Inner Ear biology is broad and multidisciplinary, ranging from studies on cell and tissue organization mechanisms during development, the causes of hearing loss, and novel treatment therapies for repair. These questions are tackled using state-of-the-art methodologies and different animal model systems, some being highlighted in this Research Topic.Jones et al., the power of zebrafish genetics and live imaging are combined to uncover the requirement of sulfate proteoglycans for projection outgrowth necessary for semicircular development. The zebrafish mutant exostosin (ext2) that impairs heparan sulphate synthesis has defects in the formation of the semicircular canals, together with deficiencies in cartilage, bone, and teeth development. This paper highlights the relevance of ECM in inner ear morphogenesis. During cochlear maturation, a dynamic interplay between cell death and epithelial remodeling is revealed by Borse et al., who show that the appearance of macrophages in the cochlea correlates with the time of greater epithelial ridge (GER) cell death. How macrophages are attracted to the GER is an open question since experiments indicate that they need another attractant than the CX3CL1 chemokine. Despite the link between macrophage invasion and GER remodeling, macrophages are indispensable for GER regression.During embryogenesis, the inner ear acquires a highly complex 3D structure, with very apparent dorsal semicircular canals and the ventral coiled cochlea. In this Research Topic, several papers investigate the tissue rearrangements that help acquiring the adult shape of the inner ear. In Renauld et al. In the developing cochlea, melanocytic cells of neural crest origin extend protrusions, ingress, and migrate on top of the marginal cells. These melanocytes have a dual origin; on the one hand, they derive from migratory neural crest cells, and on the other, from Schwann cell precursors located at the cochlea-vestibular ganglion. It would be interesting to perform live imaging to explore in detail these cell movements.Most cochlear cells derive from otic precursor cells, but intermediate cells of the stria vascularis derive from neural crest cells that ingress into the developing cochlea. Genetic linage tracing reveals the migratory path of neural crest cells and shown in in vitro. Together with imaging, a better understanding of the heterogeneity of cells and molecular networks operating during cell specification is currently achieved thanks to novel single-cell sequencing technologies. As discussed in the review by Kelley, different methods of scRNA-seq are available, each with particular advantages depending on the type of data sought. Methods sequencing full-length cDNA allow the discovery of novel splicing variants, while bar-coding approaches allow sequencing a greater number of cells and can improve the description of cellular diversity within a tissue. The author suggest that the difficulty of isolating cells of the inner ear at late postembryonic stages makes isolation of single nuclei the preferred option. In these cell dissociation experiments, unfortunately, spatial information is lost. New techniques of scRNA-seq on top of frozen sections, although still expensive, are the future for capturing the transcriptome while maintaining the knowledge of the position of each cell.Recent advancements in understanding cochlear development have been made by improving the culturing conditions of cochleae Filova et al., the effects of early NeuroD elimination are analyzed in mice using Foxg1 cre that is expressed in the otic placode but not the brainstem. As expected, the auditory ganglion is severely reduced in the NeuroD KO, but interestingly the cochlea and vestibular sensory patches are also severely developmentally affected. In particular, the cochlea is shorter, with ectopic and disorganized hair cells within and outside the sensory epithelium. Further work should elucidate the underlying mechanisms of the different phenotypes.One of the master regulators of inner ear neuronal lineage specification is the proneural factor NeuroD. In Schwarzer et al. establish a lesion paradigm to investigate whether the cochlea-vestibular ganglion regenerates after cell death upon damage. While NeuroD-positive cells are proliferative during development, they become postmitotic later on. NeuroD cells reenter the cell cycle after lesion and new neurons are generated. It will be interesting to analyze the cell identities of newly generated cochleo-vestibular neurons, perhaps with scRNA-seq, and to determine whether the neurons are functional. The zebrafish lateral line is an excellent system to image hair cell and neuronal activity in vivo with light sheet fluorescent microscopy (LSFM). Zhang et al. reveal that spontaneous activity of hair cells happens in two distinct domains, at the bundle and the presynaptic compartment, where it drives the synapse activity. This work highlights the detection of spontaneous activity in several cells, such as supporting cells and efferent nerves, being uncoupled with the spontaneous activity of hair cells. In another set of experiments, Le Prell et al., explore whether Calcitonin-gene-related-peptide (CGRP) modulates sound-evoked auditory nerve (AN) activity or affects the AN spontaneous activity. Infusion of CGRP into the pig cochlea and measurements in the round window suggest that can do both. The work provides new information of the role of olivocochlear efferent neurotransmitters in modulating auditory nerve activity.The zebrafish is a well-established model for studying tissue regeneration, and, in contrast to mammals, hair cells regenerate. However, whether inner ear sensory neurons can regenerate upon damage is unknown. This topic also presents advances in research to understand the genetic and molecular bases of hearing and deafness to study new possible targets for the prevention and treatment of hearing loss.Thulasiram et al. review how the different cell types that compose the stria vascularis contribute to maintaining adult hearing with a focus on how its alterations contribute to cochlear degeneration and, finally, hearing loss.The stria vascularis, an important tissue for maintaining the adult endocochlear potential is becoming more and more interesting for its roles in cochlear metabolic homeostasis, ageing, and its roles in response to insults including noise and ototoxic drugs. Rousset et al. addresses the study of the deficiency in NOX3, one of the enzymes involved in controlling the redox response. After studying a mouse deficient in Nox3, the authors propose that inhibition of this mechanism protects against cochlear insult caused by excessive exposure to noise. This finding confirms that during the response to noise, the over-activation of NOX3 increases the release of reactive oxygen species (ROS), ultimately resulting in cell damage and death. It would be interesting to know if temporary inhibition of NOX3 in the acute phase of injury has similar otoprotective effects since NOX3 activity is necessary for cellular functioning.The control of redox balance is key to the survival of hair cells and, therefore, of the rest of cochlear cell types and for hearing. This control also regulated from the stria vascularis, although not only, has been the subject of numerous studies whose ultimate goal is the design of otoprotective treatments. On this topic, the original article by Draf et al. show how by using a collection of small molecules designed for the study of autophagy, they were able to dissect the participation of the proteasome in the cochlear response to the insult caused by toxic drugs. Specifically, they study the deterioration and loss of hair cells caused by treatment with broad-spectrum aminoglycoside antibiotics. This study provides relevant data on the proteasome’s role in maintaining the delicate cochlear homeostasis. It opens new perspectives for the development of new strategies in the prevention of ototoxicity.Blanc et al. establish a novel delivery route for adeno-associated virus delivery to both inner ears of mice. This relevant study shows that a single virus injection into the cisterna magna, a cerebrospinal fluid-filled opening close to the brain, is sufficient for efficient binaural transduction of inner hair cells and, to a lesser extent, spiral ganglion neurons. Reaching both inner ears with a single and straightforward delivery strategy is innovative but likely requires the development of specific viruses for their respective inner ear target cell types.Finally, We hope that the reader will find in this Research Topic a flavor of the emerging questions in the exciting field of inner ear biology and hearing loss.


