ability of oat β-glucan , which u® SI 320 and β-glucan in both cases increases the viscosity, but the highest value was observed for the ice cream mix with oat β-glucan, which can be explained by its high degree of free moisture adsorption and corresponding structuring [® SI 320 and β-glucan decreases slightly, but falls within the generally known limits [The minimal value of the coefficient of dynamic viscosity of 140 mPa·s  was chosucturing ,27,28. Tn limits .® SI 320 is a typical defect of low-fat ice cream due to the recrystallization of free water during freezing [The overrun of ice cream without stabilizers is quite low . Accordifreezing ,32.Aljewicz et al.  reportedApproaching the average value 0.990 for the sample with β-glucan  indicateIn the absence of stabilizing substances, the control ice cream has a high content of free water, which leads to rapid melting of the product .® SI 320 somewhat inhibits the process of ice crystal growth and, as a result, the rate of melting slows down, which is especially noticeable for the samples that were studied after 1 M of storage. The use of β-glucan ensures a significant decrease (p ≤ 0.05) in the melting rate, in particular, due to the high adsorption capacity of oat β-glucan. Such data confirm the available information [In the process of storage, such ice cream acquires a snowy consistency due to the growth of ice crystals that form a coarse crystalline structure. The introduction of Cremodanormation , that thormation ,33, no s® SI 320 (15.7 ± 0.3 μm) and with oat β-glucan (12.5 ± 0.5 μm). Photographs of the microstructure of soft ice cream samples after freezing are presented in the Analysis of the microstructure of ice cream samples indicates that the average diameter of air bubbles was larger for the ice cream sample without stabilizers (25.2 ± 1.1 μm), compared to the sample with Cremodan® SI 320 and β-glucan at the molecular level and ensures the maximum technological effect.What is interesting that the small air bubbles in the β-glucan ice cream sample form connecting elements with the larger air bubbles. The aggregation of air bubbles contributes to a more even distribution of the air phase in the thickness of the product, and forms a plastic and homogeneous structure ,27. The Clear changes in the diameter of ice crystals in the different formulations of ice cream can be seen in ® SI 320, it is obvious that the addition positively influences ice crystals diameters just after production and in a short period of storage. For two investigated variants with additives, the smallest ice crystals were created in milky ice cream with a β-glucan addition  were at the level of 20 µm and, after 1 month of storage, it was noticed at the level of 22.32 µm . It was addition  and an X17.69 µm .Small ice crystals ranging in size to 20 µm of equivalent diameter give the product desired smoothness, while ice crystals larger than 50 μm give the product an undesirable lumpy texture ,7,39,40.Muzammil et al.  reportedIt was previously examined that the shape of ice crystals is strictly dependent on the type of added stabilizers, while the diameter is also influenced by the ice cream composition ,10,39. Scr) determines the patterns of crystallization of the aqueous phase in ice cream. The value of this parameter is affected by the type and concentration of substances dissolved in water [cr of ice cream mixes with different chemical composition is quite contradictory, and its values calculated according to the methods [The cryoscopic temperature ; the further crystallization process at temperatures of minus −15 to −20 °C slows down (up to 88.88%), reaching a maximum at the upper limit, which is 94.44%. In the control and ice cream with CremodanThe prospect of further research is the scientific explanation of the use of natural ingredients, which, in combination with oat β-glucan, will reduce the rate of freezing of free water during all technologically important stages down to a temperature of −40 °C.A sensory evaluation of ice cream samples was carried out using the descriptive-integral method, the results of which are given in p ≤ 0.05) difference in the sensory indicators of control samples and with β-glucan.Oat β-glucan in low-fat ice cream improves its form; overrun, foaming, gives it a rich color and a milky-creamy taste , which cKaur et al.  reportedThe polysaccharides effectively mimic the absence of fat, and their combination with protein ingredients during their further special processing leads to the formation of effective substitutes for milk fat . AljewicIn addition, oat β-glucan exerts a pronounced effect on the formation of the consistency of ice cream, preventing the defect of presence of ice crystals perceptible when consuming the product, which was noted in the Cre sample. It is known that oat β-glucan is able to prevent consistency defects, increase viscosity, and stabilize product acidity .