Synchronization of network oscillation in spatially distant cortical areas is essential for normal brain activity. Precision in synchronization between hemispheres depends on the axonal conduction velocity, which is determined by physical parameters of the axons involved, including diameter, and extent of myelination. To compare these parameters in long-projecting excitatory and inhibitory axons in the corpus callosum, we used genetically modified mice and virus tracing to separately label CaMKIIα expressing excitatory and GABAergic inhibitory axons. Using electron microscopy analysis, we revealed that (i) the axon diameters of excitatory fibers (myelinated axons) are significantly larger than those of nonmyelinated excitatory axons; (ii) the diameters of bare axons of excitatory myelinated fibers are significantly larger than those of their inhibitory counterparts; and (iii) myelinated excitatory fibers are significantly larger than myelinated inhibitory fibers. Also, the thickness of myelin ensheathing inhibitory axons is significantly greater than for excitatory axons, with the ultrastructure of the myelin around excitatory and inhibitory fibers also differing. We generated a computational model to investigate the functional consequences of these parameter divergences. Our simulations indicate that impulses through inhibitory and excitatory myelinated fibers reach the target almost simultaneously, whereas action potentials conducted by nonmyelinated axons reach target cells with considerable delay. The formation and recall of sensory, motor, and cognitive representations require coordinated fast communication among multiple cortical areas. The mechanisms underlying long-range coordinated timing of neuronal activity are currently debated. Excitatory principal cells monosynaptically innervate both glutamatergic principal cells and local inhibitory interneurons in each target area and these gamma aminobutyric acid (GABA) expressing inhibitory cells play a key role in the local synchronization of principal cells . InterneThe fiber composition of the corpus callosum has been investigated extensively in number of animal species including rodents, rabbits, cats, monkeys, and humans . Cross-sHowever, it is not clear how highly coherent activity, at a precision of milliseconds, is achieved over areas covering hundreds of millimeters by richly branched axons with a wide range of conduction velocities. Studies involving conduction velocity and myelinated nerve fibers started long ago in which more emphasis was given to axoplasmic conductivity and fiber diameter . ConductAlthough previous studies have investigated the fiber diameter composition of the mouse corpus callosum, the existing methodology then did not allow the distinction between excitatory and inhibitory fibers . The purAll mice used in the present study were obtained by time-mating wild-type C57BL/6 mice  with transgenic mice expressing Cre under the CamKIIα promoter , Cre B R (5′-AGCTACACCAGAGACGG-3′).An adenovirus construct was produced by inserting a DNA fragment, CA-promoter (CMV-enhancer+chicken actin-promoter)-loxP-mRFP-loxP-GAP43-palmitoylation-signal -GFP-polyA into SwaI site of pAxcw . The adenovirus was produced by transfecting HEK 293 cells with the cosmid DNA. The titer of the adenovirus was raised to 1 × 109 PFU/mL before use by ultracentrifugation. The adenovirus was designated AdlmRFPlGGFP .BDA injection Biotinylated dextran amine, molecular weight 10,000 (BDA) was dissolved in 0.01-M sodium phosphate buffer at 10% concentration. Micropipettes with tip diameter 20–30 μm were filled with the BDA solution and 0.5 μL was injected into the cortex (coordinates are below).Virus injection from the virus stock of 1X 109 PFU/mL, 0.5 μL was taken and mixed with 0.5 μL of NaCl (2M) at a ratio of (1:1). We standardized the volume of viral solution to be injected with or without NaCl and found that 0.5 μL of the mixed viral solution with NaCl (1:1) was the best dilution to be administered for each injection. The weights of the mice individually were taken and 0.01 mL of anafen was administered intra-peritoneally before surgery. The animals were anesthetized with 1.4% Isofuran. Adenovirus solution (0.5 μL) was injected into the cortex mediolaterally 3.40 mm, anterior–posterior 0.5 mm and dorsoventrally at 0.5 and 1 mm. After the surgery, the animals were back to full consciousness within a few minutes with normal movement and locomotion. The animals had normal survival periods and none died of viral toxicity or infection.Animals of the same age (3 weeks old) were injected with tracers. One month after virus injection or 1 week after BDA injection animals were perfused transcardially with 0.9% NaCl, followed by 4% paraformaldehyde in phosphate buffer (pH 7.4). Fixed brains were removed sectioned sagittally at a thickness of 50 μm using a vibrating microtome . After fixations, the tissues were washed again 3 times with PB 0.1 M followed by rinsing 3 × 5 min in Tris Buffer Saline  and blocked for 45 min with 10% normal goat serum  containing 0.5% Triton X . Primary antibodies like rabbit-anti-GFP , mouse-anti-GFP , mouse-anti-GAD; , mouse-anti-GABA , rabbit-anti-RFP , and mouse-anti myelin-associated glycoprotein  were diluted in TBS and sections were incubated overnight as the first layer of the immunoreaction. After 3 washes in TBS, sections were incubated for 3 h into either CY2-conjugated goat anti-mouse or CY2-conjugated goat anti-rabbit antibodies to detect GFP , or CY3-conjugated goat anti-rabbit antibodies  to detect RFP, or biotinylated anti-mouse IgG or biotinylated anti-rabbit IgG (both Vector Laboratories) followed by streptavidin-conjugated Alexa 405-conjugated  to detect GAD or GABA. Appropriate controls by omitting primary antisera were performed resulting in no detectable immunosignal except at the injection site (not shown). BDA was detected with streptavidin-conjugated Alexa 546 . Immunoreactions were analyzed under fluorescent (Olympus AX 70) and confocal microscopy . FV5-LD405, Ar488, and HeNe1 543 lasers were used to visualize blue, green, and red signals, respectively. To filter suppression of crosstalks, images were taken sequentially in averaged frames using Kalman Filtering.Tissue sections were cut sagittally, washed in Tris-buffered saline , cryoprotected in 30% sucrose overnight and permeabilized over liquid nitrogen by freeze-thawing. Sections were washed again in TBS and blocked in 5% NGS in TBS for 45 min, then tissues were kept overnight with primary antibodies. The following primary antisera were used: rabbit anti-GFP , mouse-anti-GFP , and rabbit anti-RFP . Sections were thoroughly washed TBS 0.05 M and incubated at room temperature with biotinylated secondary antibody diluted 1:1,000 in TBS for 1.5 h. After washing with TBS tissue were incubated with Avidin biotinylated horseradish peroxidase complex  for 1.5 h at room temperature, washed with PB (0.1 M) and the immunoperoxidase reaction was carried out using Ni2 + −intensified 3,3′ diaminobenzidine 4-HCl  as chromogen and 0.03% H2O2. After osmication (1% osmium tetroxide in PB) for 30 min, sections were washed in PB, dehydrated in a graded series of ethanol, counterstained with 1% uranyl acetate in 70% ethanol, cleared in propylene oxide, subjected to overnight infiltration in Durcupan , flat embedded, and polymerized at 57°C for 48 h. Areas from the anterior (genu) region of the corpus callosum contralateral to the injection site were re-embedded in such a way that the cutting surface was perpendicular to the surface of the sagittal section. Thus, we maximized the chance to sample axons cut across. Serial sections were cut with diamond knife and collected onto Formvar-coated single slot copper grids and counterstained with lead citrate before examination on a Tecnai T12 electron microscope (Thermo Fisher Scientific Inc.) equipped with a Megaview II digital camera . For DAB labeling, a non-biotinylated secondary antibody was used  and further developed with Peroxidase anti peroxidase (PAP) conjugated tertiary antibody.In the sections prepared with the electron microscopy protocol, it was estimated the shrinkage for sections to be symmetrical and 20% in each of the axes; a correction factor of 1.25 was applied to all 3 dimensions for these cells  and axon diameter without myelin were measured.Diameters of the cross section of axons were measured in U test after Bonferroni correction and Kruskal–Wallis H-test was carried out to analyze differences between axon diameter and myelin thickness of excitatory and inhibitory neurons. Because the group size was often different, significance level was adjusted to avoid sampling bias. Null hypotheses were rejected if the significance level was equal or below 0.05. All statistical analysis was performed using SPSS v21  and Microsoft Excel v 2010 .To determine the difference in axon diameter and thickness of myelination in excitatory and inhibitory axons, first outliers were removed using Outliers labeling rules, then the distribution normality was analyzed with Saphiro–Wilk test, Q–Q plot, and Detrended normal Q–Q plot. Mann–Whitney Computational models of the axons were developed using the compartmental modeling technique on NEURON simulation platform, as illustrated in We identified the callosally projecting axons by injecting BDA 10k  cells, whereas Cre negative cells  expressed RFP . Becausex and y axonal diameter ratio fell between 0.8 and 1.2. The number of axons that fulfilled the ratio requirement and used for the analysis is indicated in The electron microscopy images of sagittally cut sections from the corpus callosum of wild-type noninjected animals showed normal morphology. Most of the axons with varying diameter were myelinated, intermingled with smaller diameter nonmyelinated axons. The axon diameter and myelination were greatly varied . To distQualitative inspection suggested that the diameter of excitatory axons was larger than inhibitory and we often observed only thin myelin layers around the excitatory axons. In the case of DAB-Ni labeled inhibitory axons, the myelin layer was thicker. These findings suggested that inhibitory axons had smaller axon diameter and thicker myelin sheath in comparison with excitatory axons.n = 100) than the diameter of nonmyelinated excitatory axons . The diameter of excitatory myelinated fibers were also significantly larger than inhibitory myelinated axons without including myelin sheath thickness . There was no statistical difference between excitatory and inhibitory nonmyelinated axons, nor between the bare axons (myelin thickness subtracted from the diameter of the fiber) of myelinated inhibitory fibers and nonmyelinated inhibitory axons . The myelin thickness and number of layers of myelin sheets of excitatory and inhibitory neurons were remarkably different. We used additional high-magnification images to count the number of myelin sheath layers . The thickness of inhibitory fibers myelin sheath was significantly larger than excitatory fibers (P < 0.001). The g ratio was determined by dividing the diameter of the axon by the diameter of the fiber (axon with myelin). Excitatory fibers had g ratio of 0.9 ± 0.027 (n = 94) while the g ratio value of inhibitory fibers was significantly smaller 0.8 ± 0.034 . Interestingly, the myelin thickness of inhibitory neurons increased significantly more with the fiber diameter than the excitatory fibers (P < 0.001). Unsurprisingly, the thickness of individual lamellae of myelins ensheathing excitatory and inhibitory axons  was also statistically significant  followed by inhibitory fibers (0.551 m/s at 12-mm distance), whereas the nonmyelinated axons conduction velocity was much slower ; therefore, they differ in cross-sectional area and volume by nearly 10,000-fold. For methodological reasons we used transgenic mice to reveal differences between excitatory and inhibitory callosal axons. These experiments cannot be performed in human samples therefore the findings presented here need to be used with caution when translated to human brain functions. Functional lateralization is a fundamental