The characteristic temporal relationship between actions and their sensory outcomes allows us to distinguish self- from externally generated sensory events. However, the complex sensory environment can cause transient delays between action and outcome calling for flexible recalibration of predicted sensorimotor timing. Since the neural underpinnings of this process are largely unknown this study investigated the involvement of the cerebellum by means of cerebellar transcranial direct current stimulation (ctDCS). While receiving anodal, cathodal, dual-hemisphere or sham ctDCS, in an adaptation phase, participants were exposed to constant delays of 150 ms between actively or passively generated button presses and visual sensory outcomes. Recalibration in the same  and in another sensory modality (auditory outcome) was assessed in a subsequent test phase during which variable delays between button press and visual or auditory outcome had to be detected. Results indicated that temporal recalibration occurred in audition after anodal ctDCS while it was absent in vision. As the adaptation modality was visual, effects in audition suggest that recalibration occurred on a supra-modal level. In active conditions, anodal ctDCS improved sensorimotor recalibration at the delay level closest to the adaptation delay, suggesting a precise cerebellar-dependent temporal recalibration mechanism. In passive conditions, the facilitation of inter-sensory recalibration by anodal ctDCS was overall stronger and tuned to larger delays. These findings point to a role of the cerebellum in supra-modal temporal recalibration across sensorimotor and perceptual domains, but the differential manifestation of the effect across delay levels in active and passive conditions points to differences in the underlying mechanisms depending on the availability of action-based predictions. Furthermore, these results suggest that anodal ctDCS can be a promising tool for facilitating effects of temporal recalibration in sensorimotor and inter-sensory contexts. Despite the multitude of sensory signals that we are exposed to during daily interactions with the environment, we can effortlessly distinguish between those caused by our own actions and those with external origin. This ability relies heavily on the characteristic and highly predictable temporal relationship between actions and their sensory outcomes. For example, when clapping hands, the clap sound is strongly expected to be perceived near-instantaneously after the hands touch, by considering inherent delays in sound transmission and in sensory pathways. Such predictions about sensory action-outcomes are assumed to be generated by an internal forward model based on a copy of the motor commands . SensatiNotably, the complexity of the sensory environment can cause transient violations in the temporal relationship between action and outcome. For instance, additional delays can be imposed on the interval between an action and the perception of its outcome by changes in environmental conditions  by repeatedly inserting a constant delay between a participant’s action (like a button press) and its sensory outcome in form of a light flash or a brief tone . After rNotably, while sensorimotor temporal recalibration finds support in a wide range of behavioral studies, evidence for the neural basis of this process remains sparse. The sensorimotor TRE could for instance be associated with in a shift of readiness potentials closer to movement onset indicating the involvement of action-based predictive mechanisms . MoreoveThe cerebellum has emerged as an important brain area regarding the generation of predictions about sensory action-outcomes. Since it receives and combines afferent inputs from sensory areas and efferent inputs from motor areas, its anatomy and location seem ideal for performing predictive forward model computations. It has consequently been proposed as critical brain structure for forward model related processes or even as the site of internal forward models itself . It is fIt could be argued that the effects of sensorimotor temporal recalibration emerge simply due to recalibration of the perceived inter-sensory timing  which is also known to be an important mechanism for dealing with differential and varying delays in the transmission and processing of sensations from different sensory modalities . ImportaThese results also suggests that the involvement of regions known for action-outcome processing, such as the cerebellum, in temporal recalibration is specific to the sensorimotor context, but direct evidence for this claim is missing.A recent MEG study reported first evidence for cerebellar contributions to temporal recalibration by investigating the M100 component known to reflect an early response to auditory stimuli. This component is typically attenuated for the processing of tones that occur in synchrony with a self-generated action compared to passive listening to externally presented tones. Here, after repeated exposure to a delayed tone, M100 attenuation also emerged for the delayed tones, but this effect was abolished after inhibition of the right cerebellum by TMS . While tInterestingly, the sensorimotor TRE could frequently be shown to transfer from one modality to another, such that the temporal perception of the action-outcome of one modality recalibrated to a delay previously inserted between the action and the outcome of another modality . This suIn this study, we therefore used transcranial direct current stimulation (tDCS) on the cerebellum to address the question of whether it is related to temporal recalibration. We were specifically interested in the question of whether the relationship is unique to the sensorimotor context and whether the cerebellum contributes to the modality-transfer of recalibration effects. tDCS is a non-invasive brain stimulation technique that has already been used by a range of previous studies that investigated whether the stimulation of mainly frontal and parietal brain areas can influence the processing of sensory action outcomes. Frontal tDCS for instance facilitated the detection of delays between an action and its sensory outcome ,2020 whiParticipants engaged in adaptation phases during which the temporal relationship between a button press and a visual sensory outcome was manipulated by introducing constant delays between button press and outcome. A subsequent delay detection task assessed if and to what extent these temporal incongruencies triggered temporal recalibration. Here, participants were asked to detect varying temporal delays between the button press and its sensory outcomes. The TRE was expected to manifest in decreased delay detection performance  indicating a shift of predicted stimulus timing in the direction of the constant delay participants were previously exposed to. Importantly, button presses were either performed actively by the participants or passively by an electromagnetic passive button device. Since both passive and active movements were associated with similar tactile and proprioceptive sensations, this manipulation allowed us to disentangle effects of sensorimotor temporal recalibration due to adaptation of action-based predictions from effects due to inter-sensory temporal recalibration. Based on previous findings we expected a stronger TRE in active compared to passive movement conditions due to the additional involvement of predictive signals based on the motor commands . As actiWhile the constant delay was always inserted between button presses and visual outcomes during adaptation , the sensory modality in the delay detection task could be either visual or auditory. Based on the assumption that action-outcome predictions are generated for multiple sensory modalities  and baseThis experimental paradigm was applied in four separate sessions for each participant during which they received 20 min of either anodal, cathodal, dual-hemisphere or sham ctDCS to investigate whether the TRE can be facilitated or impaired, respectively, depending on stimulation polarity, which might be attributed to changes in the sensitivity to temporal prediction errors or in the speed or precision of sensorimotor learning.Anodal tDCS has frequently been shown to increase cortical excitability while it is decreased by cathodal tDCS . AlthougSD = 4.59). All participants had normal or corrected-to-normal vision and normal hearing. They reported no history of psychiatric, neurological or movement disorders or of drug or alcohol abuse. Additionally, no one reported any contraindications for tDCS . According to a power analysis, this sample size should have been sufficient to reproduce effects of similar size as reported in previous studies with a similar experimental design  and two rubber electrodes (5 × 7 cm) covered in saline-soaked sponges (0.9% NaCl).For anodal and cathodal ctDCS, the center of the respective active electrode was placed on the midline 2 cm below the inion to target the bilateral cerebellum while the return electrode was attached to the right upper arm (onto the deltoid muscle). For dual-hemisphere ctDCS, electrodes were placed with their centers 2 cm below and 3 cm lateral to the inion targeting the right (anode) and the left cerebellar hemisphere (cathode), respectively. All electrodes were attached with rubber bands.In each session, a current of 2 mA was applied for 20 min (+10 s fade in and fade out periods). For sham stimulation, sinus (HW) mode was used for a duration of 30 s. The stimulation parameters are in accordance with established tDCS safety guidelines e.g., .Participants were seated in a dimly lit room in front of a computer screen with a refresh rate of 60 Hz at a standardized distance of approximately 55 cm. During the experiment button presses were performed with the right index finger using a custom-made electromagnetic passive button. In active conditions, participants pressed the button actively themselves. In passive conditions, the button was pulled down automatically by an electromagnet with a maximum force of 4N. Participants’ fingers were tied to the button with an elastic fabric band to ensure that it would smoothly follow the movement of the button in passive conditions.When a button press was registered by the computer  the presentation of a visual or an auditory stimulus was triggered. The visual stimulus was composed of a Gabor patch  presented at the center of the screen. The auditory stimulus was a brief sine-wave tone  played through headphones. Both stimuli were presented for a duration of 33.4 ms each. Stimuli were created and presented using Octave and the Psychophysics Toolbox .To ensure that sensory outcome perception would not be influenced by direct visual or auditory feedback of the actual button press movements, the button was covered by a black box and pink noise was applied through the headphones at individually adjusted volume during the whole experiment.movement type). Importantly, the visual outcome occurred either directly after the button press was registered (0 ms delay) or after a constant delay of 150 ms (factor adaptation delay). While the 0 ms condition was assumed to match the natural expectation of temporal congruence between action and visual action-outcome (in active conditions) or between tactile or proprioceptive and visual sensory signals (in passive conditions), the constant delay was assumed to induce a prediction error and thus to trigger sensorimotor or inter-sensory temporal recalibration, respectively.In each session, participants underwent multiple pairs of adaptation and test phases. Adaptation phases consisted of 18 consecutive button presses each followed by the presentation of the visual sensory outcome. Throughout an adaptation phase all button presses had to be performed either actively or were elicited passively . In each trial one of six temporal delays  was inserted between button press and stimulus presentation. Thus, each delay appeared once in each test phase. Participants’ task was to report whether they detected a delay between the button press and its sensory outcome by pressing one of two keys on the keyboard with their left hand. The order of delays was counterbalanced across test phases and the assignment of keys was counterbalanced across participants. The TRE was defined as the difference in the percentage of detected delays in this task following an adaptation phase with vs. without constant outcome delay. Worse delay detection performance after adaptation with the delay of 150 ms would reflect a shift of the expected timing of the sensory stimulus in the direction of the delay and thus temporal recalibration.Each adaptation phase was followed by a test phase assessing the impact of the preceding adaptation phase on sensory perception. A test phase was composed of six individual test trials. In each trial the button was pressed once, either actively or passively . The button press triggered the presentation of either the visual or the auditory stimulus in each of the six test trials (factor adaptation delay (0 vs. 150 ms), movement type (active vs. passive) and test modality  were