® SI 320  was used as a stabilization system, as a fully integrated mixture of food emulsifier and stabilizers, consisting of homogeneous beads and used for hardened and soft ice cream, in particular low-fat or non-fat.Cremodan® SI 320 in the composition of the ice cream was chosen at the level of 0.5% in accordance with the manufacturer’s recommendations.The mass fraction of Cremodanβ-glucan , extracted from oats with a degree of purification 72%  has the form of a finely dispersed light yellow powder with high solubility. The rational amount of β-glucan for use in low-fat ice cream was chosen according to the recommendations of Aljewicz et al. .Pasteurized cow’s milk 3.2% fat , skimmed milk powder , sugar, vanillin, and drinking water were also used as basic ingredients of low-fat ice cream .The samples of low-fat ice cream (2% fat) with and without stabilizing additives were made according to the compositions given in the Milky mixes were obtained by mixing recipe ingredients  at a tem-−1 for 120 s;at the first stage, the mix was cooled in a cooling cylinder (capacity—7 L) to a temperature of −1 °C at a rotation frequency of a scraper-type stirrer of 4.5 s-−1 for 180 s to a temperature of −5.0 ± 0.5 °C.at the second stage, the mix was frozen at a rotation frequency of 9 sPasteurization was carried out at a temperature of 85 ± 2 °C and holding for 120 s. The mixes were homogenized under a pressure of 12 ± 1 MPa using a laboratory homogenizer-disperser model 15M-8TA “Lab Homogenizer & Sub-Micron Disperser”  with further cooling to a temperature of 2 °C and maturating before freezing for 12 h. Experimental samples of ice cream (4 kg for each sample) were obtained in a periodic freezer FPM-3.5/380-50 “Elbrus-400” . The temperature of the mixes before freezing was 4 ± 2 °C. The temperature of soft ice cream at the exit from the freezer was −5.0 ± 0.5 °C. Freezing was carried out in two stages:The ice cream samples (4 kg for each sample) were additionally cooled and stored in a freezer chamber “Caravell” A/S  at a temperature of −18 ± 1 °C for 1 M Samples of the same chemical composition were prepared at least 3 times. For the study, test samples of ice cream were prepared according to the compositions given in Chemical composition of the samples: fat—2%, MSNF—at least 10%, sugar—15%, total solids—27.01–27.51%.3 of distilled water, and three drops of a 1% phenolphthalein solution of 0.1 mol/dm3 NaOH until a light pink color appeared, which did not disappear within 1 min. The amount of alkali used for neutralization was multiplied by a factor [Active acidity was determined by inserting the electrodes of a potentiometric analyzer into prepared ice cream samples . The acia factor .A Hepler viscometer with a set of 6 glass and metal balls of different diameters was used to determine the coefficient of dynamic viscosity. The ball №3 was used to study ice cream mixes. The glass tube of the device was filled with ice cream mix, cooled after pasteurization to 20 °C . After i3/g; ρ1 and ρ2 are the density of the ball materials and mix, respectively, g/cm3; and t is the duration of the passage of the ball between the ring marks, s.The coefficient of dynamic viscosity of the mix µ (mPa·s) was calculated according to the formula ,61:µ = kIn total, 5 g of each ice cream sample was taken from the center of the portion in, at least, three different places and at a distance of 3 cm from the surface of the product, placed at a temperature of 19 ± 1 °C in a Goryaev chamber, covered with glass, and immediately subjected to microscopy at a magnification of 160 times. At the same time, the ice crystals melted, but the foam remained, because under these conditions the shells of the air bubbles did not dehydrate. Photomicrographs were obtained using an Olympus CX41 light microscope  and a camera .The overrun of ice cream samples was determined by the weight method ,63, accoa) is a technological value interdependent on the overrun of ice cream and was calculated according to the following formula [The volume fraction of air , a small piece of ice cream was taken from the center of the plastic box, from at least 3 different locations, a minimum of 3 cm away from the ice cream surface, and placed on an object slide by using a spatula; and then covered by the cover slip placed on the top of the sample. Pressing the sample reduced the overlap of ice crystals to discern them individually and destroy the air cell structure to provide excellent clarity. Samples were prepared in a freezing chamber and transferred into a microscope with the cooling system . This system eliminates the influence of ambient temperature.The recrystallization process was analyzed based on the images of ice crystals taken after preparation. A microscope Olympus BX53 with the cooling system Linkam LTS420  (temperature range from −196 °C to 420 °C) and a camera Olympus SC50 were used. The obtained images were then analyzed using an NIS Elements D software. From 300 to 500 crystals were marked for a particular sample; and then area, equivalent diameter and standard deviation were calculated using the NIS Elements D Imaging software . The frequency distribution of crystal size was computed using Microsoft Excel 2011 macro data analysis. The relative frequency of any