principle of the human brain  that is Although there might be a sampling bias in our method (i.e. virus uptake or GFP/RFP expression of myelinated neurons differs from nonmyelinated ones) our finding that more nonmyelinated excitatory axons run in the corpus callosum than myelinated suggest a functional segregation of myelinated versus non/myelinated axon population. The prediction is that myelinated axons are responsible for cross-hemisphere synchronization, the information traveling through nonmyelinated axons represent a fundamentally different modality. In contrast, inhibitory axons were almost always myelinated thus likely playing an important role in interareal synchronization. A recent paper has shown that the locally-projecting, relatively short-axon inhibitory interneurons are a major source of myelinated axons within cortical gray matter, in contrast to the myelin in white matter that forms almost exclusively on the axons of long-distance projecting excitatory neurons . In partOur study also demonstrated that the long-projection axon’s myelination thickness was linearly dependent on its axon diameter. Interestingly, the relationship of myelination and axon diameter, while linear in both cases, was different for excitatory and inhibitory fibers. The functional explanation might lie in the importance of inhibitory neurons in network synchronization. Since a small number of inhibitory neurons are needed for network synchronization but their efficient information transfer is essential for the normal function of the network it is expected that all long-projection inhibitory axons are myelinated, and the myelin sheath thickness is larger compared with the excitatory axons with the same diameter.g ratio number  for inhibitory compared with the excitatory axons. Previous studies have suggested that the practically feasible ways of increasing conduction velocity to compensate for larger brain sizes in a biological system, is via myelination or increasing the axon diameter. The majority of axon diameters remain more or less similar across species (~ 0.1–0.2 μm). A small minority of axons were found to have increasingly larger diameters (thereby larger conduction velocities) with increase in brain size. However, the increase in diameters does not sufficiently compensate for the delay in interhemispheric transmission time that accompanies larger brain sizes. Such biophysical constraints on the conduction velocity in larger brain sizes might have favored the development of hemispheric asymmetry, i.e. functional lateralization. Studies from our simulations illustrate how the various biophysical parameters affect axonal conduction. The various parameters influence the conduction velocities to varying extents, and even in the nature of these effects. The model built here was based on data obtained experimentally from mice, and thus it most accurately can be described as a mouse model for callosal axons. Nevertheless, we have additionally made a tentative attempt to utilize this model to simulate for larger brain sizes, and thereby extrapolate data as applicable to the human brain. Studies in the past have proposed maximal conduction distances in the human brain ~180 mm (Although our finding that the mean myelin thickness of excitatory axons is much larger than the inhibitory seems in contradiction to previous reports on the hippocampus , it is o ~180 mm . We have ~180 mm . Accordi ~180 mm .The ultrastructural difference of myelin layers between excitatory and inhibitory axons was intriguing. Previous studies have demonstrated the heterogeneity of oligodendrocytes based on their morphology , gene ex


The study aimed to reveal physical exercise conditions and catecholamine response-dependent differences while an individual experiences a flow state (FS) following noncompetitive and competitive running drills. Urine laboratory catecholamine levels were measured using a standard clinical method during pre- and post-physical exercises. The noncompetitive task involved intermittent running drills, from an absolute beginning up through exhaustion. Initially, the drill is performed individually then later competing alongside other runners. Twenty-two males  were selected in accordance to the following criterion: healthy status without using medication, routine forms of training  ideally performed with regularity, at least three times per week, 45 min per session. During the noncompetitive task, a high FS experience was associated with a low level of catecholamines,  while in parallel, the high FS was associated with a low concentration of homovallinic acid. During competitive conditions, the FS-related catecholamine level changes have not yet been found. In conclusion, the low concentration of the circulating catecholamines supports the transient hypofrontality hypothesis regarding the FS experiences. Furthermore, synchronized noradrenaline and adrenaline neurosecretion play an essential role in the manifestation and the prolongation of FS in noncompetitive exercise conditions. Attention does not respond to environmental cues, and the social demands become marginal, however, realization remains goal-oriented and self-confident4. The general description of FS contains a positive affective state involving high cognitive flexibility to integrate complex visual scenes and motor actions. The initial starting point of the flow-guided action is a confidently running mental schema. In most cases, the FS manifests in conditions associated with high-level mental or physical performances and intensive physiological reactions while the outcome of the flow-guided behavior remains individually positively evaluated5. The flow state definition originates from several interviews among artists, athletes, outdoor enthusiasts, scientists, explorers, and drug-dependent populations. Its factors were measured by self-reported questionnaires. The challenge-skills balance, action-awareness merging, clear goals, concentration on the task execution, withdrawn self-consciousness, experiences of intrinsic reward, and independence in the sense and interpretation of the emotional state are the main characteristics of the FS. The FS features a dual nature; one side is considered a cognitive working model, and the other side is a desired affective state, the flow experience per se1.According to Nakamura et al., the flow state manifests as an unexpectedly euphoric, uplifting sensation in which a distal environmental event fuses with an individual’s body and personal space. The spatial and temporal cues of the currently executed actions withdraw from the foreground regarding conscious control ushering to a place consumed with an automatic sequence of successive actions. They flow without strong attention control, worrying, and unpleasant efforts6. Similar results were reported in EEG, transcranial direct current brain stimulation, and near-infrared spectroscopy studies9. Additionally, FS was associated with higher theta activities in the frontal brain areas10. These results support the transient hypofrontality neurocognitive theory (THT), in which FS was introduced by Dietrich11. Due to the current evidence in support of THT, the components of FS can be articulated in a detailed network wherein the dorsolateral prefrontal cortex via the working memory information buffer plays an essential role in the regulation of environmental direct triggers and the affording automatisms from the striatal system. These areas are engaged in the construction of self-reflective references and the conflict-based arousal system of the brain. While individuals maintain their drive toward a goal-directed action, this transient top-down inhibitory system supports them in remaining focused on the task independently from the redundant environmental signals without intensive conscious effort or control12. The FS plays a vast role in innovative artistic creativity including several areas of successful athletic performance. However, rapid changes in the circulation of neurotransmitters and the limitation in fMRI use during intensive physical activity restrict revealing the exact relationship of neurotransmitter background regarding FS.Under fMRI recording, increased levels of FS were associated with increased activity in the inferior frontal gyrus and putamen, and in contrast, decreased activity was detected in the medial frontal cortex and amygdala14. On the other hand, in consideration of the dopaminergic mesolimbic-mesocortical pathway connecting with medial prefrontal cortex is engaged with motivation and regulation of the reward-oriented behavior in which the approaching (to do something) and the anticipated reward  are concomitant agents15. However, it is worthy to note the component of the catecholamines (CATs) in any case showed a reciprocal regulation from one another depending on the location of the receptors of the brain16. Notably, the conditions of the ongoing exercises influence the outcome of the performance, and the manifestation of the FS7.Human and animal studies demonstrate how physical exercises induce changes in dopamine (D), noradrenaline (NA) and adrenaline (A) while contributing to the form of acute cognitive and behavioral responsibility. The catecholamines, NA, A, and dopamine D are the main regulators in the exercise-induced physical, emotional, and cognitive response and the motivation of the goal-directed execution. The dopaminergic system originates from the substantia nigra and the ventral tegmental nucleus and, via the nigrostriatal pathway, assumes its role in the acquisition and the recalling of the subsequent movement schemata17. A minimal condition during sports activity is to keep the movement and cognitive strategy for automatized control of the action and is the self-confidence and anxiety-free realization of an idea18. Following the consciously controlled movements, drilling the conscious reappraisal training in hypnosis or mindfulness mediations are very beneficial to apply for transmitting the part of the movements into a global gestalt. During the reevaluation, the focus is fixed on the automatic running of the acts while the environmental triggers are desensitized or depersonalized. This mental prolusion plays an essential role in the manifestation of the FS, both in rhythmic, physical drills and the construction of motor sequences. Results from fMRI studies support the specific role of the prefrontal attention network in the induction of FS among athletes and outdoor enthusiasts19. The FS manifests during several types of athletic activities and in both younger and older athletes20. Attention allocation, a relatively effortless deep innovation into the task-related current activity, is the major area in which to understand the FS, the optimal arousal, and athletic performance success21.The exercises are considered a behavioral intervention which enhances brain health and plasticity while providing neuroprotection and mental and physical fitness. Successful performance demands exact goal description, personal persistence, and excessive training of motor sequences, all of which serve as the main prerequisite to higher-quality sports performance24. An earlier study focused on hobby runners found marked catecholamine changes in serum concentration and urine metabolites during physical challenges, namely, the exposition to a noncompetitive or competitive exercise; the D, NA and A concentration remains enhanced until task completion26. The main proposed question is the way in which this catecholamine undergoes change during exercise among individuals with different degrees of FS following the completion of a noncompetitive or competitive physical exercise. While the body and the mental apparatus adapt to the physical challenges, the regulation of the NA, A and D play a pivotal role in the emotional and motivational aspect of the adaptation and influence the integration of affective, cognitive and motoric functions27. The definition of FS, optimal arousal, mental and emotional state to reach excellent physical or mental performance2 has different stages during intensive sport activity. The