combined to eight different experimental conditions. Together with the factor stimulation  this resulted in a 4 × 2 × 2 × 2 within-subjects design.In summary, the factors Each participant went through all four stimulation conditions in four separate sessions. Intraindividual sessions were performed at least 24 h apart to prevent spill-over effects from the previous session. The order of stimulation conditions was counterbalanced across participants.Each adaptation phase started with written instructions on the screen about the movement type of the upcoming button presses displayed for 1500 ms see . The insEach test phase also started with the fixation cross and instructions about the movement type and outcome modality of the upcoming test trials (displayed for 1500 ms). The disappearance of the fixation cross initiated the first of six test trials. In active conditions, participants had 1500 ms to press the button once. Yet, they were instructed to press the button not immediately after the fixation cross had disappeared but to withhold their button press action for about 700 ms. This was to ensure that the button press was not triggered as a reflex upon a starting signal but as a self-initiated action . The samIn one session, each of the eight experimental conditions was presented in eight pairs of adaptation and test phases. Since each of the six delay levels was presented once in a test phase, this procedure provided us with eight trials per delay and condition for the analyses. Four adaptation-test pairs of each condition occurred in sequence in a first task block during which tDCS stimulation was applied. After the first task block, tDCS electrodes were detached during a short break. The remaining four adaptation-test pairs of each condition were presented in a second task block after the break in the same order as in the first task block. The second task block was completed without tDCS stimulation. Within each task block conditions with 0 and 150ms adaptation delay were blocked to prevent potential spill-over effects due to rapid switching. Whether the block of 0 or 150 ms delay was presented first as well as the order of conditions within these blocks was counterbalanced across participants.After each session potential side effects of the tDCS stimulation  were assessed with a custom-designed questionnaire of 28 items using ratings on a scale from one (no side effect) to five (strong side effect). During the first session, participants additionally went through a training procedure before the beginning of the stimulation in order to familiarize with the task. They were instructed to place their finger loosely on the button and not to apply any counter-pressure in passive movement conditions. For the adaptation phases, they were trained to perform the button presses in intervals of approximately 750 ms and for a duration of 500 ms while receiving feedback about their performance. For the test phases, participants were trained in each of the experimental conditions, once with the undelayed and once with the maximally delayed sensory outcome (417 ms) to familiarize with the stimuli and the delays. Here, they were provided with feedback about the actual presence of a delay in the respective trial. Participants were asked to answer as accurately, not as fast as possible. Finally, they went through a 10-min training of the experiment to further familiarize with the task and the alternation of adaptation and test phases.Test trials for which no button press  or response  was registered were excluded from the analyses.1). Detection thresholds  and slopes  were derived as summary measures from the psychometric functions. While the detection thresholds serve as a measure for the general delay detection performance , the slopes represent the increment in detected delays when the amount of delay increases and thus indicate the discrimination ability between delay levels.The proportion of detected test delays as a measure for delay detection performance was calculated separately for each participant and experimental condition. These data were then modeled by fitting psychometric functions in form of a cumulative Gaussian distribution function using the Psignifit toolbox version 4  for Pyth2, repeated-measures ANOVAs were performed to examine effects of temporal recalibration. But to get a more direct insight on the influence of the experimental manipulations on recalibration, the adaptation delay was not included as a factor in the analysis, but the TRE was used as a dependent variable. The TRE was quantified as the difference in detection thresholds in conditions with the adaptation delay of 150 ms vs. 0 ms. Positive values indicate a rightward shift of the psychometric function which corresponds to a decrease in delay detection performance after adaptation to the 150 ms delay as expected for temporal recalibration. Further, to better differentiate between the influence of ctDCS on the TRE in both test modalities, two repeated-measures ANOVAs were performed separately for the visual and auditory test modality, each including the factors stimulation and movement type. Similarly, repeated-measures ANOVAs were performed on the difference in slopes of conditions with 0 ms vs. 150 ms adaptation delay since temporal recalibration might also result in a lower ability to discriminate between delay levels represented in flatter slopes of the psychometric functions. Post hoc two-samples t-tests were used for significant main and interaction effects to inspect differences between movement types and stimulation conditions compared to the sham control condition. Additionally, in case of significant differences in the TRE between conditions, one-sample t-tests were further used to examine whether the respective TREs differed significantly from zero. As the TRE is defined as decreased delay detection performance after exposure to the adaptation delay of 150 ms, one-tailed t-tests were used. For effects pointing in the negative direction, only descriptive results are reported. All ANOVAs and t-tests were conducted with JASP , movement type , and test delay . A full factorial model was used with all main and interaction effects of the included factors and the TRE was modeled with a linear link function. If indicated, post-hoc tests for differences in TRE between active and passive conditions as well as between stimulation conditions and the sham control condition were then calculated to further explore the direction of effects. Since this latter analysis was only done exploratory to investigate the potential influence of the test delay in the emergence of the TRE and post hoc tests were only calculated for significant main and interaction effects, the p-values reported here are not corrected for multiple comparisons.In that sense, we further explored the distribution of the TRE across the tested delay levels by generalized estimating equations (GEE) analyses using IBM SPSS Statistics (Version 25.0). Here, the difference in the percentage of detected delays between 150 vs. 0 ms adaptation delay conditions served as measure for the TRE which can be computed separately for each test delay level. For the regression coefficients an AR (1) working correlation structure and robust (sandwich) covariance estimators were used. The analysis was performed separately for visual and auditory test modalities and the respective models were composed of the factors t-tests using the average score of all questionnaire items.Finally, differences in perceived side effects between the ctDCS stimulation conditions were assessed by paired-samples p > 0.43) suggesting that ctDCS did not influence the visuomotor or visuo-tactile TRE. Notably, according to one-sample t-tests, the TREs in the visual modality were also not significantly greater than zero in the sham control condition, neither the visuomotor TRE in active , nor the visuo-tactile TRE in passive conditions .Repeated-measures ANOVAs were performed on the TRE (difference in detection thresholds of 150 ms vs. 0 ms adaptation delay conditions) for each test modality  = 2.810, p = 0.047, ηp2 = 0.118; see t-tests, in passive conditions, anodal ctDCS increased the audio-tactile TRE compared to sham ctDCS , but was absent after sham ctDCS .For the auditory test modality, the interaction of t(21) = 1.522, p = 0.143, d = 0.324; dual-hemisphere vs. sham ctDCS: t(21) = 1.464, p = 0.158, d = 0.321] nor active movement conditions .No other stimulation condition elicited a TRE significantly different from the sham control condition for neither passive  indicating that stimulation-dependent effects on the TRE differed here depending on the amount of temporal delay presented in the test phase. Post hoc tests specified that the facilitation of the TRE by anodal ctDCS compared to sham manifested for the test delay closest to the adaptation delay . Again, the TRE was significantly greater than zero after anodal ctDCS for this delay level  which was absent in the sham condition . Moreover, adaptation to the constant delay of 150 ms was associated with fewer false alarms  after anodal  and cathodal ctDCS  compared to the sham control condition. TREs were significantly greater than zero after anodal as well as cathodal ctDCS at this delay level , but not present after sham ctDCS .For the visual test modality, again, none of the interaction effects reached significance  = 37.183, p = 0.001]. Thus, we inspected the post hoc comparisons of anodal compared to sham ctDCS to investigate whether the faciliatory effects of anodal ctDCS differed across the range of test delays depending on the movement type. Accordingly, the facilitation of the effect for the 167 ms test delay by anodal ctDCS was specific to the audiomotor TRE in active movement conditions , and did not differ significantly from the audio-tactile TRE induced for this delay level in passive conditions .Finally, the interaction p = 0.014]. According to a one-sample t-test the audio-tactile TRE was significant here after anodal ctDCS  and differed also weakly from the audiomotor TRE for this delay level in active conditions .In contrast, in passive movement conditions, the facilitation by anodal ctDCS occurred at a larger test delay level of 333 ms . The comparison of perceived side effects between the ctDCS conditions compared to the sham control condition revealed a significant difference for anodal ctDCS . There were no significant differences for the comparison of sham stimulation against cathodal  or dual-hemisphere ctDCS . Furthermore, ratings for perceived side effects were significantly higher after anodal compared to dual-hemisphere ctDCS . However, control analysis including side effects as covariate of no interest suggest that main results cannot be explained by side effects alone , but at a larger delay level (333 ms) for the audio-tactile TRE in passive conditions.These findings suggest a general role of the cerebellum in potentially supra-modal temporal recalibration across sensorimotor and perceptual domains, but the differential manifestation of the effect across tested delay levels in active and passive conditions point to differences in the underlying mechanisms depending on the availability of action-based predictions.We expected ctDCS to influence action-based prediction mechanisms in the cerebellum and therefore the TRE in both visual and auditory modalities specifically in active movement conditions. Indeed, in the auditory test modality anodal ctDCS facilitated the occurrence of the TRE compared to the sham control condition. Surprisingly however, this stimulation-dependent effect was not specific to active conditions, but an audio-tactile TRE appeared across the tested delay levels only in passive conditions. However, an inspection of the faciliatory effect of anodal ctDCS for the individual test delay levels revealed that in active conditions an audiomotor TRE could be elicited by anodal ctDCS specifically at the 167 ms delay which was the delay level closest to the constant delay of 150 ms used during the adaptation phases. In contrast, in passive conditions, a significant facilitatory effect of anodal ctDCS on the audio-tactile TRE occurred at the later 333 ms delay level, at which the influence of the same stimulation on the audiomotor TRE had already decreased.This raises the question of why the effect of ctDCS generally extended to inter-sensory temporal recalibration and therefore also appeared in passive conditions, and what reason might underlie the different manifestation of the effects across the range of tested delays depending on the movement type.Since anodal tDCS is known to enhance cortical excitability  the stroIndeed, multiple studies support the involvement of the cerebellum in a variety of processes beyond sensorimotor functions ranging from perceptual processing  and perfThus, in our study anodal ctDCS could have facilitated temporal recalibration particularly in an inter-sensory context by promoting the updating of the expected relative timing between tactile and auditory sensory signals. This adds evidence to the importance of the cerebellum for temporal recalibration