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Age-related hearing loss (ARHL) is a common sensory impairment with complex underlying mechanisms. In our previous study, we performed a meta-analysis of genome-wide association studies (GWAS) in mice and identified a novel locus on chromosome 18 associated with ARHL specifically linked to a 32 kHz tone burst stimulus. Consequently, we investigated the role of Formin Homology 2 Domain Containing 3 (Fhod3), a newly discovered candidate gene for ARHL based on the GWAS results. We observed Fhod3 expression in auditory hair cells (HCs) primarily localized at the cuticular plate (CP). To understand the functional implications of Fhod3 in the cochlea, we generated Fhod3 overexpression mice (Pax2-Cre+/-; Fhod3Tg/+) (TG) and HC-specific conditional knockout mice (Atoh1-Cre+/-; Fhod3fl/fl) (KO). Audiological assessments in TG mice demonstrated progressive high-frequency hearing loss, characterized by predominant loss of outer hair cells, and a decreased phalloidin intensities of CP. Ultrastructural analysis revealed loss of the shortest row of stereocilia in the basal turn of the cochlea, and alterations in the cuticular plate surrounding stereocilia rootlets. Importantly, the hearing and HC phenotype in TG mice phenocopied that of the KO mice. These findings suggest that balanced expression of Fhod3 is critical for proper CP and stereocilia structure and function. Further investigation of Fhod3 related hearing impairment mechanisms may lend new insight towards the myriad mechanisms underlying ARHL, which in turn could facilitate the development of therapeutic strategies for ARHL., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Boussaty et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pulsed radiofrequency (PRF) is a safe method of treating neuropathic pain by generating intermittent electric fields at the needle tip. Resiniferatoxin (RTX) is an ultrapotent agonist of transient receptor potential vanilloid subtype-1 (TRPV1) receptors. We investigated the mechanism of PRF using a rat model of RTX-induced neuropathic pain. After administering RTX intraperitoneally, PRF was applied to the right sciatic nerve. We observed the changes in TRPV1, calcitonin gene-related peptide (CGRP), and brain-derived neurotrophic factor (BDNF) in the dorsal root ganglia by western blotting. Expressions of TRPV1 and CGRP were significantly lower in the contralateral (RTX-treated, PRF-untreated) tissue than in control rats (p<0.0001 and p<0.0001, respectively) and the ipsilateral tissues (p<0.0001 and p<0.0001, respectively). BDNF levels were significantly higher in the contralateral tissues than in the control rats (p<0.0001) and the ipsilateral tissues (p<0.0001). These results suggest that, while TRPV1 and CGRP are decreased by RTX-induced neuronal damage, increased BDNF levels result in pain development. PRF may promote recovery from neuronal damage with concomitant restoration of TRPV1 and CGRP, and exert its analgesic effect by reversing BDNF increase. Further research is required to understand the role of TRPV1 and CGRP restoration in improving mechanical allodynia., Competing Interests: No potential conflict of interest relevant to this article was reported.