optimal state is depending on trained motoric and cognitive skills that influence the way of attention allocation. In non-competitive conditions, the dominant mental states are enjoyment, enhanced motivation, perceived control, altered perceptions, absorption, and confidence. However, in a competitive environment the concentration on a fixed goal, the quick decisions, increasing effort, and heightened awareness may be beneficial to reach the win18. In this condition, the race stress behaves as a clutch, that induces a change in the rate of arousal, and modification in the focus of the attention allocation. The present study is focusing on special motoric, emotional and cognitive changes which are subjectively manifested as an intensive FS experience. Considering the Transient Hypofrontality Theory regarding the FS14, the exercise-induced FS may be a manifestation of prefrontal cortex hypoactivity. This suggestion is supported by EEG studies, however, the neurotransmission research, as of yet, has not revealed a coherent concept regarding FS-related CATs associations. The associations between neurotransmitters in the central nervous system and excreted catecholamines in the urine are not fully proven, however there are clinical data that suggests urinary neurotransmitter testing could be a tool to estimate the nervous system function, as in the case of ADHD, PTSD and depression30. In this study, we suppose excreted CATs  and their metabolites; Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are associated with the degree of FS which may be manifested both in noncompetitive exercise and competitive exercise. On the other hand, a manifestation of FS varies in rate and patterns dependent upon other personality predispositions including levels of anxiety, the degree of self-confidence, emotional intelligence31, and absorption capability-related parasympathetic psychophysiological activation32. The FS and the excreted CATs associations will be measured in pre-and post-exercise in noncompetitive and competitive running conditions. The introduction of the condition-dependent flow is grounded in Csikszentmihalyi’s3 and Rakei’s et al.31 suggestion in which levels of elevated stress, such as competition or anxiety, results in elevated attention to the environmental demand33 and in parallel, attenuates the emergence regarding FS experiences. We suppose (Hy1): The high score in FS will be higher in noncompetitive than when compared with competitive running conditions. (Hy2): The high score in the FS may be attributed with lower scores in anxiety and higher scores in self-confidence. Furthermore, (Hy3): as a previous study suggested26, the circulated amount of the CATs is associated with the demand for physical effort during exercise in noncompetitive and competitive conditions and will be increased to the end of the task completion, independently from the applied different conditions. The last yet not least hypothesis (Hy4): we suppose the amount of the excreted CATs will be lower in non-competitive than when compared with competitive conditions.Sports and regular physical training make use of the multiple mechanisms of the brain’s hemodynamic and neurotransmitter system, which is beneficial in the enhancement of the motoric skills, social and cognitive capabilities and support the physical and psychic health26.The measured blood parameters did not show significant correlation between the non-competitive and the competitive running test as published beforeThe obtained data alludes to a difference with a large effect size between noncompetitive and competitive conditions in reference to the action-awareness merging (AAM) flow scale. In competitive conditions, the score of the AAM flow was lower than in the noncompetitive condition Table .Table 1F34. Furthermore, higher FS were associated with lower perceived life stress. Moreover, the post-running administered self-reported anxiousness in most of the cases was lower in both noncompetitive and competitive conditions among participants with high flow state experiences. Considering the post-running emotional state, the low somatic anxiety and the high self-confidence was associated with a higher flow state in noncompetitive and competitive situations , depression , and perceived life stress  personality traits compared to the mean scores of a sample of the national population indicated these participants could be considered a healthy sampleRepeated measures ANOVAs were used to test the effect of time (pre- vs. post-task), condition (noncompetitive vs. competitive), and their interaction (time × condition) on individual and summarized catecholamine levels. Results are presented in Table 2 values indicated large effect sizes with regard to all significant results.Concerning the combined level of CATs Table , a signi26.Levels of HVA were also higher in the competitive condition, as compared to the noncompetitive condition, however, its levels generally showed a significant decrease from pre-task to post-task. Similar data were reported in a previous study by Nagy et al.Pearson’s correlations Table . demonstHy1, in which the competitive situation and the manifestation of FS, namely the action-awareness merge (AAM) cognitive function is partially inhibited. This result is consonant with earlier findings regarding stress situations (competition) in which the flow in most cases is lower than when compared with anxiety-free conditions3. The lower flow in competitive exercise is not inevitable.This study aimed to explore the relationship between the flow state experience and the excreted catecholamines while participants underwent noncompetitive or competitive standard running tasks. The obtained results indicate that FS manifests both in noncompetitive and competitive tasks yet differs to varying degrees. In comparing noncompetitive vs competitive intermitted running tasks, the degree of the FS and challenges-skills balance CSB scales have not shown any measurable difference. However, the AAM flow state experience showed a lower score in competitive conditions. Interestingly, the obtained results support the 36.Among athletes, mainly during elite sports training, successfully applied methods include hypnosis, meditation, mindfulness, and relaxation techniques to improve the task focusing abilities and also to keep calm during periods of stress in an anxiety-provoking situation. This intervention enhances the chance of the FS while parallels the improvement of task performance and enhancement regarding the FSHy2 and previous data33. These correlations indicate anxiety plays a negative while self-confidence play a positive role in the manifestation of the FS. Therefore, anxiety is disadvantageous, however, self-confidence bears an advantageous effect regarding flow embodiment.In another course of results, the association analysis between the FS and health-related trait variables revealed FS is associated with low-level general life stress. In examining the relationships between the degree of the FS and positive and negative affective states in post-measured noncompetitive and competitive conditions, the obtained results illustrated both in noncompetitive and competitive conditions, the FS and levels of self-confidence are equally high. On the other side, the high FS is accompanied with a lower level of somatic and in a partially cognitive anxiety state. The FS and state anxiety associations support the 26 and show how in all conditions, the pre-and post-measured degree of the CATs concluding task completion significantly increased. The time and condition interaction indicate both NA and A play a vast role in exercise-induced neurotransmitters response. These are supporting results regarding Hy3.The change in pre-and-post-measured CATs in the noncompetitive and competitive conditions is identical to previously reported data25, however, presently, the nature of the uncontrolled sex-dependent reaction and other cardiovascular and glucose metabolite effects of these systematic changes has not been exactly interpreted.Considering the urinary collected metabolites, it can be stated, changes regarding VMA showed a consonant enhancing pattern, both in noncompetitive and competitive situations. This results in support by others37 which significantly decreases following mental stress38, however, in this population, considering the small number of enrolled participants precludes thoroughly investigating mental stress. Seemingly, our data indicates exercise-induced changes in the urine concentration of the CATs, with the exception of HVA, are working in a unidirectional and synchronized means. Clearly, the HVA depicted a reverted line.Distinctively, the HVA concentration was attenuated after performing noncompetitive and competitive conditions. The role of the manifestation of HVA is thought to be a peripheral indicator regarding the central dopaminergic activityHy4 which indicates the elevated stress attenuates the manifestation of the FS and inhibits the synchronized release of CATs.The main aim of this study was to reveal the associations between CATs and the intensity of FS intensity. The conducted analyses indicated in noncompetitive conditions, the lower rate of NA and A, and HVA low concentration in urine samples predicts elevated FS systematically associated with challenge-skill balance and action-awareness merging cognitive functions. A similar association in competitive conditions occurs, in which elevated environmental triggered stress has not yet been detected. This data is aligned with our Hy1.Notably, an result obtained by ANOVA revealed NA + A is higher in competitive conditions. We can underline the importance of this data. We suppose the higher NA + A together may be an essential role in the inhibition of the manifestation of FS in competitive conditions. This difference has been demonstrated during the control of the 39. The present research focused on mainly urine samples among healthy males, who underwent different exercise conditions in which trait and state biochemical and behavioral and personality traits were assessed. The CATs sample taking, and the personality stated data, were precisely scheduled early in the evening including all participants. Consequently, the collected data defined in this standard environment and timescale intervals describes characteristics in reference to both the humoral and the psychological state. The neurophysiologic signature of FS as a psychobiological phenomenon is presently developing and raises numerous questions regarding neurocognitive examinations40. The discussion in the present context focuses on the explication of the correlative data since the data gathering in this domain is of yet, considerably limited. Competition in front of an audience triggers elevated effort stress which influences performance and potentially alters personal experiences during an ongoing task. Earlier studies showed the intensity of the FS is inhibited in conditions in which negative emotions are present, in most cases, the pressure of the excessive achievement distracts the control of the allocation of attention. Consequently, the attention focus turns from inner actions schemata and instead remains fixed on the environmental requirements and events3.The interaction of FS and CATs changing follow diurnal rhythms, situation-specific effects, sex, illness- and stress-dependent and their effect are different in the cortical and mesolimbic areas of the brain14, implies exercise induces a schema-like regulation unit in which the motor pattern, the sensory inputs, the cognitive design and the autonomic regulation by the intensive activity of the working memory are collected into an integrated schema. These mechanisms reflect the “second nature or deep structure” regarding human cognition and behavior, wherein the goal-directed action is running with low self-awareness, of which, is a beneficial condition for the manifestation of the FS. Other research results provide a detailed description of a variation in the biochemical mechanism of this prefrontal hypofunction manifested with a low serum concentration