of predictive mechanisms, however not exclusively within the motor domain as previously suggested e.g., , but alsIf anodal ctDCS facilitated the TRE identically in passive and active conditions, then this would suggest that the TRE in active conditions can be explained by the facilitation of inter-sensory recalibration mechanisms alone, since the additional availability of the motor commands would not provide any further explanatory contribution to the emergence of the effect in this condition. But notably, although anodal ctDCS facilitated the TRE in the auditory modality for both movement types, differences emerged in the manifestation of the effect when it is considered separately for the different delay levels used in the test phases. Thus, there may be differences in the exact underlying temporal recalibration mechanisms depending on the movement type.For active conditions, the fact that the facilitation of the TRE by anodal ctDCS compared to sham occurred precisely at the delay level closest to the adaptation delay may point to a particularly accurate recalibration mechanism. This could be related to the fact that sensory action-outcomes are considered as particularly well predictable due to the availability of information on the motor commands . It has Such a precise recalibration mechanism could be particularly important in a sensorimotor context, because in real-life situations, changes in the relative timing between actions and their outcomes have direct implications for motor behavior, as movements may need to be adjusted accordingly. Thus, precise sensorimotor temporal recalibration could be important to still enable accurate motor behavior under flexibly changing environmental conditions.For passive conditions on the other hand, faciliatory effects of anodal ctDCS on the audio-tactile TRE appeared at larger delays . In part, this could be related to the fact that detection performance was generally worse in passive compared to active conditions. It might thus be that the perceptual performance at medium-size delay levels was generally too low for a strong recalibration effect to manifest here. Only at larger delay levels the recalibrated inter-sensory timing leads to significant decreases in delay detection performance. Nonetheless, the audio-tactile TRE induced by anodal ctDCS appears somewhat more distributed across the delay range, which becomes also apparent in the fact that the audio-tactile and audiomotor TRE do not differ significantly at the earlier 167 ms delay. But since the audiomotor TRE after anodal ctDCS decreases across delays after its early peak at the 167 ms delay, the relative impact of anodal ctDCS grows stronger for the audio-tactile TRE, resulting in significant differences between the audiomotor and audio-tactile TRE at the larger delay level of 333 ms. This somewhat broader distribution of the audio-tactile TRE after anodal ctDCS could also explain why it is overall, i.e., across all delay levels, stronger than the audiomotor TRE.post hoc tests of this exploratory analysis are uncorrected for the multiple testing at each of the six delay levels and should therefore be interpreted with caution. Although the exact explanation for and interpretability of the differential manifestation of the sensorimotor and inter-sensory TRE as well as the overall rather weak recalibration effects in this study remains open, our findings nevertheless highlight the importance of not just focusing on the overall shift in perceptual thresholds  during the investigation of temporal recalibration, but also at the exact manifestation of the effect at different delays.In conclusion, anodal ctDCS facilitated both the audio-tactile and the audiomotor TRE, but the differential manifestation of the effect across delay levels suggests the additional involvement of action-based predictive mechanisms for the emergence of the effect in active conditions potentially leading to a more precise audiomotor TRE. It should be noted though that these delay-dependent effects appeared to be rather weak and Since predictions about sensory action-outcomes are assumed to be generated on a supra-modal level, we expected that a TRE would manifest after the visual recalibration procedure in the visual, but also in the auditory test modality in active movement conditions, and that anodal ctDCS would further boost these effects.Surprisingly, however, without the influence of ctDCS, i.e., in the sham control condition, a TRE did not manifest for the visual modality in either active or passive conditions and did not transfer to the auditory modality either. Only after anodal ctDCS recalibration occurred in the auditory modality which was still absent in visual conditions. This raises the question of the reason for the resistance of the visual modality to behaviorally relevant recalibration in our study, as well as why the constantly delayed visual stimulus during adaptation phases nevertheless produced an auditory TRE after anodal ctDCS.Although recalibration of the perceived timing between actions and their visual sensory outcomes could be demonstrated in a range of previous studies , a certaSimilarly, in the context of inter-sensory timing, synchrony perception of visuo-tactile stimuli (as it is relevant for passive conditions in our study) has been shown to have rather low resolution as compared to, for example, audio-tactile stimuli . This imThus, in our study, the short temporal lag of 150 ms during the adaptation phase could have caused too noisy or unreliable prediction errors in the visual modality to induce recalibration of upcoming sensorimotor predictions or recalibration of inter-sensory timing and thus a behaviorally observable TRE in active or passive movement conditions. Nevertheless, we cannot rule out the possibility that the effects of temporal recalibration in general, as well as the effect of ctDCS on temporal recalibration, were so small for the visual modality that we are lacking sufficient statistical power in this study to detect these effects.The emergence of an audiomotor and audio-tactile TRE even though the sensory modality during the adaptation phase was visual would point to the recalibration of predictive or perceptual mechanisms that operate on a supra-modal level and not in modality-specific circuits. The fact that a TRE occurred indeed only in the auditory modality after anodal ctDCS in our study might be related to a generally more precise temporal perception and discrimination performance for auditory stimuli . This alThus, our results suggest that the delayed visual stimulus in the adaptation phases still resulted in an updating of the perceived relative timing between the active or passive button movement and the feedback presentation, even though its effects were not measurable as TRE in the visual modality possibly because of the rather imprecise temporal perceptual acuity. However, with the faciliatory influence of anodal ctDCS, prediction error processing increased and resulted in a TRE now assessable in the auditory modality possibly due to the temporally more accurate perception.Notably, we originally expected that anodal ctDCS would mainly promote the modality-transfer of the TRE in active conditions due to the facilitation of supra-modal sensorimotor recalibration mechanisms in the cerebellum, but the effect extended to passive conditions and thus to inter-sensory recalibration. Cross-modal transfer of the TRE in an inter-sensory context has also been shown in previous studies  and speaNevertheless, it should be noted that due to the absence of effects in the visual modality conclusions about supra-modal recalibration mechanisms should be made with caution. Still, the fact that the visuomotor and visuo-tactile recalibration procedure could trigger an audiomotor or audio-tactile TRE, respectively, points to recalibration mechanisms in the cerebellum evoked by anodal ctDCS that are not modality specific.Some limitations of the study design and interpretability of the results should be considered. The potential of ctDCS in affecting performance in a variety of tasks has been demonstrated in an increasing number of studies . Also inFurthermore, although anodal tDCS is generally thought to increase cortical excitability, the evidence for polarity-dependent effects of tDCS on the cerebellum is heterogeneous. While some studies find polarity-dependent effects in the expected direction , others It has to be noted that the recalibration effects in our study were overall rather small and not present without the facilitating influence of anodal ctDCS, i.e., in the sham control condition. The audio-tactile TRE in passive conditions seems to point even into the opposite direction than one would expect. Even though the reason for this remains unclear based on our data, considerably smaller shifts in temporal perception during recalibration compared to the magnitude of the actual adaptation delay has been reported previously e.g., . The parFirst, the size of the recalibration effect, or even the occurrence of the effect itself, could be related to the amount of attention deployed to the sensory stimuli during adaptation. For example, it has been reported that awareness of the constant delay between action and visual outcome is necessary to trigger the visual sensorimotor TRE . FurtherSecond, it could be argued that recalibration effects that have built up over the course of an adaptation phase decay relatively quickly over the course of a test phase, so that the last trials of the test phase are less affected by recalibration, resulting in lower overall recalibration effects. To counteract this, previous studies have used so-called top-up trials, in which subjects were quickly exposed to the adaptation delay again before each test trial e.g., . HoweverLastly, within our experimental procedure, conditions with the same adaptation delay (0 vs. 150 ms) were each presented in a block during and after stimulation, resulting in multiple changes of the adaptation delay within a session. While it was intended to challenge the flexibility of recalibration mechanisms with this procedure, it is conceivable that the more frequent switching between adaptation delays in our experiment caused spill-over effects, thereby decreasing the overall size of the TRE or the time window available for the effect to build up before the adaptation delay changed again might have been too short. However, the rapid recalibration effects that could be observed even on a trial-by-trial basis at least in the inter-sensory domain  argue agThe prospect that anodal ctDCS may improve sensorimotor adaptation, has implications for neurological and psychiatric disorders known to have impairments in this process and potentially also in related processes like motor adaptation and learning. Patients with cerebellar ataxia for instance show impairments in the ability to adapt to visuomotor perturbations  and exhiBeyond that, patients with schizophrenia spectrum disorder have been shown to have dysfunctions in forward model based predictive mechanisms . Such a The present study investigated the impact of ctDCS on visuomotor and visuo-tactile temporal recalibration and the manifestation of its effect on visual and auditory delay detection performance. We demonstrated that anodal ctDCS facilitated the TRE in the auditory modality in both active and passive conditions which points to a general role of the cerebellum in temporal recalibration across sensorimotor and perceptual domains. The fact that the effect occurred in the auditory modality even though the adaptation modality was visual further suggests that this temporal recalibration mechanism operates on a supra-modal level. The differential manifestation of the effect across tested delay levels in active and passive conditions, however, indicates a more precise cerebellar-dependent temporal recalibration mechanism in a sensorimotor context possibly due to the additional recalibration of action-based predictions.Together these findings emphasize the role of the cerebellum in potentially supra-modal temporal recalibration mechanisms in both perceptual and sensorimotor domains, but there might be differences in the precision of recalibration depending on the availability of action-based predictions. Additionally, despite the complex pattern of observed effects, our findings suggest that anodal ctDCS can be a promising tool for facilitating effects of temporal recalibration in the sensorimotor, but explicitly also in the inter-sensory domain.10.5281/zenodo.6861087 (Zenodo), https://osf.io/qhryx (OSF preregistration).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: doi: The studies involving human participants were reviewed and approved by Ethics Commission of the Medical Faculty of University of Marburg, Germany. The patients/participants provided their written informed consent to participate in this study.BS and CS conceived and designed the study, and discussed and interpreted the data. CS was responsible for data acquisition and analysis, and wrote the manuscript. Both authors revised and approved the submitted version of the manuscript.