class interval was calculated as the number of the crystals in that class (class frequency) divided by the total number of crystals, and expressed as a percentage. The parameter X50 was analyzed as a mean diameter (DA) for 50% of crystals in the sample. The mean diameter (DA) and standard deviations (SD) of each class was also calculated. The method of determination has been shown in the scientific works ,42,68.The cryoscopic temperature and osmolality were determined using a Mercer osm 3000 osmometer  The device measures the freezing temperature with an accuracy of 0.002 °C.The sensory evaluation was carried out on the 28th day of ice cream storage by an expert group (ten members) using the descriptive-integral method ,70. The Analysis of variance (ANOVA) was performed using STATISTICA 13 NIS Elements D software. Test significance was set at α = 0.05. Data are expressed as mean with standard deviations (±SD), and differences between groups were assessed using Tukey’s HSD test. The study of physicochemical indicators of ice cream samples was conducted three times to ensure the reliability of the data obtained.® SI 320. After one month of storage at −18 °C, the ice crystal sizes in in the sample with oat β-glucan did not exceed 12.96 ± 1.92 μm, while in the control sample, they were at the level of 22.32 ± 2.28 μm, and in the sample with Cremodan® SI 320—17.69 ± 1.98 µm.Oat β-glucan (0.5%) reduces the recrystallization of free water in low-fat milky ice cream by more effectively inhibiting the growth of ice crystals compared to Cremodan® SI 320 with the same content (a decrease of 0.166 °C vs. 0.078 °C), significantly (p < 0.05) inhibits the process freezing of free water and, accordingly, prevents the uncontrolled formation of ice agglomerates in ice cream during technological processing. Oat β-glucan also increases overrun and slows down the melting rate of ice cream due to the formation of a specific foam structure. According to the results of the sensory evaluation by the descriptive-integral method, it was established that the sample of ice cream with β-glucan has the highest taste properties.Oat β-glucan in the specified amount has a greater effect on the cryoscopic temperature of ice cream mixes (2% fat), compared to Cremodan® SI 320 with oat β-glucan.The obtained research results indicate the possibility of the complete replacement of Cremodan


In contrast, the MSLT screw using insertion strategies A and C had a greater pullout strength than the SLT and DLT screws both with and without pre-loading. In conclusion, the MSLT pedicle screw exhibited the greatest pullout strength of the screws tested under all insertion strategies and loading conditions, except for insertion strategy B with a cyclic pre-load. While all screw types showed a reduced pullout strength when using insertion strategy B (screw-out depth adjustment), the MSLT screw had the largest reduction in pullout strength when using a pre-load before testing. Based on these findings, during the initial screw insertion, it is recommended to not fully insert the screw thread into the bone and to leave a retention length for depth adjustment to avoid the need for screw-out adjustment, as with insertion strategy B.Pullout strength is an important indicator of the performance and longevity of pedicle screws and can be heavily influenced by the screw design, the insertion technique and the quality of surrounding bone. The purpose of this study was to investigate the pullout strength of three different pedicle screws inserted using three different strategies and with two different loading conditions. Three pedicle screws with different thread designs (single-lead-thread (SLT) screw, dual-lead-thread (DLT) screw and mixed-single-lead-thread (MSLT) screw) were inserted into a pre-drilled rigid polyurethane foam block using three strategies: (A) screw inserted to a depth of 33.5 mm; (B) screw inserted to a depth of 33.5 mm and then reversed by 3.5 mm to simulate an adjustment of the tulip height of the pedicle screw and (C) screw inserted to a depth of 30 mm. After insertion, each screw type was set up with and without a cyclic load being applied to the screw head prior to the pullout test. To ensure that the normality assumption is met, we applied the Shapiro–Wilk test to all datasets before conducting the non-parametric statistical test . All screw types inserted using strategy A had a significantly greater pullout strength than those inserted using strategies B and C, regardless of if the screw was pre-loaded with a cyclic load prior to testing. Without the use of the cyclic pre-load, the MSLT screw had a greater pullout strength than the SLT and DLT screws for all three insertion strategies. However, the fixation strength of all screws was reduced when pre-loaded before testing, with the MSLT screw inserted using strategy B producing a significantly lower pullout strength than all other groups ( Screw loosening is a common complication associated with pedicle screws used for spinal fixation, particularly in osteoporotic patients ,2,3. EarThe design of pedicle screws, the