of CATs. It has been suggested that a routine induces an attenuation in the concentration of NA and A at the same time, the prefrontal activation after finishing the schema configuration is decreased42. Our results support this theoretical context and are consonant with the basic idea of the THT hypothesis.As a result, personal achievement including goals are interrupted. The biopsychological examination regarding this attention process evolved into a different yet partially fitting theory in support of the neuropsychological mechanism of the FS. The transient hypofrontality theory (THT)1 wherein the intrinsic reward is the free running of the constructed schema with an adequate adjustment in a real environment.Transient hypofrontality could be a decreasing ability to perceive the risk or identify the negative consequences of the ongoing flow-associated behavior. A higher degree in flow state experiences detected during non-competitive conditions results in challenges in which skills balance activity is dominant. This is a preparatory mental schema forming the training phase of the planned action and has been running at different times and conditions. To paraphrase, it has mentally been executed before it has been implemented in a defined environmental context. In the course of the schema construction, the schema becomes an abstract “blueprint” for execution. Consequently, the implementation automatically runs down if the environmental cues and conditions are available. In this case, an inner motivated scheme is adjusted to a well-defined environmental condition in which the agent is confident and focuses on conducting and integrating a trained design and an environmental demand. This process is defined as a FSThe relevance of the low CATs level of urine in the manifestation of the FS appears in our results from the noncompetitive condition. We suggest low circulation catecholamines (CATs) are associated with an intrinsic motivation state wherein a self-relevant, well-laid mental schema fits the environmental challenges. In contrast, during competitive conditions in which an individual’s attention focuses on the environmental challenges, the rivals and to win accolades, the noradrenaline and adrenaline-releasing augment and the FS becomes, in part, inhibited. Parallel with this is the synergistic release of CATs, of which, is disrupted. Our data emphasizes how FS may be manifested in a competitive situation as well, however, its embodiment is dependent upon decreasing the amount of the excretion of NA + A. The intervention which decreases the CATs disruption and the level of NA + A neurotransmitters may originate from a psychological source, such as induced by mindfulness and relaxation techniques or other mental training methods deemed advantageous for the generation of the elevated FS experiences.In consideration of the above-mentioned interactions between several components of CATs and the limited amount of association with an exercise-induced FS, the hypofrontality hypothesis is partially supported. However, the description of the exact nature of interactions is not yet clearly articulated nor fleshed out.43 Setting the threshold for rejecting the null hypothesis to α = 0.05, a sample size of at least 23 participants was calculated to detect even small effects  with an adequate power of 0.85. During the preselection phase of the investigation, from a group of recreational athletes, volunteers were invited to participate in a physical training-related experiment. The participant’s current health status was screened using the hemodynamic labor assessment and was duly performed by a Doctor of Medicine. ECG and blood pressure were monitored, and heart rhythm detector was implemented to explore potential risk markers for cardiovascular stress. Furthermore, a psychical health-related questionnaire packet was administered regarding depression, perceived life stress and general health status. All participants’ health-related scores remained in the normal range of the normative population sample. Hemodynamic risks were not revealed. Twenty-two males  were recruited for the study, of whom, were selected based on routine forms of exercise  at least three times per week for a minimum of 45 min per session. Additional criteria included a healthy status (no known acute or chronic disease) and no use of prescription medications. To ensure homogeneity of the group and considering many gender differences, including hormonal system that may affect the outcome, our study was restricted to men. All volunteers were asked to avoid consumption of hormone-containing foods, avoid alcohol and the use of drugs 24 h prior to conducting the experiment. The participants were asked to restrain from exercising during the day prior to their assessment and only water consumption was allowed. All running tests were executed during the evening hours. Questionnaires were also completed within 15 min prior to and immediately following the running tests. Informed consent was obtained from all subjects. The investigation was a part of a larger set of experiments that approved by the Regional Committee for Research Ethics of the Locale State University (ref. No.: 7162/2018). All methods were performed in accordance with the Declaration of Helsinki, the relevant guidelines and regulations.Required sample size for repeated measures ANOVAs was calculated using G*Power 3.126. All exercises were performed in the evening approximately at the same time in all cases. It took on average 15 min.The blood and urine laboratory health indicators were measured using standardized clinical method pre- and post-noncompetitive and competitive physical exercises. The noncompetitive task involved a single intermittent indoor run, from an initial starting point up through complete exhaustion. The competitive task contained the same exercise, however, the running was conducted in a competitive environment including other runners and it was hosted in front of an audience. Neither the baseline nor pre- and post-conditions exercise and the health state-related anamnesis did not show illness-related deficiencies among the participants. The heart rate was registered in the pre- and post-phase of the conditions by a Polar Team Pro System. Volunteers were fitted with transmitters positioned just beneath the xiphoid. Blood and urinary samples were taken immediately in both pre- and post-conditions while completing noncompetitive and competitive tasks. The procedure was identical to one used in an earlier study26.The sample taking and evaluation follow a standardized method used systematically throughout our investigations. Prior to and following the running tests within a few minutes, venous blood collections were performed and stored in suitable vacutainers for testing cellular blood parameters, plasma glucose, lactate analysis and routine laboratory blood tests. The subjects were in sitting position while the blood sampling was taken. Plasma and serum parameters were measured as published beforePrior to and following the running test, middle stream urine was collected and stored in native vials. All samples were checked using a rapid test (Cybow 10) and were transferred to the laboratory where it were aliquoted and frozen at − 80 ° until further needed. Following thaw, catecholamines and metabolites  levels were detected by Shimadzu Prominence High-Performance Liquid Chromatography (HPLC) system with an Antec Decade SDCTM electrochemical detector. Chromsystems® kit ) and reverse phase column  were used in full compliance to the manufacturer’s recommendations.VMA and HVA were detected by the same system using Chromsystems® kit ) and reverse-phase column (ref. No.: 1100/B) in full adherence to the manufacturer’s recommendations. All data were interpreted utilizing the LabSolution program.44 was established by the phenomenological definition of FS1 and contains twenty items including a response sheet with a five-point Likert scale, which ranges from strongly disagree (1), to strongly agree (5). Following the noncompetitive and competitive running tasks, participants were asked to annotate their flow experiences using the response scales. Example for items: “I was absorbed with the task”. “I felt control in the use of my hand.” The total FS score involves two basic elements: the challenges-skills balance (CSB) (Cronbach’s α = 0.921) and the absorptions regarding the task which is the action-awareness merging (AAM) (Cronbach’s α = 0.907) indicating high FS capability in the defined situation. The CSB grounded a stable self-coherence and self-knowledge wherein the individuals possess clear ideas regarding their talents and skills. The motivation to cope with challenges are poised and aligned to their skills. The AAM refers to changes in time and space experiences, the feeling of the absorption of the task completion and the self-perception, automatisms and the diminishing loss in self-control. The internal consistency in the present sample: Cronbach’s α CSB = 0.893; AAM α = 0.833; FS total α = 0.890.The Flow State QuestionnaireWell-Being Scale46 involving five aspects which measure the degree of general social, psychical, and physical health Cronbach’s ɑ = 0.793). Beck Depression Inventory BDI Short form-H48, contains nine characteristics to measure the level of depression (Cronbach’s ɑ = 0.745). Furthermore, the Perceived Stress Scale50 was used which contains fourteen aspects to assess the degree of perceived life stress in the recent past (Cronbach’ ɑ = 0.859). Post-Race Questionnaire PRQ52 contains eleven items to assess the somatic anxiety, the cognitive anxiety and the current state of self-confidence pre- and post- measurement of athletic forms of exercise (Cronbach’s ɑ = 0.736); somatic anxiety, cognitive anxiety, and self-confidence ɑ = 0.912–0.656.Other personality trait and state assessment methods are the WHO-5 54. Running induces an exact, well-documented catecholamine secretion which takes part in the regulation of physiological, behavioral and psychological outcomes regarding provoked stress. The concentration of catecholamines in the plasma or their metabolites in urine is a strong indicator of the individual’s stress sensitivity among trained and untrained individuals including animals56. Previous research has documented increasing levels of NA and A are involved in the emotional factor regarding social behavior57. Furthermore, NA is considered a dominant modulator in social behavior, and exercise also induces stress linked to D functions58.The Multi-Stage 20-m Shuttle Run test (Beep test) was used as a standardized exercise in our study2) values were computed as measures of effect size. According to Cohen, benchmarks for small, medium, and large effects were set at 0.01, 0.06, 0.14, respectively59. Lastly, Pearson’s correlations were used again to test the association between reported flow experience and levels of catecholamines after the non-competitive condition. SPSS version 22.0 was used for all statistical analyses.Means and standard deviations were used to describe variables. Levels of flow experience were compared with paired sampled t-tests across varied conditions. Associations between measured and computed trait and state variables were tested using Pearson’s correlations. Repeated measures ANOVAs were used to test the effect of time (pre- vs- post-task), condition (non-competitive vs. competitive), and their interaction on measured and summed values of catecholamine levels. Partial eta-squared (ηThis interpretation remains considerably open to potential questions regarding the role of the D, of which, in association with the CATs, is not yet clearly defined. The role of the HVA during the manifestation of the flow experiences impacts the amount of the metabolite of D, however, this process is not visible. The hemodynamic stress response has not been controlled and we plan to do so, in the upcoming phase of this study. In addition, the present experimental data only represent population of men, thus further studies may be necessary for women.Supplementary Figure 1.Supplementary Figure 2.