The Giant Vesicle Book”  might appear as a rather niche technique, but it has literally revolutionized experimental approaches to bottom-up Artificial Cells (ACs) . The DTmle Book” .Frontiers on Bioengineering and Biotechnology by gene expression  thanks to the presence of about 100 different macromolecules and dozens of small MW compounds co-captured in the GV lumen. Proteins will be produced in cell-like fashion, i.e., from within.The timely investigation presented in This commentary is an opportunity to briefly add some considerations on the DTm. We will highlight its major historical developments, the pros and cons, and the still missing mechanistic details.asymmetric membrane. This is in principle possible when the two lipid leaflets of a vesicle membrane are assembled sequentially. Membrane asymmetry was also the goal of more recent studies, due to the Weitz group (Demetrios Papahadjopoulos (1934–1998), one of the fathers of liposome research, reported that attempts to prepare liposomes from water-in-oil emulsion droplets date back to the 1970s . The aimtz group  and to ltz group .isotonic density gradient strategy is employedThe shift of interest from LUVs to GVs, and from membrane asymmetry to high encapsulation yield coincided with the above-mentioned Subsequent investigations have refined, optimized , scaled-The advantages of the DTm have been mentioned above see also . A furthThe most-often repeated criticism pertains to the possible residual presence of hydrocarbons in the GVs membrane. Researchers interested in studying phenomena strictly dependent on biophysical/biochemical properties of membranes are understandably worried about this issue  e.g., . The “mispontaneous formation of cell-like systems from lipids and solutes. Therefore, when ACs are intended as primitive cell models and the experimental focus is on spontaneous assembly mechanisms in an aqueous environment, other preparation methods should be considered.A less frequently mentioned aspect is that DTm is not the best choice in studies intended to explore the et al. (2003ab) reported several insightful observations, but because their protocol is quite different from the ones commonly used today, more investigations are needed : does the composition of the resulting GV membranes mirrors the overall bulk composition of lipids used in the preparation? .For example, open questions concern the mechanism of formation and the structure of the w/o droplets  and the distribution of solutes therein. The dynamics of lipids at the interface between the emulsion phase and the aqueous outer phase and the very mechanism of bilayer formation


As sex hormone-dependent tumors, it remains to be clarified whether there is a common genetic signature and its value between breast and endometrial cancers. The aim of this study was to establish the shared sex hormone metabolism-related gene prognostic index (SHMRGPI) between breast and endometrial cancers and to analyze its potential role in the therapeutic and prognostic assessment of endometrial cancers.Using transcriptome data from TCGA, tumor-associated gene modules were identified by weighted gene co-expression network analysis, and the intersection of module genes with female sex hormone synthesis and metabolism genes was defined as sex hormone metabolism-related gene. SHMRGPI was established by the least absolute shrinkage and selection operator and Cox regression. Its prognostic value of patients with endometrial cancer was validated, and a nomogram was constructed. We further investigated the relationship between SHMRGPI groups and clinicopathological features, immune infiltration, tumor mutation burden, and drug sensitivity.P< 0.05). Furthermore, we revealed significant differences between SHMRGPI groups as regards tumor immune cell infiltration, somatic mutation, microsatellite instability and drug sensitivity. Patients with low SHMRGPI may be the beneficiaries of immunotherapy and targeted therapy.A total of 8 sex hormone metabolism-related gene were identified as key genes for the construction of prognostic models. Based on SHMRGPI, endometrial cancer patients were divided into high and low SHMRGPI groups. Patients in the low SHMRGPI group had longer overall survival (OS) compared with the high group (The SHMRGPI established in this study has prognostic power and may be used to screen patients with endometrial cancer who may benefit from immunotherapy or targeted therapy. Breast and endometrial cancers are the first and fourth most common tumors in women and pose a serious threat to women’s health . As tumoThe morbidity and mortality of endometrial cancer have increased annually in recent years, which is probably due to the combined effects of an aging population, the decline in benign hysterectomies, and the obesity epidemic . DespiteIn this study, we performed a weighted gene co-expression network analysis (WGCNA) using transcriptome data from the TCGA database for breast and endometrial cancers to identify the sex hormone metabolism-related gene (SHMRG) associated with the synthesis and metabolism of female sex hormone, and subsequently established the SHMRG prognostic index (SHMRGPI) in patients with endometrial cancer and analyzed the value of SHMRGPI in survival assessment and therapeutic modality selection. With this study, we hope to identify novel biomarkers that can be used for screening and treatment and provide a basis for the search for potential beneficiaries of immunotherapy and targeted therapy.The transcriptome data for the breast cancer samples were downloaded from TCGA. Samples from ER and PR positive female patients were selected for subsequent analysis. Transcriptome, somatic variation and clinical data for endometrial cancer samples were downloaded from TCGA, and EEC samples were selected. Transcriptome data for normal breast and uterine tissues were downloaded from the Genotype-Tissue Expression (GTEx) dataset from XENA . Gene seWeighted gene co-expression network analysis (WGCNA) was established to explore the relationship between expression and phenotype data based on correlation coefficients . In thisThe EEC sample was randomly divided into training and testing cohorts in a ratio of 7:3. SHMRG associated with the prognosis of EEC patients was screened in the training cohort using univariate Cox regression. Subsequently, the SHMRGPI was established using the least absolute shrinkage and selection operator (LASSO) and stepwise multivariate Cox regression. The SHMRGPI was calculated according to the following equation:The distributions of SHMRGPI and survival status were plotted as scatter plots. The correlation between SHMRG and SHMRGPI was shown as heat maps. Patients with EEC were divided into two groups based on the median SHMRGPI  in prognostic prediction. We then use the regplot R packages to develop a nomogram. The accuracy of the nomogram was assessed using calibration curves. Furthermore, we performed a stratified analysis to examine the predictive power of SHMRGPI in different clinical subgroups.To assess immune cell infiltration in the different SHMRGPI groups, we performed single sample gene set enrichment analysis (ssGSEA). Then we investigated the differential expression of immune checkpoint genes between the SHMRGPI groups. Tumor immune dysfunction and exclusion (TIDE) scores predict the response of immunotherapy by model primary mechanisms of tumor immune evasion . We calcWe used the maftools R package to collate and analyze the somatic mutation data from patients with EEC. Fifteen genes with the highest tumor mutation frequency in each SHMRGPI group were visually analyzed. The tumor mutation burden (TMB) was subsequently calculated for each sample to analyze the discrepancy in TMB levels between SHMRGPI groups. After establishing subgroups based on the median TMB, we analyzed the prognostic value of SHMRGPI in the TMB subgroups. The microsatellite instability (MSI) status of EEC patients is downloaded by invoking the cBioPortalData R package. Thereafter, differences of MSI status in the SHMRGPI group and differences of SHMRGPI in the MSI subgroup were analyzed.The half-maximal inhibitory concentrations (IC50) of common antitumor drugs were predicted for both SHMRGPI groups based on data from Cancer Drug Sensitivity Genomics . The difP< 0.05 was considered statistically significant.The WGCNA was analyzed and visualized using the WGCNA and limma R packages. Cox regression and survival analysis was performed by the survivor and survminer R packages. Differences in survival between groups were visualized using Kaplan-Meier survival curves. The Wilcoxon signed-rank test was used to test the differences between quantitative data. The entire analysis was conducted in R (version 4.0.3). 802 ER/PR positive tumor tissue samples and 79 normal samples were selected from the TCGA-BRCA dataset, and 179 normal samples from the GTEx database were combined for WGCNA after batch effects were removed. The gene clustering dendrogram was shown in The female sex hormone synthesis and metabolism related-gene sets downloaded from MSigDB were shown in P< 0.1, Total 399 samples from the TCGA database were included in this study and were randomly divided into training and test groups in a 7:3 ratio. The univariate regression analysis of SHMRG combined with expression and clinical data revealed a total of 19 SHMRG were potentially associated with prognosis in patients with EEC .Drug sensitivity analysis based on the oncopredict R package revealed significant differences between the SHMRGPI groups in olaparib, niraparib, and talazoparib , proliferation, and malignant transformation  polymerase inhibitors (PARPi) in maintenance therapy for ovarian cancer can be replicated in the management of patients with endometrial cancer is a common concern among gynecologic oncologists , 32, 33.A limitation of this study is that the data used to build the model were obtained from a retrospective database and the findings are potentially susceptible to bias. Inferences based on the results of immune and drug sensitivity analysis need to be supported by additional experimental evidence.In this study, we developed a prognostic model and analyzed it with respect to clinical, somatic mutation, immune and drug sensitivity. Focusing on biomarkers shared by endometrial cancer and breast cancer, it provides a new idea for the precise treatment of patients with EEC.The original contributions presented in the study are included in the article/JD and CL participated throughout the conception, data analysis, and manuscript writing of the study. JY was primarily involved in the writing of the study. YW was involved in the conception of the study and review of the manuscript. All authors contributed to the article and approved the submitted version.