In small and large spinal dorsal root ganglion neurons, subtypes of voltage-gated sodium channels, such as NaV1.7, NaV1.8, and NaV1.9 are expressed with characteristically localized and may play different roles in pain transmission and intractable pain development. Selective stimulation of each specific subtype in vivo may elucidate its role of each subtype in pain. So far, this has been difficult with current technology. However, Optogenetics, a recently developed technique, has enabled selective activation or inhibition of specific neural circulation in vivo. Moreover, optogenetics had even been used to selectively excite NaV1.8-expressing dorsal root ganglion neurons to induce nocifensive behavior. In recent years, genetic modification technologies such as CRISPR/Cas9 have advanced, and various knock-in mice can be easily generated using such technology. We aimed to investigate the effects of selective optogenetic activation of NaV1.7-expressing afferents on mouse behavior. We used CRISPR/Cas9-mediated homologous recombination to generate bicistronic NaV1.7-iCre knock-in mice, which express iCre recombinase under the endogenous NaV1.7 gene promoter without disrupting NaV1.7. The Cre-driver mice were crossed with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7iCre/+;Ai32/+, NaV1.7iCre/iCre;Ai32/+, NaV1.7iCre/+;Ai32/Ai32, and NaV1.7iCre/iCre;Ai32/Ai32 mice. Compared with wild-type mice behavior, no differences were observed in the behaviors associated with mechanical and thermal stimuli exhibited by mice of the aforementioned genotypes, indicating that the endogenous NaV1.7 gene was not affected by the targeted insertion of iCre. Blue light irradiation to the hind paw induced paw withdrawal by mice of all genotypes in a light power-dependent manner. The threshold and incidence of paw withdrawal and aversive behavior in a blue-lit room were dependent on ChR2 expression level; the strongest response was observed in NaV1.7iCre/iCre;Ai32/Ai32 mice. Thus, we developed a non-invasive pain model in which peripheral nociceptors were optically activated in free-moving transgenic NaV1.7-ChR2 mice., Competing Interests: The authors have declared that no competing interests exist.

Movements synchronized with external rhythms are ubiquitous in our daily lives. Despite the involvement of the cerebellum, the underlying mechanism remains unclear. In monkeys performing synchronized saccades to periodically alternating visual stimuli, we found that neuronal activity in the cerebellar dentate nucleus correlated with the timing of the next saccade and the current temporal error. One-third of the neurons were active regardless of saccade direction and showed greater activity for synchronized than for reactive saccades. During the transition from reactive to predictive saccades in each trial, the activity of these neurons coincided with target onset, representing an internal model of rhythmic structure rather than a specific motor command. The behavioural changes induced by electrical stimulation were explained by activating different groups of neurons at various strengths, suggesting that the lateral cerebellum contains multiple functional modules for the acquisition of internal rhythms, predictive motor control, and error detection during synchronized movements., (© 2022. The Author(s).)

Comprehensive pharmacology education in nursing based on the "Patient-oriented Pharmacology" is effective against the improvement of quality of pharmacotherapy and patient satisfaction. Two active learning programs of practical pharmacotherapy for nursing students have been performed in School of Nursing, University of Miyazaki; (1) pharmacotherapy role-play for interprofessional education (IPE) and (2) practical excise for Kampo medicine. Pharmacotherapy role-play for IPE was performed as joint lecture both medical students and nursing students. This pharmacotherapy role-play is named Case & Communication based approach (C&C approach), since it is studied through communication between physicians, nurses and patients based on cases presented beforehand. In the practical excise for Kampo medicine, nursing students studied Kampo medicines and tried to taste 9 frequently used Kampo medicines. These active-learning programs in nursing pharmacology education may be effective for better understanding of pharmacotherapy and patient's feeling, and improvement of students' motivation as a nurse.

Pulsed radiofrequency (PRF) therapy is one of the most common treatment options for neuropathic pain, albeit the underlying mechanism has not been hitherto elucidated. In this study, we investigated the efficacy and mechanism of PRF therapy on resiniferatoxin (RTX)-induced mechanical allodynia, which has been used as a model of postherpetic neuralgia (PHN). Adult male rats were intraperitoneally injected with a vehicle or RTX. Furthermore, PRF current was applied on a unilateral sciatic nerve in all RTX-treated rats. On both ipsilateral and contralateral sides, the paw mechanical withdrawal thresholds were examined and L4-6 dorsal root ganglia (DRG) were harvested. In the DRG of rats with RTX-induced mechanical allodynia, Na V 1.7, a voltage-gated Na + channel, was upregulated following the enhancement of extracellular signal-regulated kinase phosphorylation. Early PRF therapy, which was applied 1 week after RTX exposure, suppressed this Na V 1.7 upregulation and showed an anti-allodynic effect; however, late PRF therapy, which was applied after 5 weeks of RTX exposure, failed to inhibit allodynia. Interestingly, late PRF therapy became effective after daily tramadol administration for 7 days, starting from 2 weeks after RTX exposure. Both early PRF therapy and late PRF therapy combined with early tramadol treatment suppressed Na V 1.7 upregulation in the DRG of rats with RTX-induced mechanical allodynia. Therefore, Na V 1.7 upregulation in DRG is related to the development of RTX-induced neuropathic pain; moreover, PRF therapy may be effective in the clinical management of patients with PHN via Na V 1.7 upregulation inhibition.