COMMENTARY:ANGPTL3 have on average a 35% reduction in the lifetime risk of atherosclerotic cardiovascular disease (ASCVD) events.N‐acetylgalactosamine ‐conjugated siRNA targeted at the liver, where it specifically inhibits and degrades ANGPTL3 mRNA in the RNA‐induced silencing complex within the cytoplasm.ANGPTL3 (angiopoietin‐like protein 3) is a hepatokine exclusively secreted by the liver and a key regulator of plasma lipid and lipoprotein metabolism through the inhibition of lipoprotein lipase (LPL) and endothelial lipase (EL).1AROANG1001 is the first‐in‐human, phase 1 basket trial design that evaluated the safety, tolerability, pharmacokinetic and pharmacodynamic effects of ARO‐ANG3 in adult healthy and dyslipidaemic subjects , triglyceride (median −34% to −54%), non‐high‐density lipoprotein cholesterol (non‐HDL‐C) (mean −24% to −29%), very‐low‐density lipoprotein‐cholesterol (VLDL‐C) (mean −35% to −52%) and LDL‐C (mean +4.1% to −27%) concentrations with the three highest dose  85 days after dosing Figure . Repeat 2Elevated plasma triglyceride and LDL‐C concentrations are both causally related to ASCVD risk.Given that the mechanism of action of ANGPTL3 inhibition with ARO‐ANG3 appears to be independent of the LDL receptor, this agent may be a significant advance for managing patients with severe hypercholesterolaemia, particularly in those with homozygous FH (HoFH) who have minimal or no residual LDL receptor function.ANGPTL3 inhibition with siRNA‐based therapy is not only an effective therapeutic option for severe FH and mixed dyslipidaemia, but may also be useful for mitigating hepatic steatosis associated with the insulin resistance syndrome.3Despite optimal lifestyle and statin therapies, a substantial proportion of patients, mostly those with FH, do not reach guideline‐recommended LDL‐C goals. There is also a substantial residual risk of ASCVD attributable to other atherogenic lipoproteins, such as TRLs.Gerald F. Watts has received honoraria for advisory boards and research grants from Amgen, Arrowhead, Esperion, AstraZenca, Kowa, Novartis, Pfizer, Sanofi and Regeneron. Dick C. Chan declares no conflict of interest.


In young BAs (y-BAs) mechanical stretch does not lead to spontaneous tone generation. Stretch-induced tone in y-BAs appeared only after inhibition of NO-release by L-NAME and was fully prevented by treatment with 3 μmol/L RhoA-kinase (ROK) inhibitor Y27632. L-NAME-induced tone was reduced in y-BAs from heterozygous mice carrying a point mutation of the targeting-subunit of the myosin phosphatase, MYPT1 at threonine696 (MYPT1-T696A/+). In y-BAs, MYPT1-T696A-mutation also blunted the ability of L-NAME to increase MLC20-phosphorylation. In contrast, senescent BAs  developed stable spontaneous stretch-induced tone and pharmacological inhibition of NO-release by L-NAME led to an additive effect. In s-BAs the MYPT1-T696A mutation also blunted MLC20-phosphorylation, but did not prevent development of stretch-induced tone. In s-BAs from both lines, Y27632 completely abolished stretch- and L-NAME-induced tone. In s-BAs phosphorylation of non-muscle-myosin-S1943 and PAK1-T423, shown to be down-stream effectors of ROK was also reduced by Y27632 treatment. Stretch- and L-NAME tone were inhibited by inhibition of non-muscle myosin (NM-myosin) by blebbistatin. We also tested whether the substrate of PAK1 the thin-filament associated protein, caldesmon is involved in the regulation of stretch-induced tone in advanced age. BAs obtained from heterozygotes Cald1+/− mice generated stretch-induced tone already at an age of 20–21 months old BAs (o-BA). The magnitude of stretch-induced tone in Cald1+/− o-BAs was similar to that in s-BA. In addition, truncation of caldesmon myosin binding Exon2 (CaD-▵Ex2−/−) did not accelerate stretch-induced tone. Our study indicates that in senescent cerebral vessels, mechanisms distinct from MLC20 phosphorylation contribute to regulation of tone in the absence of a contractile agonist. While in y-and o-BA the canonical pathways, i.e., inhibition of MLCP by ROK and increase in pMLC20, predominate, tone regulation in senescence involves ROK regulated mechanisms, involving non-muscle-myosin and thin filament linked mechanisms involving caldesmon.Stretch-induced vascular tone is an important element of autoregulatory adaptation of cerebral vasculature to maintain cerebral flow constant despite changes in perfusion pressure. Little is known as to the regulation of tone in senescent basilar arteries. We tested the hypothesis, that thin filament mechanisms in addition to smooth muscle myosin-II regulatory-light-chain-(MLC We have recently shown that aging of cerebral arteries is associated with a hypercontractile state of smooth muscle . The hypVascular smooth muscle tone is primarily the result of the cyclic interaction of thick myosin filaments with filamentous actin (F-actin) in a cycle driven by ATP hydrolysis . Althoug20) on serine19 (S19)  in response to an increase in cytosolic [Ca2+]. The rise in Ca2+ is triggered by various extracellular stimuli such as neurotransmitters, metabolites and mechanical forces   . Phosphol forces . MLC20 ise (ROK) .20 is catalyzed by a constitutively active type 1 phosphatase (MLCP) the activity of which is modulated by phosphorylation of its myosin targeting (regulatory) subunit MYPT1 at several serine and threonine residues  stretch induced tone is augmented in senescent basilar arteries in a ROK-dependent manner, 2) ROK is upregulated in senescence and serves as a central signaling hub, which not only increases inhibitory phosphorylation of MYPT1 but also of non-muscle myosin and PAK1 3) the PAK1-target caldesmon acts as a molecular brake to attenuate stretch induced tension rise.Ethic statement and detail description of all materials and methods can be found in the online supplement.Three different types of muscle tone were examined in this work: 1) stretch-induced tone refers to tone that is present after stretching y-BA to IC90 ; see expandet “Methods” section in online supplement) and in s-BA it refers to the slow tension rise that develops in 20–25 min after stretching the preparations to IC90 in the absence of treatment. This tone is maintained throughout the experiment. 2) L-NAME-induced tone develops in response to addition of L-NAME, a pan-NOS inhibitor; 3) agonist-induced tone occurs after stimulation with contractile agonist, U46619. Our first goal was to determine the effect of ROK on each of these types of muscle tone.Tone in the absence of L-NAME amounted to ∼1.7 mN, present after the normalization procedure. To demonstrate that this increase in tone involves ROK, ring preparations from BAs were treated after the normalization procedure (20–25 min equilibration in PSS see “Methods”) with 3 μmol/L Y27632 for 10 min, followed by addition of 100 μmol/L of the pan-NOS inhibitor L-NAME for another 20 min in the presence of Y27632. Pretreatment with Y27632 did not alter stretch-induced tone (1.69 ± 0.1 mN in the presence of Y27632 versus 1.73 ± 0.28 in non-treated time matched controls), but prevented the increase in L-NAME induced tone, which was 1.66 ± 0.1 mN in the presence of Y27632 and 2.7 ± 0.5 mN in the absence of Y27632 .2 analogue, U46619. In the presence of Y27632, the cumulative concentration-response relation was shifted to the right; the pEC50 value was significantly lower with than without Y27632 in time-matched controls . Maximal force was also lower  under basal conditions. For technical reasons, the vessels were mounted on two wires (ø 25 µm) but not stretched as for mechanical experiments. Basal phosphorylation levels of MLC20-S19 in WT and MYPT1-T696A/+ were similar  was higher in BA from WT-mice than in those from MYPT1-T696A/+-mice . The preparations from both genotypes were then incubated with 100 μmol/L L-NAME for 20 min, followed by stimulation with increasing concentrations of U46619 . However, unlike the case with y-BA, the mutation did not blunt L-NAME induced tone  a slow spontaneous rise in force was observed  after stced tone . U46619 ced tone ; Table 3max after Y27632 treatment were similar between s-BAs from both groups  or (+) were similar to those measured previously in Y27632 treated group  . Blebbised group ; Table 320-S19 phosphorylation or inhibitory phosphorylation of MYPT1. Phosphorylation of pMLC20-S19, pMYPT1-T696, and pMYPT1-T853 in the presence of blebbistatin (−) was determined in s-BAs from WT and from MYPT1-T696A/+ mice using phosphospecific antibodies. Neither pMLC20-S19, nor pMYPT1-T696/T853, were reduced by blebbistatin (−) in BAs from both mouse lines  at S1943 . Here, wIn these experiments, phosphorylation of MYPT1-T853 as a marker for ROK-activity was monitored in parallel. Phosphorylation of T853 was significantly higher in s-BAs than in y-BAs. In both age groups, Y27632 strongly reduced the immunoreactivity of pMYPT1-T853 . These ePAK1 ist another target of ROK, which phosphorylates PAK1 at threonine 432 and thereby activates the enzyme . We asse20 phosphorylation when phosphorylated by PAK1  were employed . At this age, o-BAs from WT mice did not exhibit stretch-induced tone yet . There was no difference between both genotypes considering L-NAME- and U46619-induced tone (+/− mice (12–14 weeks old)  .−/−, in which expression of the strong myosin binding domain, which is encoded by Exon 2 of the Cald1 gene, was ablated (−/−)  . Bindinged (−/−) . o-BAs fobserved .The G/F-actin decreased in a ROK-dependent manner by agonist stimulation of airway smooth muscle cells  or underIn the next series of experiments, we investigated whether maximal stimulation with calyculin resulted in a redistribution of F-actin in favor of subcortical actin. Calyculin treatment induced a maximal increase in force, starting 7–10 min after application and reached its maximum after 30 min (not shown). In the previous experiments, we showed that in BAs, calyculin treatment maximally activates ROK as seen by phosphorylation of MYPT1-T853 . Alexa F20 phosphorylation was lower in MYTP1-T696A/+ than in WT BAs in both age groups. Based on this, we propose that regulation of stretch-induced tone in senescent arteries involves alternative mechanism. 5) The basal phosphorylation level of NM-II and PAK1, which phosphorylates and inhibits caldesmon, were higher in s-BAs than in y-BAs, and were reduced by the ROK inhibitor, Y27632. 6) Ablation of caldesmon augmented stretch-induced tone in old BAs suggesting that it acts as a molecular brake on stretch-induced tone in aged BAs. We propose that in senescent cerebral vessels, mechanisms distinct from MLC20 phosphorylation contribute to regulation of tone in the absence of a contractile agonist.Stretch-induced vascular tone is an important element of autoregulatory adaptation of the cerebral circulatory system. It maintains constant cerebral flow, despite changes in perfusion pressure, and thus ensures adequate brain perfusion. The present work provides novel insights into the mechanisms of regulation of the stretch-induced tone of murine BA in senescence. 