Synaptogenesis is the final phase of axonal pathfinding. Its sequences of spatial and temporal development in the immature nervous system are precisely timed and consistent. Synaptophysin, a principal structural glycoprotein of synaptic vesicle membranes regardless of the chemical transmitter substance within, is a reliable means of demonstrating sequences of synaptogenesis in human fetal brain tissue at autopsy and is resistant to postmortem autolysis. Furthermore, synaptophysin molecules are demonstrated during axoplasmic flow before being assembled into membranes in immature axons and also mature axons of neurons with a high metabolic rate. In brain malformations these sequences often are altered both in distribution of synapses and in timing, often delayed but sometimes precocious, with postnatal clinical manifestations such as epilepsy and cognitive development. Synaptogenesis is fundamental to nervous system circuitry and networks that are the basis of functional expression. Though the concept had been expressed decades earlier , chemotrSynaptophysin is a structural glycoprotein, a major constituent of synaptic vesicular membranes, regardless of the identity of neurotransmitter within the vesicles in axonal terminals. Other structural glycoproteins of these vesicular membranes include synaptobrevin and synaptotagmin. The latter is particularly important for rapid resupply of synaptic vesicles by micromolar presynaptic calcium signals . SynaptoTiming is a key determinant not only of normal brain development but aIso cerebral dysgeneses . In malfth century . In our studies of more than 200 normal human fetal and neonatal brains at various gestational ages from 6 to 42 weeks, we found reliable consistency between brains of similar age in distribution and timing of appearance of synaptophysin reactivity. Other systematic surveys of temporal and spatial sequences of synaptogenesis are sparse, though many neuropathologists in other centers in Canada and Europe have anecdotally confirmed to us their similar findings. Ongoing studies in our laboratory include the spinal cord, retina and detailed nuclei of the brainstem and thalamus.The first part of the human fetal hippocampus to express synaptophysin immunoreactivity is the molecular zone of the dentate gyrus, as early as 12 weeks gestation, followed by the CA2 sector of Ammon’s horn at 14 weeks, CA2 at 14 weeks, CA3 and CA4 at 15-16 weeks and finally CA1 at 19 weeks . ReactivIn the frontal lobe, synaptophysin was detected in a laminar pattern above and below the neocortical plate at 12 weeks and around Cajal-Retzius neurons of the molecular zone at 14 weeks gestational age. Pyramidal neurons of the future deep layers of cortex were surrounded by synaptophysin reactivity at 16 weeks and at the surface of neuronal somata of layers 2 and 4 at 22 weeks. Immunoreactivity remained more intense in the molecular zone and in deep cortical layers until about 36 weeks and in late gestation and at term was uniform throughout the cortex . Other lFrom 16 weeks gestation synaptophysin immunoreactivity is demonstrated at the somata of mitral and tufted neurons and also many but not all olfactory bulb glomeruli. Glomeruli are uniformly strongly reactive by 20 weeks. The granular layer of the core of the olfactory bulb exhibits concentric alternating layers of neurons and sheets of dendro-dendritic synapses which are synaptophysin immunoreactive in the most peripheral layers from 16 weeks but the deepest third of the multiple layers are still not reactive at term and acquire synaptophysin reactivity in the first few postnatal weeks . NeuN, aThe corpus striatum (caudate nucleus and putamen) shows a unique patchy pattern of synaptophysin reactivity, neither laminar nor uniform, not seen in the normal neocortex but found also in the nuclei of the basis pontis (see cerebellar system below). This patchy synaptophysin pattern of the corpus striatum is first detected at 13 weeks gestation, is prominent before midgestation and is still subtle even at term . This syAscending thalamocortical axons were detected with axoplasmic synaptophysin reactivity as early as 12 weeks but most intense and best demonstrated at 26 weeks gestation . ThalamiThe cerebellar cortex is best considered not in isolation but as part of circuits that comprise complex cerebellar systems. One loop, limited to brainstem structures, is the “Guillain-Mollaret triangle” or dentato-rubro-olivary-cerebellar circuit. Another is the ascending dentato-ventrolateral thalamo-cortical-pontine-cerebellar circuit. Prominent dorsal and ventral ascending spinocerebellar tracts are present, but a reciprocal direct cerebello-spinal pathway is lacking, though the cerebellum can influence the spinal cord via small vestibulospinal, reticulospinal, olivospinal and tectospinal tracts. The principal influence of the cerebellum on the spinal cord for muscle tone and coordination is via the corticospinal tract.neuronal maturation and synaptogenesis can occur before the mature anatomical form of the nuclei develops. Crenation of these nuclei begins just before mid-gestation and is complete by 33 weeks, similar to the adult morphology.The dentate and other smaller deep cerebellar nuclei , as well as the inferior olivary nuclei, initially are rather amorphous gray matter masses without much form; only with maturation do they convolute to form the “crenated” characteristic form of the mature brain. Synaptophysin reactivity appears in these nuclei before crenation occurs, highlighting a fundamental principle of developmental neuroanatomy that Accessory olivary and deep cerebellar nuclei are intensely reactive for synaptophysin earlier than the principal olivary and dentate nuclei . The dorThe red nucleus matures in synaptophysin reactivity later than the bulbar nuclei. Before 18 weeks, the red nucleus contrasts with the intensely reactive surrounding tegmental gray matter because of its lack of reactivity. Later in gestation it also exhibits high contrast. The transition is at about 18 weeks. Synaptophysin reactivity in the red nucleus is initiated in the periphery, with scattered nests of reactive small neurons also in its more central and inferior region where large neurons (magnocellular) predominate. The lesser reactivity seen in the center than the periphery persists until term but part of this appearance is because neurons normally are less densely clustered in the center than the periphery of the red nucleus . HistoloPontine nuclei of the basis pontis receive corticopontine axons from neocortex and project their axons to the contralateral cerebellar cortex. As noted above, as with the corpus striatum, these nuclei have patchy synaptophysin reactivity not explained by intervening axonal projections of the corticospinal and corticopontine pathways. Its synaptic vesicle reactivity appears at 20 weeks and is uniform by 34 weeks gestation .In the cerebellar cortex, the vermis matures sooner than the cerebellar hemispheres and the paravermal portions earlier than the lateral folia. The earliest synapses occur around the somata of Purkinje neurons and later in the internal granular layer, but synaptic glomeruli are not well formed until after 26 weeks . In the The area postrema is a small but important nucleus in the dorsomedial medulla oblongata near to the caudal end of the floor of the 4th ventricle , with raThe tractus solitarius is a paired small descending pathway in the tegmentum of the medulla oblongata, surrounded by neurons of its nucleus . TogetheThe U-fiber layer is a rim of white matter just beneath layer 6 of the neocortex and follows the contours of the gyri. U-fibers consist of short association axons of neurons in layer 6 that locally interconnect different parts of the same gyrus and also immediately adjacent gyri, but are not long descending projections or commissural fibers, thus differ significantly from the deep white matter of the cerebral hemisphere. The U-fiber layer develops in fetal life when gyration and sulcation of the cortex begin, shortly after mid-gestation. Within the U-fibers of normal mature brains are a few heterotopic neurons with axonal connections between them. These axons can be demonstrated by synaptophysin immunoreactivity. Beneath focal cortical dysplasias, types I and II, the number of heterotopic neurons is greatly increased and complex synaptic plexi form between them and also extending into the overlying cortex to integrate with epileptic networks . These pSynaptophysin immunocytochemistry is a reliable and valuable method of demonstrating sequences of normal and abnormal synaptogenesis in paraffin sections of the various structures of human fetal and neonatal brain. It adds a dimension of understanding to neuropathological examinations by light microscopy of immature brain tissue not duplicated by histological stains, particularly useful in studying cerebral malformations. It is useful both for research and in diagnostic neuropathology.The author confirms being the sole contributor of this work and has approved it for publication.