Genetic mutations in actin regulators have been emerging as a cause of cardiomyopathy, although the functional link between actin dynamics and cardiac contraction remains largely unknown. To obtain insight into this issue, we examined the effects of pharmacological inhibition of formins, a major class of actin-assembling proteins. The formin inhibitor SMIFH2 significantly enhanced the cardiac contractility of isolated frog hearts, thereby augmenting cardiac performance. SMIFH2 treatment had no significant effects on the Ca 2+ sensitivity of frog muscle fibers. Instead, it unexpectedly increased Ca 2+ concentrations of isolated frog cardiomyocytes, suggesting that the inotropic effect is due to enhanced Ca 2+ transients. In contrast to frog hearts, the contractility of mouse cardiomyocytes was attenuated by SMIFH2 treatment with decreasing Ca 2+ transients. Thus, SMIFH2 has opposing effects on the Ca 2+ transient and contractility between frog and mouse cardiomyocytes. We further found that SMIFH2 suppressed Ca 2+ -release via type 2 ryanodine receptor (RyR2); this inhibitory effect may explain the species differences, since RyR2 is critical for Ca 2+ transients in mouse myocardium but absent in frog myocardium. Although the mechanisms underlying the enhancement of Ca 2+ transients in frog cardiomyocytes remain unclear, SMIFH2 differentially affects the cardiac contraction of amphibian and mammalian by differentially modulating their Ca 2+ handling., (© 2021 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

The cerebellum is thought to have a variety of functions because it developed with the evolution of the cerebrum and connects with different areas in the frontoparietal cortices. Like neurons in the cerebral cortex, those in the cerebellum also exhibit strong activity during planning in addition to the execution of movements. However, their specific roles remain elusive. In this article, we review recent findings focusing on preparatory activities found in the primate deep cerebellar nuclei during tasks requiring deliberate motor control and temporal prediction. Neurons in the cerebellum are active during anti-saccade preparation and their inactivation impairs proactive inhibitory control for saccades. Experiments using a self-timing task show that there are mechanisms for tracking elapsed time and regulating trial-by-trial variation in timing, and that the cerebellum is involved in the latter. When predicting the timing of periodic events, the cerebellum provides more accurate temporal information than the striatum. During a recently developed synchronized eye movement task, cerebellar nuclear neurons exhibited periodic preparatory activity for predictive synchronization. In all cases, the cerebellum generated preparatory activity lasting for several hundred milliseconds. These signals may regulate neuronal activity in the cerebral cortex that adjusts movement timing and predicts the timing of rhythmic events., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

Sequential movements are often grouped into several chunks, as evidenced by the modulation of the timing of each elemental movement. Even during synchronized tapping with a metronome, we sometimes feel subjective accent for every few taps. To examine whether motor segmentation emerges during synchronized movements, we trained monkeys to generate a series of predictive saccades synchronized with visual stimuli which sequentially appeared for a fixed interval (400 or 600 ms) at six circularly arranged landmark locations. We found two types of motor segmentations that featured periodic modulation of saccade timing. First, the intersaccadic interval (ISI) depended on the target location and saccade direction, indicating that particular combinations of saccades were integrated into motor chunks. Second, when a task-irrelevant rectangular contour surrounding three landmarks ("inducer") was presented, the ISI significantly modulated depending on the relative target location to the inducer. All patterns of individual differences seen in monkeys were also observed in humans. Importantly, the effects of the inducer greatly decreased or disappeared when the animals were trained to generate only reactive saccades (latency >100 ms), indicating that the motor segmentation may depend on the internal rhythms. Thus, our results demonstrate two types of motor segmentation during synchronized movements: one is related to the hierarchical organization of sequential movements and the other is related to the spontaneous grouping of rhythmic events. This experimental paradigm can be used to investigate the underlying neural mechanism of temporal grouping during rhythm production., Competing Interests: The authors have declared that no competing interests exist.