1) In young BAs (y-BAs) mechanical stretch did not lead to spontaneous tone generation, while the senescent-BAs (s-BAs) developed stable stretch-induced tone. 2) Stretch-induced tone in y-BAs appeared only after inhibition of NO-release by L-NAME, whereas in s-BAs such an inhibition led to an additive effect. 3) In BAs from both age groups, stretch-induced tone was fully inhibited by treatment with a ROK-inhibitor, Y27632. 4) Lowering of the phosphorylation propensity of MYPT1 in heterozygous mice carrying the point mutation MYPT1‐T696A/+ prevented stretch-induced tone in y-BAs but not in aged BAs. Basal MLC2+-concentration and activation of the specific Ca2+/calmodulin-dependent protein kinase, MLCK, and 2) Ca2+-sensitization via inhibition of MLCP  the plasma membrane depolarization, leading to a rise in intracellular Ca of MLCP , both in2+-influx, which then via tyrosine kinase Pyk2 might activate Rho-ROK signaling [, which encodes the strong myosin binding domain, resulted in expression of a truncated caldesmon but the expression level did not differ from WT mice. In vitro binding of the truncated caldesmon, isolated from these mice, had lost myosin but not actin binding (−/− mice was not different from that in WT mice. This indicates that the C-terminal domain is sufficient for inhibition of force.Caldesmon, which is expressed in smooth muscle cells of the walls of visceral organs as well as in the vasculature including the medial layer of human cerebral vessels , has two binding . However20, predominate tone regulation, while in senescence regulation of tone involves ROK related thin filament linked mechanisms.Taken together, our work corroborated the notion of the central role ROK has as a signaling node that orchestrates several downstream processes, essential for regulating vascular tone both in y- and s-BAs. Our observations expand previous reports that inhibition of ROK reduced vascular tone of pulmonary, cerebral, coronary, and mesenteric arteries from different species including humans, typically performed in young arteries . Howeverin vivo studies in animal models demonstrated that ROK inhibition prevented coronary and cerebral artery vasospasm and neointimal formation after balloon injury of carotid arteries (1. Therefore, one goal of the present work was to delineate the ROK substrates NM-II and PAK1-phosphorylation as regulators of spontaneous tone generation in the senescent cerebrovascular system. Based on our study, it is reasonable to conduct further studies, investigating whether targeting of these phosphoproteins would be an approach to treat cerebrovascular dysfunction in the elderly.2. Genetically lowering caldesmon expression leads to early onset of spontaneous contractions in o-BAs, typical for senescence. Therefore, it will be intriguing to investigate, at least in animal models, whether re-expression or overexpression of caldesmon would be able to prevent hypercontractility in cerebral vasculature.Several arteries . Howeverarteries .1. There


Magnaporthe oryzae, the MEN pathway, consisting of conserved protein kinases MoSep1 and MoMob1-MoDbf2, is important in the development and pathogenicity of the fungus. We found that deletion of MoSEP1 affects the phosphorylation of MoMob1, but not MoDbf2, in contrast to what was found in the buddy yeast Saccharomyces cerevisiae, and verified this finding by in vitro phosphorylation assay and mass spectrometry (MS) analysis. We also found that S43 residue is the critical phosphor-site of MoMob1 by MoSep1, and proved that MoSep1-dependent MoMob1 phosphorylation is essential for cell division during the development of M. oryzae. We further provided evidence demonstrating that MoSep1 phosphorylates MoMob1 to maintain the cell cycle during vegetative growth and infection. Taken together, our results revealed that the MEN pathway has both distinct and conservative functions in regulating the cell cycle during the development and pathogenesis of M. oryzae.The mitotic exit network (MEN) pathway is a vital kinase cascade regulating the timely and correct progress of cell division. In the rice blast fungus  The cell cycle is a complex and ubiquitous process involving numerous regulatory proteins during the growth and development of eukaryotesSchafer . The corSaccharomyces cerevisiae, the spindle position checkpoint (SPOC) curbs mitotic exit and cytokinesis until the mitotic spindle is properly oriented to ensure the normal generation of progeny cells  phosphorylates Dbf2 to regulate the MEN pathway. To test whether a similar mechanism occurs in M. oryzae, we constructed MoMob1-GFP and MoDbf2-GFP fusion genes and introduced them into the wild-type strain Guy11 and the ΔΜosep1 mutant. We extracted total proteins from ΔΜosep1/MoDBF2-GFP, Guy11/MoDBD2-GFP, ΔΜoSep1/MoMOB1-GFP, and Guy11/MoMOB1-GFP, respectively. We then estimated phosphorylation levels of MoDbf2 and MoMob1 by visualizing migrating patterns of Mn2+-Phos-tag SDS-PAGE. The result showed that no significant difference in the band migration rate of MoDbf2-GFP between Guy11 and the ΔΜosep1 mutant, suggesting that the deletion of MoSEP1 does not affect MoDbf2 phosphorylation , more septa were observed in the ΔΜosep1/MoMOB1S43D strain than in the ΔΜosep1 mutant, but fewer septa in the ΔΜosep1/MoMOB1S43A strain than Guy11, suggesting that the activation of this key phosphorylation site dependent on MoSep1 is important for septum formation. In addition, normal hyphae have one nucleus per cell; however, after inactivation of the specific phosphorylation site on MoMob1, more than one nucleus was observed per hyphae cell  decreased significantly. Furthermore, we found that S43 residue of MoMob1 is the key phosphorylation site dependent on MoSep1, and it is important for vegetative growth, conidiation, and pathogenicity. These results indicate that although the MEN pathway is conservative in regulating cell division and exit in eukaryotes, it also has a relatively evolutionary specific regulatory mechanism in different species. And as the important MEN pathway protein kinase, MoSep1 also plays an important role in coordinating other signaling pathways during cell division. As our previous studies demonstrated, MoSep1 links the MEN pathway to Cell Wall Integrity (CWI) signaling through the phosphorylation of the CWI component MoMkk1  in this study. All strains were cultured on complete medium (CM) agar plates in the dark at 28°C, unless indicated otherwise  at 80 V at 4°C, and then the membrane was analyzed by Western blotting analysis using the anti-GFP antibody  cells and purified. A rapid Fluorescence Detection in Tube (FDIT) method using Pro-Q Diamond Phosphorylation Gel Stain was used to analyze protein phosphorylation. First, 2 μg MoMob1 (or the unphosphorylated mutations) was mixed with MoSep1 in a kinase reaction buffer , with 50 μM ATP at 25°C for 1 h. Ten folds of cold acetone was then added to terminate the reaction. Casein was homogenized and suspended in Mili-Q water at a concentration of 0.2 μg/ml to stain the proteins. Briefly, 100 μl of Pro-Q Diamond was mixed with 10 μl of casein, and the mixture was kept in the dark for 1 h. Ten folds of cold acetone were then added, and the mixture was allowed to incubate overnight at − 20°C. Proteins were precipitated by centrifugation at 14,000 rpm for 1 h at 4°C. The supernatants were discarded, and protein pellets were washed twice using 500 μl cold acetone. The pellets were dissolved in 200 μl of Mili-Q water and transferred to a 96 well plate. The fluorescence signal at 590 nm (exited at 530 nm) was measured using a Cytation 3 microplate reader . For CFW staining, mycelia were stained with 10 μg/ml for 5 min in the dark to visualize nuclei  gelation solution, and 5 ml was sprayed on 2-week-old rice seedlings (Oryza stative cv. CO39). Inoculated plants were kept in a chamber at 25°C under 90% humidity for the first 24 h, followed by a light/dark cycle for 4–7 days.Conidia were adjusted to 5 × 10


The magnetoresistive anomaly also captures the characteristic angular and temperature dependence of this ice-rule-breaking transition, which has been understood as magnetic monopole condensation. These results demonstrate a novel heteroepitaxial approach for electronically probing the transition between exotic insulating spin states, laying out a blueprint for the metallization of frustrated quantum magnets.While geometrically frustrated quantum magnets host rich exotic spin states with potentials for revolutionary quantum technologies, most of them are necessarily good insulators which are difficult to be integrated with modern electrical circuit. The grand challenge is to electrically detect the emergent fluctuations and excitations by introducing charge carriers that interact with the localized spins without destroying their collective spin states. Here, we show that, by designing a Bi Spin ice compounds are typically insulating and introducing carriers can destroy the spin ice state, making integration into electronic devices problematic. Here the authors report a transport response to an ice-rule-breaking transition in a heterostructure of a pyrochlore spin ice and a nonmagnetic metal. Such a spin lattice has a large degree of degeneracy due to an enormous number of configurations at the same or similar energy, leading to exotic ground states, entangled fluctuations, and collective excitations7, such as magnetic monopole and spinon, which may afford revolutionary quantum technologies10. The so-called dipolar spin ice is one of the most iconic representatives of such frustrated magnets. It was first discovered on pyrochlore lattices of Ising spins constrained along the local [111] direction, such as Ho2Ti2O7 and Dy2Ti2O713. While the spin-spin correlation is dominated by the dipolar interaction, the ferromagnetic instability is geometrically frustrated by the local Ising anisotropy1. As a result, the ground state of each tetrahedron settles in one of the six-fold degenerate 2-in-2-out configurations, forming a spin ice network throughout the lattice following the ice rule in analog with the proton displacement in the water ice14. The non-zero residual entropy has indeed been observed with a value close to that in the water ice15. Breaking the ice rule could lead to rich spin dynamics, including emergent magnetic monopoles16. For instance, applying a magnetic field along the pyrochlore [111] direction turns the three-dimensional spin ice into a Kagome spin ice17, and eventually stabilizes the 3-in-1-out/1-in-3-out configuration by condensing the monopoles18, giving rise to a jump from ~3.3 µB/Dy to ~5 µB/Dy in Dy2Ti2O7 between two magnetization plateaus19. The 3-in-1-out/1-in-3-out state