Small and medium enterprises (SMEs) in India are suffering from the long-standing challenges related to asymmetric information, high transaction costs, SMEs’ opacity and limited access to credit. Blockchain technology, which is still in its infancy in terms of adoption in India, can facilitate SMEs to counter these challenges. Fuelled by this motivation, the study aims to investigate the significant barriers to blockchain adoption in supply chain finance practices by Indian SMEs. Using fuzzy-analytic hierarchy process, sensitivity analysis, and fuzzy-decision-making trial and evaluation laboratory this paper identifies the blockchain barriers, prioritises them and examine their cause and effect relationships. The results of the study indicate that technology barriers are the most influential barriers that impede blockchain adoption. The findings will help the policymakers and practitioners to take suitable measures to overcome these barriers and fuel the adoption of blockchain in Indian SMEs. Supply chain finance (SCF) is an approach for two or more supply chain partners and an external service provider to create value by planning, steering, and controlling the flow of financial resources on an inter-organizational level . It is lBlockchain technology can bring new levels of collaboration among the supply chain actors and accelerate the cash flows through the supply chain . It is aTraditional SCF processes suffer from inefficiencies in the financial settlements in the supply chain. Although SMEs have digitised their processes to some extent, the processing of financial transactions remains in traditional paper form in most organisations, leading to delay in payment, greater days of sales outstanding, and ultimately raising the demand for working capital . BlockchThe commercial application projects in this area are already gaining traction. The technology giant IBM has teamed up with one of the largest logistics service providers, Maersk Line, to create a blockchain-based solution to digitise the global, cross-border supply chains. China-based fintech firms Dianrong and FnConn have launched the blockchain-based SCF platform to secure funding for SMEs in China . MoreoveBlockchain technology has good potential to transform the SCF practices in SMEs, but its adoption is still in its infancy . AlthougIdentifying the adoption barriers would help understand the steps for the successful adoption of blockchain in SCF processes by SMEs. Such information will be helpful to the policy-making bodies, supply chain partners, and government to prepare an appropriate strategy for adopting blockchain in SCF by SMEs. The following section presents a review of literature explaining the relevance of blockchain technology in SCF processes. The research methodology is explained in Sect. Blockchain is a decentralised, public database that is shared across a network of computers . This neMaintaining trust among a large number of anonymous members on the blockchain is a challenging task due to its open and decentralised architecture. This is where the consensus algorithm comes into play. A consensus algorithm is a mechanism through which all the members of a blockchain network validate the transactions and ensure its accuracy. In this way, blockchain participants can trust the unknown peers in a distributed network and maintain the integrity and security of the data. When new intended transactions are validated and accepted by the network, it is added to a block. A cryptographic algorithm called hashing provides a unique hash value to each block . These bSuccessful implementation of SCF programs depends on improving software and technology solutions. The automated processes can speed up the cash flows throughout the supply chain. The advent of enterprise resource planning (ERP) systems and the rise of e-invoicing brings a certain level of automation and dematerialisation to B2B processes, enabling faster and more efficient SCF solutions .At present, most supply chain operations rely heavily on centralised and stand-alone information management systems, such as ERP systems . These sAlthough ERP systems partially integrate different layers of supply chain processes into one wide application system, isolated operating units can still exist within the organisation . This leOn the other hand, if the organisations use blockchain as the underlying technology, information can be recorded and broadcast across all the participants while maintaining a single source of truth . Each paBlockchain enhances the automation of SCF processes by bringing fully digital and signed documents which leads to faster invoice approval , 30. EarThe supply chain partners face two key risks in pre-shipment SCF solutions, such as inventory finance and purchase order finance. First, the performance risk of the supplier in fulfilling the purchase order and second, the credit risk of the buyer . CombinaIn order to develop a comprehensive understanding of blockchain adoption barriers, it is imperative to develop the theoretical foundation of the barriers discussed in the study. Previous studies discussing blockchain adoption barriers in supply chains have lacked theoretical background. While popular theories such as the technology acceptance model and institutional theory have been used in the literature to explain the underlying motivations of an organisation to adopt a technology, such theories do not explain how managers develop knowledge of the new technology, which eventually shape their actions . There iMost of the existing literature on technology adoption emphasises the implementation phase of the technology adoption process, focusing less on the pre-adoption phase . AdoptinThe lack of adoption of blockchain technology can also be explained with the help of force field theory . Force fIn pursuit of a higher level of traceability and visibility in supply chain finance transactions, blockchain is a promising technology to drive operational efficiency and competitive advantage. According to Koh et al.  resourceInformation processing theory (IPT) complements the understanding of the effects of blockchain on the SCF processes. The SCF information is used to reduce the firms’ capital and investment risk within the supply chain and improve their financing decisions. The uncertain environment and inadequate management of information processing may jeopardise the integration of the financial supply chains . InformaThe study applies a hybrid methodology combining fuzzy-AHP, sensitivity analysis, and fuzzy- DEMATEL. Fuzzy-AHP is used for ranking the barriers and sub-barriers based on their significance, whereas fuzzy DEMATEL highlights the cause and effect relationships among them. In order to evaluate the ranks of particular barriers by making small changes in their weights, the study employs sensitivity analysis. Sensitivity analysis is a crucial tool to ensure the validity of a developed model . The AHPIn order to collect data, academicians and practitioners knowledgeable in blockchain and supply chains were approached to identify the key barriers, prioritise them and evaluate their cause and effect relationship. A questionnaire-based survey was conducted amongst 58 supply chain professionals to identify the key barriers and sub-barriers to blockchain adoption in supply chain finance. The experts were asked to rank the barriers based on their significance on a five-point rating scale in this survey. The experts were also asked to suggest any new barriers relevant to the study. We shortlisted 22 sub-barriers grouped in 6 categories based on the survey responses. The experts also added three new barriers to the study. Finally, we got 25 sub-barriers categorised under six barrier categories, which are given in Table In the next step, 30 experts in supply chains and blockchains were contacted to know the priority and the relationships between these barriers and sub-barriers. However, 15 experts agreed to participate as decision makers in this task. The group size of experts can affect the results of data analysis in the study. According to Gumus , 5–50 exFuzzy AHP is an advanced analytical method developed from the traditional AHP proposed by Saaty . It is aStep 1 Identifying the key barriers and sub- barriers to the adoption of blockchain in SCF: To identify the key barriers, past studies in this area have been reviewed, and six main barriers and 26 sub-barriers were identified. These barriers were then put for deliberation of practitioners and academicians to add or eliminate any barrier. Finally, the experts shortlisted 22 sub-barriers and added three new barriers, giving us 25 sub-barriers classified into six main categories.Step 2 Employ fuzzy set theory to deal with the imprecision in the experts’ judgments: Fuzzy set theory, proposed by Zadeh  can be represented as follows:by Zadeh , helps sby Zadeh . The fuzStep 3 Build a hierarchical structure of the barriers: In this step, a hierarchical structure of the problem is presented, comprising three levels. Level 1 indicates the goal of the given problem; level 2 shows the major barriers to the adoption of blockchain in SCF, and level 3 describes the sub-barriers categorised within each main barrier. Figure Step 4 Compute the pairwise comparison matrices for the barriers and sub-barriers: Fuzzy pairwise comparison matrices are prepared based on the judgement of the experts through the fuzzy linguistic scale provided in Table ij]mxn where the fuzzy entries in the matrices are represented by aij = . These fuzzy numbers fulfils the following property:Step 5 Calculate the significance weights of the barriers: In this step, the triangular fuzzy numbers of the fuzzy pair-wise comparison matrices are processed to calculate the weights of the barriers’ significance to establish their priority. The calculation requires specific algebraic operations. Therefore, Chang’s extent analysis method is used to calculate the significance weights of the barriers and sub-barriers [barriers , 68, 69.In order to establish the weight vectors for main barriers, the obtained values are normalised. The resultant significance weights of the barriers are shown in Table The sub-barriers were ranked based on their relative weights and global weights in the next level. The global weights of the sub-barriers were obtained by multiplying the relative weights of each sub-barrier with the preference weights of the barriers. The global ranking of the barriers to blockchain adoption in supply chain finance is summarised in Table Sensitivity analysis is an essential tool to check the validity and robustness of the proposed framework , 69. It The DEMATEL technique helps analyse complex cause and effect relationships among factors in multi-criteria decision-making problems , 70. It Step 1 Collect inputs from the experts on the effect of one barrier on others: The responses from the experts are collected through a fuzzy-DEMATEL questionnaire. The experts made pairwise comparisons among the barriers, recorded their responses using the fuzzy linguistic scale  using Eq.  is calculated using Eq. I represents the identity matrix.In the total relationship matrix, the sum of all rows (represented by R) and all columns (represented by C) has been calculated using Eq. The results of fuzzy-AHP reveal that technology barriers (TB) obtain the first rank, and therefore, occupy the highest priority among all the barriers. This category includes the barriers stemming from the limitations of blockchain technology. It incorporates the technical capability, difficulty, complexity, and availability of blockchain technology . There aOrganisational barriers come second in the priority list and play a critical role in the adoption of blockchain technology. This category identifies the barriers stemming from the internal activities of organisations and supply chain partners’ relationships. On an inter-organisation level, its challenging to manage the relationships between supply chain partners when adopting innovative technologies . This caSecurity barriers (SB) occupy third