Changes in the shape and size of the dendritic spines are critical for synaptic transmission. These morphological changes depend on dynamic assembly of the actin cytoskeleton and occur differently in various types of neurons. However, how the actin dynamics are regulated in a neuronal cell type-specific manner remains largely unknown. We show that Fhod3, a member of the formin family proteins that mediate F-actin assembly, controls the dendritic spine morphogenesis of specific subpopulations of cerebrocortical pyramidal neurons. Fhod3 is expressed specifically in excitatory pyramidal neurons within layers II/III and V of restricted areas of the mouse cerebral cortex. Immunohistochemical and biochemical analyses revealed the accumulation of Fhod3 in postsynaptic spines. Although targeted deletion of Fhod3 in the brain did not lead to any defects in the gross or histological appearance of the brain, the dendritic spines in pyramidal neurons within presumptive Fhod3-positive areas were morphologically abnormal. In primary cultures prepared from the Fhod3-depleted cortex, defects in spine morphology were only detected in Fhod3 promoter-active cells, a small population of pyramidal neurons, and not in Fhod3 promoter-negative pyramidal neurons. Thus, Fhod3 plays a crucial role in dendritic spine morphogenesis only in a specific population of pyramidal neurons in a cell type-specific manner., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Molecular recognition to preorganize noncovalently polymerizable supramolecular complexes is a characteristic process of natural supramolecular polymers, and such recognition processes allow for dynamic self-alteration, yielding complex polymer systems with extraordinarily high efficiency in their targeted function. We herein show an example of such molecular recognition-controlled kinetic assembly/disassembly processes within artificial supramolecular polymer systems using six-membered hydrogen-bonded supramolecular complexes (rosettes). Electron-rich and poor monomers are prepared that kinetically coassemble through a temperature-controlled protocol into amorphous coaggregates comprising a diverse mixture of rosettes. Over days, the electrostatic interaction between two monomers induces an integrative self-sorting of rosettes. While the electron-rich monomer inherently forms toroidal homopolymers, the additional electrostatic interaction that can also guide rosette association allows helicoidal growth of supramolecular copolymers that are comprised of an alternating array of two monomers. Upon heating, the helicoidal copolymers undergo a catastrophic transition into amorphous coaggregates via entropy-driven randomization of the monomers in the rosette.

Moving objects are often occluded behind larger, stationary objects, but we can easily predict when and where they reappear. Here, we show that the prediction of object reappearance is subject to adaptive learning. When monkeys generated predictive saccades to the location of target reappearance, systematic changes in the location or timing of target reappearance independently altered the endpoint or latency of the saccades. Furthermore, spatial adaptation of predictive saccades did not alter visually triggered reactive saccades, whereas adaptation of reactive saccades altered the metrics of predictive saccades. Our results suggest that the extrapolation of motion trajectory may be subject to spatial and temporal recalibration mechanisms located upstream from the site of reactive saccade adaptation. Repetitive exposure of visual error for saccades induces qualitatively different adaptation, which might be attributable to different regions in the cerebellum that regulate learning of trajectory prediction and saccades.