is effectively a magnetic charge-ordered state, which is also called the saturated ice20 where the ice rule is broken everywhere.Novel emergent states of quantum magnets often arise from geometric frustration, where local spins residing on the corners of a triangle or tetrahedron cannot agree on any alignment that simultaneously minimizes their energies11, where the spins are hosted by highly localized electrons in the absence of itinerant carriers, such as the f-electrons of Ho3+ or Dy3+ ions in combination with the empty d-shell of the Ti4+ valence state in the pyrochlore titanate13. When introducing itinerant carriers, simply replacing the B-site with another element with a metallic configuration often ends up interfering the spin ice state. For instance, the correlated d-electrons in pyrochlore iridates not only exhibit an all-in-all-out ordering of their own at much higher temperatures, but also force the same magnetic order onto the A-site sublattice regardless of the rare earth element21. In some cases, the interactions within the rare earth sublattice may modify the transport properties of the d-electron24 at lower temperatures, such as the fragmented monopole crystal ground state25 as well as the monopole-density-related magneto-transportation26. However, the spin state of the rare earth sublattice is often impaired by the energy scale of the d-electrons, which is much larger than the interaction. This situation indeed applies to a broad class of frustrated magnets beyond spin ice.However, the spin-ice behaviors have been only observed in highly insulating compounds2Ir2O7/Dy2Ti2O7 (BIO/DTO), where DTO hosts the spin ice state and BIO, the only nonmagnetic/time-reversal-invariant member of the pyrochlore iridate series, provides the correlated carriers. We directly grow ultrathin BIO films on DTO single crystals as illustrated in Fig. In this work, we report the observation of an anomalous magnetoresistance (MR) in epitaxial pyrochlore heterostructures of Bi27. The crystals were first oriented and cut into substrate pieces along the DTO (111) lattice plane. The substrate surfaces were then polished through a five-step process  to achieve atomically flat surfaces. Figure 28. The BIO/DTO interface was examined with the scanning transmission electron microscopy (STEM). A typical cross-section image in Fig. DTO single crystal substrates were prepared by the floating zone method the DTO 11 lattic29, BIO is a bad metal on the paramagnetic side of the quantum critical point30. No magnetic ordering or spontaneous time-reversal-symmetry-breaking was found down to 50 mK31. Moreover, it has been shown that, although the BIO metallicity will degrade when grown as ultrathin films likely due to weak localization and/or disorder-enhanced electron-electron interaction in two-dimensions, the MR remains rather isotropic when the field is applied along different crystallographic axes32. Our ultrathin BIO films on DTO indeed show a weakly increasing resistivity with lowering temperature as well as an isotropic MR  plane, while the current is always along the [11-2] axis perpendicular to the field  to [1–10] (θ = 90°). One can see a sharp increase of the resistivity around the critical angle θc (~20°) due to the anomaly. Interestingly, the resistivity on both sides of θc shows a much smaller but visible difference, which can be attributed to the fact that DTO remains a spin ice at 5 T with θ > θc while becoming a saturated ice with θ < θc. The remnant spin disorder in the former is likely to elevate the resistivity compared with the latter.Moreover, as we rotate B is applied in the (1-10) plane with current along the [1-10] axis . As illustrated in Fig. This result demonstrates that forming a heterostructure can effectively introduce interactions between the charge carriers from one side and the frustrated spins on the other side. The excellent consistency in the critical field between MR and the bulk-sensitive AC susceptibility reveals the robustness of this method. Note that the field, regardless of its direction, always locally flips the moments to partially lift the 2-in-2-out degeneracy of the Dy tetrahedron within the spin ice state. Yet the MR anomaly only occurs when the transition to the saturated ice state is induced, indicating that the charge carriers are particularly sensitive to the ice-rule-breaking spin flips. Given that the Kagome spin ice-to-saturated ice transition is a first order transition2Ti2O7 substrate as a reference sample and observed isotropic MR as functions of temperature and magnetic field without any anomaly  in metals may also produce anisotropic MR responses through the so-called spin hall magnetoresistance when in direct contact with an insulating magnet37. However, SHE does not require the field-induced transition and does not account for the fact that the anomaly only occurs near the critical field of the transition, excluding SHE and any other process irrelevant to the field-induced transition from being the underlying mechanism. In fact, spin hall magnetoresistance requires an orthonormal geometry among the surface normal, current direction and magnetization, which is obviously unnecessary for the observed anomaly in the BIO/DTO heterostructure. In addition, the observed anomaly could not be attributed to the magnetocaloric effect as it is expected to cause responses of opposite signs when sweeping the magnetic field upward and downward, which was not observed in our experiment.To further verify this picture, we measured a 3.7 nm BIO film on nonmagnetic Y19. This behavior has been understood with an analogy to the liquid–gas transition in the context of a transition between a low-density phase and a higher-density phase of magnetic monopoles19. For comparison, we studied the thermal evolution of the MR anomaly with B//[111] between 0.45 K and 1 K. The obtained result displayed as the solid lines in Fig. 1. The dashed lines were measured with B//[1-10] and serve as a reference for comparison at each temperature. The anomaly eventually starts to vanish at ~0.9 K and becomes hardly visible. These observations are consistent with the field-temperature phase diagram as well as the disappearing of the spin ice state in DTO due to thermal fluctuation16 around 1 K, confirming the sensitivity of the charge carriers to the abrupt change at the boundary between the two spin states until they are no longer distinguishable above the critical endpoint of the transition19.Studies of the field-temperature phase diagram of spin ice have shown that the field-induced phase boundary is terminated at elevated temperature and the transition becomes a continuous crossover as there is no sharp distinction between the Kagome spin ice and the saturated ice anymore39, as well as investigating charge conversion of the monopole condensation16 excitations under non-equilibrium stimuli41. Recently, the magnetic monopole dynamics was detected by a magnetization noise spectrometer in the spin ice state of DTO45 as well as during the transition between two 2-in-2-out states of artificial spin ice46. A two-dimensional monopole gas was also proposed at the heteroepitaxial interface of the 2-in-2-out and the all-in-all-out state47. Such behaviors may manifest in electronic transport, too, when interfaced with charge carriers, providing another detection method and a potential route to application. The interfacial approach can readily be extended to other non-conducting frustrated quantum magnets, such as spin liquid and quantum spin ice10 for electronically detecting these exotic spin states and triggering electronic responses. The successful creation of functional heterostructure between DTO and BIO also showcases the potential of complex oxide heterostructures of the pyrochlore structure48.To summarize, the experimental observation of an emergent MR anomaly in BIO that responds to and closely tracks the Kagome ice-to-saturated ice transition in insulating DTO demonstrated that creating epitaxial heterostructures is a highly promising approach to introduce interactions between localized frustrated spins and correlated charge carriers. It opens the possibilities of electronically probing the spin ice states under other control parameters, such as pressure and epitaxial strain2O3 and TiO2 with the molar ratio of 1:227. Prior to weighing, Dy2O3 was preheated at 1000 °C for 10 h in order to remove the moisture. After grinding the mixture, sintering the mixture at 1000 °C and 1400 °C for 20 h each in air with intermediate grinding. The powders were then compressed to a feed rod with another additional annealing at 1400 °C for 20 h, which were used to grow the single crystal DTO by using a double elliptical mirror optical floating-zone furnace SC1-MDH from Canon Machinery. To avoid oxygen deficiency, we grew the single crystal in 5 atm flowing O2 atmosphere. In the first cycle we used a high growth rate of 50 mm/h for both feed and seed rods and in the second cycle we used a relatively slow growth rate of 4 mm/h.polycrystalline DTO was prepared by thoroughly mixing stoichiometric Dy28. A KrF excimer laser (λ = 248 nm) was used at a repetition rate of 2 Hz and the laser beam fluency is 1.7 J/cm2. The BIO films were deposited at 620 °C and 0.0667 mbar of oxygen pressure. The thickness was controlled by the nominal growth rate of BIO films at ~40 pulses/nm and verified by ex situ x-ray reflectivity. After deposition, the films were cooled down in 0.0667 mbar oxygen pressure. We focused on samples with the film thickness in the range of 3–5 nm for transport measurements.The DTO single crystal was first aligned by a HUBER Laue X-ray diffractometer and then cut into substrate pieces with DTO (111) lattice plane by using the Model 650 Low Speed Diamond Wheel Saw from South Bay Technology. The substrate surfaces were prepared by going through a five-step mechanical polishing process with monocrystalline suspension. The surfaces were characterized by an MFP-3D Atomic Force Microscope from Asylum Research. Thin film samples were synthesized by Pulsed Laser DepositionIn-house X-ray characterization was done by an Empyrean diffractometer at Copper radiation. Synchrotron X-ray diffraction was performed at beamline 33-BM at the Advanced Photon Source at the Argonne National Laboratory.TEM data were acquired using a cold field emission probe-aberration-corrected JEOL JEM-ARM200cF at 200 kV with a spatial resolution of 0.078 nm. It is equipped with Oxford Aztec SDD EDS detector for elemental analysis. TEM lamella was made by focused ion beam (FIB) in a ThermoFisher Scientific Helios G4 UC DualBeam scanning electron microscope (SEM). To avoid charging in the SEM, the thin film surface was first sputtered with a 10 nm thick layer of gold. Cross-sectional view along [1-10] was obtained by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) imaging. The