place in the priority list. In terms of security and scalability, blockchain is still considered an immature technology . TherefoExternal barriers occupy fifth rank. This barrier category presents the challenges stemming from external stakeholders, institutions, industries, and governments. In this category, ‘Legal and regulatory challenges’ (EB2) hold the highest priority. ‘Market competition and uncertainty about using blockchain’ (EB1) are ranked next to EB2 followed by ‘lack of qualified blockchain developers’ (EB3). Financial barriers hold the last place on the priority list and play a crucial role in adopting blockchain technology. It suggests that implementing blockchain technology is very costly for SMEs, and a lack of funds impedes its adoption. There are four specific barriers in this category. Among them, ‘Huge initial capital investment for infrastructure and energy resources’ (FB1) got the highest priority. ‘Lack of financial resources’ (FB2) comes next in this category followed by ‘Complex tax implications around digital assets’ (FB3) and ‘Audit concerns’ (FB4).DEMATEL traces the cause and effect relationships between the barriers. Table Blockchain has the potential to transform the functioning of SMEs. The technology can overcome SMEs’ long-standing constraints in accessing credit. However, blockchain is in the nascent stage; its adoption rate in SMEs is meagre. The study results reveal that technology and organisational barriers are the most significant barriers of blockchain adoption in SMEs’ SCF practices. Moreover, the findings of the study are in line with the two government reports on blockchain by NITI ayog (the policy think tank of the government of India) and by the ministry of electronics and information technology. Additionally, the survey conducted on blockchain by Deloitte in the year 2020 also confirms the study’s findings. Blockchain is one of the top five strategic priorities for the majority of the survey respondents. Globally, there was a substantial jump in blockchain production in 2020 . The finThis paper presents the key barriers to blockchain adoption by Indian SMEs in SCF. Managers can use the knowledge of key barriers in developing a strategy to overcome the barriers and eventually adopt blockchain in SCF practices.The fuzzy-AHP prioritises the barriers that help the managers to target the most critical barriers. Technology barriers got the highest weightage implying that SMEs’ managers should focus the most on improving the technology infrastructure of their firms.The DEMATEL-based tagging of the barriers into cause and effect groups helps the managers to control the cause group barriers to mitigate their effect for the successful implementation of blockchain technology.In India, a national-level blockchain framework can facilitate in scaling the blockchain applications and introduce shared infrastructure for the organizations . TherefoThe government needs to promote research and development in blockchain, along with skilling workforce and students . FundingSMEs need to be convinced of the advantages of blockchain technology, and the resistance to adopt this technology should be eliminated. SMEs and their supply chain partners are sceptical about the use of blockchain platforms due to the issues related to privacy, security, interoperability, and automation of invoices. Therefore, the blockchain developer community should resolve these issues to facilitate the user experience which will eventually fuel the adoption rate of blockchain in SMEs.Blockchain technology has the potential to help SMEs overcome long-standing challenges by reducing transaction costs and information asymmetry and facilitating trade and access to credit. SMEs in India face numerous barriers to adopting blockchain technology in SCF practices. Therefore, the study investigates the prominent barriers to adopting blockchain technology in SCF in Indian SMEs. Through extensive literature search and the opinion of the domain experts from academia and industry, 25 sub-barriers were identified under six main barriers. As a methodological contribution, the study employs an integrated approach of Fuzzy-AHP, Sensitivity analysis, and Fuzzy DEMATEL. Fuzzy AHP illustrates the significance of the barriers; sensitivity analysis validates the developed model, and fuzzy-DEMATEL examines the cause and effect relationship between the identified barriers.Findings of this research reveal that technology barriers have the highest priority, followed by organisational barriers and security barriers. This brings light to the fact that SCF and blockchain practitioners should work on these barriers on a priority basis so that the adoption of blockchain technology can be accelerated to enhance the efficiency of SMEs. Moreover, SMEs should reshape their organisational culture to facilitate the blockchain adoption. Additionally, by implementing the fuzzy-DEMATEL methodology, four barriers have been categorised into cause groups: organisational barriers, knowledge barriers, security barriers, and financial barriers. At the same time, technology barriers and external barriers have been identified as the effect group barriers. The categorisation of these barriers would aid the managers of SMEs to control the barriers in the cause group and reshaping them to implement blockchain technology in SCF practices successfully. In the end, the developed model is tested for its robustness by implementing sensitivity analysis.The novelty of the research is twofold; first and foremost, this is the first study investigating the blockchain adoption barriers in SCF practices in the Indian context. The second aspect lies in the integrated research methodology used in the study. The three methods complement each other and comprehensively analyse the identified barriers.The present study bears some limitations, which open the prospects for future research. First, the study has been conducted concerning SMEs in India. The research implications may vary to a certain extent in the context of SMEs in developed nations. Therefore, future researchers may investigate the barriers faced by SMEs of developed nations and compare the results with this study. Second, it is a qualitative study based on experts’ opinions. The researchers may undertake empirical research and validate this research in the future. Third, although the study identifies the most comprehensive set of barriers, in the future, additional barriers may appear, and some of the existing barriers may also become obsolete with rapidly changing technology. Therefore, future researchers have an opportunity to identify new barriers that affect the adoption of blockchain by Indian SMEs in SCF.


Cellulose nanocrystals (CNCs) have received a significant amount of attention due to their excellent physiochemical properties. Herein, based on bioinspired layered materials with excellent mechanical properties, a CNCs-graphene layered structure with covalent linkages (C-C bond) is constructed. The mechanical properties are systematically studied by molecular dynamics (MD) simulations in terms of the effects of temperature, strain rate and the covalent bond content. Compared to pristine CNCs, the mechanical performance of the CNCs-graphene layered structure has significantly improved. The elastic modulus of the layered structure decreases with the increase of temperature and increases with the increase of strain rate and covalent bond coverage. The results show that the covalent bonding and van der Waals force interactions at the interfaces play an important role in the interfacial adhesion and load transfer capacity of composite materials. These findings can be useful in further modeling of other graphene-based polymers at the atomic scale, which will be critical for their potential applications as functional materials. Cellulose nanocrystals (CNCs) show exceptional mechanical properties, biodegradability and environmental friendliness ,2,3. TheCNCs with a high degree of crystallinity can easily be formed into smooth films with uniform thickness by appropriate treatment ,22,23. TIn this study, the mechanical properties of CNCs-graphene layered nanocomposites with covalent bonding are investigated utilizing non-equilibrium molecular dynamics (NEMD) under typical temperature and strain rate. The effects of covalent bonding on the interfacial interactions are also predicted by comparing the elastic modulus of CNCs-graphene layered structure with that of pristine CNCs. In addition, the mechanism behind the mechanical property is further explained with the help of the atomic configuration. The simulation results could provide some important connections between mechanical behavior and covalent bonding in layered nanocomposites, and it could provide some insights for optimizing the mechanical properties of other polymers.The NEMD simulations were carried out using the LAMMPS  program package . All intThe NEMD simulations were conducted by first minimizing the energy of the system using the conjugate gradient method. The time step was set to 0.5 fs for all the simulation processes. The simulation temperature was controlled by using a Nosé-Hoover thermostat. Before starting the tensile simulation, the nanostructures reached the global energy minimum using the steepest descent minimization algorithm. After equilibration, the system was stretched for 200 ps under the NVT ensemble. Energy minimization was performed after each deformation increment to maintain the lowest energy state of the system. For each initial configuration, the axial tensile loading modeling was repeated three times with different velocity initialization seeds. The elastic modulus calculated according to Hooke’s law was obtained by averaging the results of three independent simulation trials.−4 fs−1 at temperatures of 300 K, 400 K, and 500 K. As shown in The calculated stress–strain behaviors of the pristine CNCs and CNCs-graphene layered structure subject to x-direction strain are shown in In order to further verify the above results, the atomic configurations of two nanocomposites under tensile stresses with the increasing temperature are shown in The stress-strain behavior of the two nanocomposites with different strain rates at the temperature of 300 K is shown in The elastic modulus of pristine CNCs and CNCs-graphene layered composites are given in –4/fs, an obvious local stress concentration appears in the pristine CNCs. Some local fractures of graphene layer in layered composite also appear. This fact proves that the covalent bonds at the interface can strengthen the stress transfer capacity. As the displacement in the tensile direction increases, an attractive restoring force arising from the covalent bonds in CNCs-graphene layered composite attempts to hold the layers together. Moreover, van der Waals forces also provide some interface strengths due to the large specific surface area between CNCs and graphene layers [To understand the dependence of strain rate on the mechanical properties of the two nanocomposites, the configuration of atoms with the increasing strain rates are shown in e layers .The mechanical properties of the CNCs-graphene layered structure at each content level of covalent bonds are shown in In this work, the mechanical properties of the CNCs-graphene layered structure with covalent bonding are investigated by molecular dynamics simulation. Compared to the pristine CNCs, the mechanical properties of the CNCs-graphene layered structure can be prominently improved due to the covalent bond interactions and van der Waals forces at the interfaces. The elastic modulus negatively correlates with temperature but has a positive relationship with strain rates and content of the covalent bonds. The atomic structures of pristine CNCs and CNCs-graphene layered structure during the tensile process are analyzed to reveal the relationship between structure and mechanical behavior. These results have a particular incentive effect on theoretical research into polymers with layered structures and their practical applications.