quality of surrounding bone and the surgical insertion technique are the main parameters affecting the screw holding strength ,10. PediSynthetic bones are commonly used in the evaluation of implant safety and function; however, they may not accurately reflect the physical loading conditions. Nevertheless, pullout strength per ASTM standard can be used as an indicator of the holding capacity of a screw in a material during preclinical testing.The purpose of this study was to investigate changes in the pullout strength of pedicle screws with different thread designs after adjusting the insertion depth. The hypothesis was that the pullout strength would decrease after partial withdrawal of the screw, particularly for complex thread designs.3)  were used for all tests in this study [3 was chosen from the literature [Rigid polyurethane foam blocks  were tested after screw insertion, and groups 4 to 6  were first subjected to a ±5 mm caudal-cephalad displacement on the screw head, followed by a cyclic load for 5000 cycles at 3 Hz  after scp > 0.05), we checked them using the Shapiro–Wilk test before performing a non-parametric statistical test. The non-parametric statistical evaluation  was used to determine significant differences between individual means with a significance level of 0.05.To ensure that the dataset in each group follows a normal distribution (p < 0.05) greater than those inserted using strategies B and C (p > 0.05). Inserting the DLT screws using strategy B without cyclic loading resulted in a significantly lower pullout strength (p < 0.05) than strategy A. Using screw insertion strategy B in the MSLT screw group without cyclic loading did not results in a higher pullout strength than strategy A and C.For the SLT screw group without cyclic loading, the average pullout strength of screws inserted using strategy A was significantly  than the other insertion strategies for all screw types (p < 0.05). For strategies A and C without or with cyclic loading, the MSLT screw had a greater pullout strength than both SLT and DLT screws.Applying a cyclic load to the screw after insertion (groups 4 to 6) using strategy B resulted in a significantly lower pullout strength , caused further damage to the bone interface. Wadhwa et al.  reportedApplying a cyclic load to the screw before the pullout test caused a significant drop in pullout strength across all tested groups, which indicated that the screw–bone interface is affected by cyclic loading. As with the screws that were not pre-loaded, reversing the screw (strategy B) prior to the pullout test reduced the screw–bone holding capacity, with the MSLT group having the lowest pullout strength among the three groups. The considerable reduction in pullout strength between strategies A and B in the MSLT group is likely due to the part proximal path of hole being tapped twice (at screw-in and screw-out) and the cyclic load damaging the material at the screw–bone interface. As shown in n = 5 in each group) and the morphology of the pedicle bone and the cortical shell of the vertebral body was not considered, and morphology is an important factor in influencing the loading pattern physiologically. In our study, pullout strength per ASTM standard was considered the indicator to represent the initial holding capacity of pedicle screws. Other real physical simulation loading conditions, such as daily cycle loadings, were not considered. Moreover, it is important to note that this study only evaluated three thread designs of a specific commercially available pedicle screw series.This study has some limitations. First, a synthetic bone block was used instead of cadaveric bone for the pullout testing. While synthetic bone may not be truly representative of real bone, the foam models used for pullout strength only calculated the contribution of the vertebral body and did not represent the cortical-cancellous region of the pedicle, nor did they consider the non-uniform bone density and morphology of the pedicle in real conditions; however, it does have some advantages, such as negligible inter-specimen variability, low cost, ready availability and minimal specimen preparation, which complement the aim of this study to compare the pullout strength of different pedicle screws. Two important limitations of our study were that the sample size was small (Without applying a cyclic load to the screw head prior to pullout testing, the MSLT screw had the greatest pullout strength of all screws inserted using strategy A. However, applying a cyclic load before the pullout test caused a drastic reduction in the holding capacity, and the MSLT screw inserted using strategy B had the lowest pullout strength of all screws regardless of insertion method. In addition, the strategy B insertion method was found to produce a lower pullout strength than the other insertion methods both with and without cyclic loading. Based on these findings, we recommend not driving the full screw thread into the pedicle bone during the initial insertion and leaving a retention length to allow the screw depth to be adjusted.