Oxaliplatin is the first-line chemotherapy for metastatic colorectal cancer. Unlike other platinum anticancer agents, oxaliplatin does not result in significant renal impairment and ototoxicity. Oxaliplatin, however, has been associated with acute and chronic peripheral neuropathies. Despite the awareness of these side-effects, the underlying mechanisms are yet to be clearly established. Therefore, in this study, we aimed to understand the factors involved in the generation of chronic neuropathy elicited by oxaliplatin treatment. We established a rat model of oxaliplatin-induced neuropathic pain (4 mg kg-1 intraperitoneally). The paw withdrawal thresholds were assessed at different time-points after the treatment, and a significant decrease was observed 3 and 4 weeks after oxaliplatin treatment as compared to the vehicle treatment (4.4 ± 1.0 vs. 16.0 ± 4.1 g; P < 0.05 and 4.4 ± 0.7 vs. 14.8 ± 3.1 g; P < 0.05, respectively). We further evaluated the role of different mitogen-activated protein kinases (MAPKs) pathways in the pathophysiology of neuropathic pain. Although the levels of total extracellular signal-regulated kinase (ERK) 1/2 in the dorsal root ganglia (DRG) were not different between oxaliplatin and vehicle treatment groups, phosphorylated ERK (p-ERK) 1/2 was up-regulated up to 4.5-fold in the oxaliplatin group. Administration of ERK inhibitor PD98059 (6 μg day-1 intrathecally) inhibited oxaliplatin-induced ERK phosphorylation and neuropathic pain. Therefore, upregulation of p-ERK by oxaliplatin in rat DRG and inhibition of mechanical allodynia by an ERK inhibitor in the present study may provide a better understanding of intracellular molecular alterations associated with oxaliplatin-induced neuropathic pain and help in the development of potential therapeutics., Competing Interests: The authors have declared that no competing interests exist.

Neural tube closure requires apical constriction during which contraction of the apical F-actin network forces the cell into a wedged shape, facilitating the folding of the neural plate into a tube. However, how F-actin assembly at the apical surface is regulated in mammalian neurulation remains largely unknown. We report here that formin homology 2 domain-containing 3 (Fhod3), a formin protein that mediates F-actin assembly, is essential for cranial neural tube closure in mouse embryos. We found that Fhod3 is expressed in the lateral neural plate but not in the floor region of the closing neural plate at the hindbrain. Consistently, in Fhod3-null embryos, neural plate bending at the midline occurred normally, but lateral plates seemed floppy and failed to flex dorsomedially. Because the apical accumulation of F-actin and constriction were impaired specifically at the lateral plates in Fhod3-null embryos, we concluded that Fhod3-mediated actin assembly contributes to lateral plate-specific apical constriction to advance closure. Intriguingly, Fhod3 expression at the hindbrain was restricted to neuromeric segments called rhombomeres. The rhombomere-specific accumulation of apical F-actin induced by the rhombomere-restricted expression of Fhod3 was responsible for the outward bulging of rhombomeres involving apical constriction along the anteroposterior axis, as rhombomeric bulging was less prominent in Fhod3-null embryos than in the wild type. Fhod3 thus plays a crucial role in the morphological changes associated with neural tube closure at the hindbrain by mediating apical constriction not only in the mediolateral but also in the anteroposterior direction, thereby contributing to tube closure and rhombomere segmentation, respectively., (© 2019 Sulistomo et al.)

The formin family proteins have the ability to regulate actin filament assembly, thereby functioning in diverse cytoskeletal processes. Fhod3, a cardiac member of the family, plays a crucial role in development and functional maintenance of the heart. Although Fhod1, a protein closely-related to Fhod3, has been reported to be expressed in cardiomyocytes, the role of Fhod1 in the heart has still remained elusive. To know the physiological role of Fhod1 in the heart, we disrupted the Fhod1 gene in mice by replacement of exon 1 with a lacZ reporter gene. Histological lacZ staining unexpectedly revealed no detectable expression of Fhod1 in the heart, in contrast to intensive staining in the lung, a Fhod1-containing organ. Consistent with this, expression level of the Fhod1 protein in the heart was below the lower limit of detection of the present immunoblot analysis with three independent anti-Fhod1 antibodies. Homozygous Fhod1-null mice did not show any defects in gross and histological appearance of the heart or upregulate fetal cardiac genes that are induced under stress conditions. Furthermore, Fhod1 ablation did not elicit compensatory increase in expression of other formins. Thus, Fhod1 appears to be dispensable for normal development and function of the mouse heart, even if a marginal amount of Fhod1 is expressed in the heart., (© 2019 Wiley Periodicals, Inc.)