HAADF-STEM images were collected with the JEOL HAADF detector using the following experimental conditions: probe size 7c, CL aperture 30 µm, scan speed 32  µs/pixel, and camera length 8 cm, which corresponds to a probe convergence semi-angle of 21 mrad and inner collection semi-angle of 74 mrad. EDS maps were collected in the STEM mode with a probe size of 0.12 nm.Transport measurement was performed with a Physical Properties Measurement System (PPMS) from Quantum Design (QD). The current is always applied perpendicular to the field direction. MR between 0.45 and 1 K was measured with the He-3 option of a PPMS Dynacool from QD. MR at 0.03 K was measured at the National High Magnetic Field Laboratory (NHMFL), using a 0.2 T/min sweeping rate; the data were collected at field values of a 0.05 T step size, after waiting for more than 60 s at each setpoint. All MR data were measured by utilizing the lock-in technique; The typical current applied was at the scale of 10 nA, with a frequency <20 Hz. Samples used in the measurement are of 3 mm (W) × 6 mm (L) in dimension. The DTO single crystal is of 0.5 mm in thickness.AC susceptibility was measured at NHMFL. The AC susceptibility measurements were conducted with a voltage-controlled current source  and lock-in amplifier . The phase of the lock-in amplifier is set to measure the first harmonic signal. The rms amplitude of the AC excitation field is set to be 0.35 Oe with the frequency fixed to be 470 Hz. The measurement at NHMFL mainly focused on identifying magnetic transitions rather than obtaining absolute magnetic susceptibility values. The unit of the AC susceptibility data was given in arbitrary units because the AC susceptometer was not calibrated within the scope of the experiment.Supplementary InformationPeer Review File


Based on simple electrostaticand harmonic potentialconsiderations,we derive a straightforward expression linking the composition ofa Prussian blue analogue (PBA) to its propensity to undergo collectivestructural distortions. We demonstrate the existence of a thresholdvalue, below which PBAs are undistorted and above which PBAs distortby a degree that is controlled by a geometric tolerance factor. Ouranalysis rationalizes the presence, absence, and magnitude of distortionsin a wide range of PBAs and distinguishes their structural chemistryfrom that of other hybrid perovskites. A value of α = 1 impliesperfect filling, which in turn prevents framework distortion. Decreasingthe value of α generally induces volume-reducing distortionsin the structure to compensate for the poorer geometric fit. Recognizingthe relationship between tolerance factor and structural distortionshas enabled targeted control over perovskite crystal structures, asin A2CrWO6 , and is seen as a crucial design approach foroptimizing functional response in, for example, magnetoresistive14 and photovoltaic15 perovskites.In conventional perovskites, which sharethe same parent ABX17 simple bonding considerationssuggest the picture may be fundamentally different for PBAs in particular.The key distinction is that directional metal–cyanide interactionsfavor a linear linkage geometry,18 in contrastto the tilted geometry generally driven by covalency in conventionalperovskites.19 For example, Mn[Pt(CN)6], which with no A-site cations at all has a tolerance factorwell below 1, nonetheless adopts a cubic crystal structure in whichthe BX3 framework is undistorted.20 Instead it is by filling the A-site  thatdistortions are switched on.21 This behavior contrastswith that of conventional perovskites but is conceptually similarto the activation of tilts in halide hybrid perovskites through hydrogen-bondinginteractions between organic A-site cations and the anionic framework.22 An additional complication that we will addressis that PBAs are highly nonstoichiometric frameworks, and one mightexpect partial site occupancies to play a nontrivial role in the activationor otherwise of collective distortions.Here we address the obvious and important questionof whether ananalogous geometric relationship holds for PBAs. While it is alreadywell established that the tolerance factor approach might be successfullyand straightforwardly extended to molecular perovskite analogues (“hybridperovskites”),3 framework is predominantly electrostatic, andsince the corresponding attraction scales inversely with distance,it is intuitive that distortions reducing the A···Xdistance should be energetically favored  and 1/(rAX – d). For small distortions d ≪ r = rAX, we have an electrostatic stabilizationd2 . Hence for a PBA with formula Axq+B[B′(CN)6]y we can writex and y are the A-site and X-site occupancies, q is thecharge on the A-site cation, and β > 0 is a proportionalityconstant into which we have subsumed the 2/r3 factor of aSo why might fillingthe A-site of PBAs switch on structural distortionsat all? The interaction between an A-site cation and its surroundinganionic BX favored 1a. Howevk is an effectivespring constant that reflects the stiffness of the M–CN–Mlinkages to deformation. So this energy cost also scales as d2 for small distortion magnitudes. Combiningthese two terms, one expects distortions to occur whenever ECoul + Eharm <0; that is,d has now disappeared because both terms scale as d2.Acting against this electrostatic driving forceis the energy costof distorting M–CN–M linkages from their linear ground-state.The leading (harmonic) term in the corresponding energy expansionhas the form xyq values fora large rangeof PBAs of different compositions (details in the Supporting Information). With only a very small number ofexceptions (which we will come to discuss), we observe a partitioninginto undistorted structures at low values of xyq anddistorted structures at high values of xyq . The existence of a universal criticalvalue of k/(2β) for which k and Coulomb prefactors β (which depend on the A···Xdistance) do not vary too much among PBAs. From our data set thiscritical value is about 1.We show in xq+B[B′(CN)6]y is likely to be structurally distorted, based simply on thecorresponding product xyq. Immediately it rationalizeswhy low-vacancy PBA cathode materials switch between distorted andundistorted phases as A-site cations are shuttled in and out duringelectrochemical cycling, for example.23 The reduced A-site occupancy of the oxidized state means there isan insufficient electrostatic driving force to drive distortion, buton reduction the increased A-site occupancy induces long-range symmetrybreaking.24This result provides a simple butpowerful heuristic for estimatingwhether or not a given PBA of arbitrary composition Axyq ≃ 0.8 correspond to low-vacancy PBAs Rb0.90Cu[Co(CN)6]0.92 and Rb0.85Cu[Fe(CN)6]0.95 containing Jahn–Teller-active Cu2+. In the particular case of Rb-containing PBAs, which tendto show columnar Rb order at half-filling,25 the combination of A-site order and cooperative Jahn–Tellerorder from the Cu2+ ions automatically activates an additionaltilt instability through symmetry considerations alone.26 Consequently, the presence of distortions inthese two systems arises from electronic effects rather than froman electrostatic drive to reduce the volume. This point is made clearalso in the small volume strains involved (<4%). We have also madethe assumption that alkali cations are situated on (or near) the 12-coordinateA-site, which is almost universally true29 but is not necessarily the case for some Na-containingPBAs,30 nor is it for the related familyof cyanoelpasolites.31 Mindful of thesevarious caveats, we nonetheless see the value of understanding whichgeneral factors switch on and off collective distortions in differentPBAs.Our derivation is intentionallysimplistic and involves a numberof crude approximations. We have not taken into account electronicdistortion mechanisms, such as the Jahn–Teller effect, whichneeds to be treated separately. Indeed the two outliers at xyq > 1 will minimizeitsenergy by maximizing the value of d; in other words,it should distort as much as the structure allows /Vref.But what determines distortion magnitude? An implicationof theanalysis leading to e allows 1b. This 12 Our data suggest an empiricalrelation δ ≃ 2(1 – α); e.g., a tolerancefactor of 0.9 corresponds to a volume reduction of about 20%.We show in 2Mn[Mn(CN)6] andK1.7Fe[Fe(CN)6]0.9, their similardistortion magnitudes can now be rationalized in terms of their similartolerance factors . Second, becausevolume reduction is cooperative, we expect that the magnitude of distortionshould be largely unaffected by the incorporation of A- or X-sitevacancies 6]y systems  show remarkablylittle variation in distortion magnitude. The small variation in cellvolume is clearly evidenced by the 24 KxMn[Fe(CN)6]y samples in ref (2Mn[Mn(CN)6] originally flagged as the undistortedanomaly in xyq = 2, so one expects a distorted structure,but the tolerance factor is so near unity that the correspondingvalue of δ is only about 6%. We anticipate that the system iscubic because small distortion magnitudes can be accommodated in theflexible PBA structure through dynamic and/or short-range fluctuationsrather than collective symmetry breaking.We make a number of commentsregarding acancies 2b. It iss in ref . By contpΔV term to the lattice enthalpybalance, which behaves as if to reduce the effective spring constant k and so shifts the critical value of xyq to below 1. This effect is consistent with the experimental observationof pressure-driven structural distortions in some PBAs.39 The effect of temperature is formally given by the balance of entropicterms in the free energies, but one simplification is to considerthe effective radii of 24The effect of externalpressure is to include an additional 41 Hence k < 0 and the inequality of 42 formate,43 or thiocyanate44) all favor distorted geometries. Hence the existenceof compositional distortion thresholds is not universal but may nonethelessbe relevant to dicyanometallates,45 borohydrides,46 bifluorides,47 andeven metal–organic frameworks.49So the structural chemistry of PBAs differs conceptually fromthatof other hybrid perovskites as a consequence of the linear ground-stategeometry of the B–X–B linkages. In conventional perovskites,for example, the tilt modes are usually mechanically unstable in theabsence of sufficiently large A-site cations.8) then the combination of high A-site occupancies, low hexacyanometallatevacancy fractions, and a tolerance factor much reduced from unitywill collectively maximize the degree of distortion. Inverting thesecriteria then forms the design strategy for avoiding structural distortions,which may be important for improving the reversible capacity of PBA-basedcathodes.Fromthe perspective of functional PBA design, our results suggesta number of avenues. In cases where structural distortions are favorable(e.g., when targeting hybrid-improper ferroelectrics