Synchronized movements with external periodic rhythms, such as dancing to a beat, are commonly observed in daily life. Although it has been well established that some vocal learning species (including parrots and humans) spontaneously develop this ability, it has only recently been shown that monkeys are also capable of predictive and tempo-flexible synchronization to periodic stimuli. In our previous study, monkeys were trained to make predictive saccades for alternately presented visual stimuli at fixed stimulus onset asynchronies (SOAs) to obtain a liquid reward. The monkeys generalized predictive synchronization to novel SOAs in the middle of trained range, suggesting a capacity for tempo-flexible synchronization. However, it is possible that when encountering a novel tempo, the monkeys might sample learned saccade sequences from those for the short and long SOAs so that the mean saccade interval matched the untrained SOA. To eliminate this possibility, in the current study we tested monkeys on novel SOAs outside the trained range. Animals were trained to generate synchronized eye movements for 600 and 900-ms SOAs for a few weeks, and then were tested for longer SOAs. The accuracy and precision of predictive saccades for one untrained SOA (1200 ms) were comparable to those for the trained conditions. On the other hand, the variance of predictive saccade latency and the proportion of reactive saccades increased significantly in the longer SOA conditions (1800 and 2400 ms), indicating that temporal prediction of periodic stimuli was difficult in this range, similar to previous results on synchronized tapping in humans. Our results suggest that monkeys might share similar synchronization mechanisms with humans, which can be subject to physiological examination in future studies.

Mutations in cardiac myosin-binding protein C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C has been considered to regulate the cardiac function via cross-bridge arrangement at the C-zone of the myosin-containing A-band, the mechanism by which cMyBP-C functions remains unclear. We identified formin Fhod3, an actin organizer essential for the formation and maintenance of cardiac sarcomeres, as a cMyBP-C-binding protein. The cardiac-specific N-terminal Ig-like domain of cMyBP-C directly interacts with the cardiac-specific N-terminal region of Fhod3. The interaction seems to direct the localization of Fhod3 to the C-zone, since a noncardiac Fhod3 variant lacking the cMyBP-C-binding region failed to localize to the C-zone. Conversely, the cardiac variant of Fhod3 failed to localize to the C-zone in the cMyBP-C-null mice, which display a phenotype of hypertrophic cardiomyopathy. The cardiomyopathic phenotype of cMyBP-C-null mice was further exacerbated by Fhod3 overexpression with a defect of sarcomere integrity, whereas that was partially ameliorated by a reduction in the Fhod3 protein levels, suggesting that Fhod3 has a deleterious effect on cardiac function under cMyBP-C-null conditions where Fhod3 is aberrantly mislocalized. Together, these findings suggest the possibility that Fhod3 contributes to the pathogenesis of cMyBP-C-related cardiomyopathy and that Fhod3 is critically involved in cMyBP-C-mediated regulation of cardiac function via direct interaction., Competing Interests: The authors declare no conflict of interest.

Cardiac development and function require actin-myosin interactions in the sarcomere, a highly organized contractile structure. Sarcomere assembly mediated by formin homology 2 domain-containing 3 (Fhod3), a member of formins that directs formation of straight actin filaments, is essential for embryonic cardiogenesis. However, the role of Fhod3 in the neonatal and adult stages has remained unknown. Here, we generated floxed Fhod3 mice to bypass the embryonic lethality of an Fhod3 knockout (KO). Perinatal KO of Fhod3 in the heart caused juvenile lethality at around day 10 after birth with enlarged hearts composed of severely impaired myofibrils, indicating that Fhod3 is crucial for postnatal heart development. Tamoxifen-induced conditional KO of Fhod3 in the adult heart neither led to lethal effects nor did it affect sarcomere structure and localization of sarcomere components. However, adult Fhod3 -deleted mice exhibited a slight cardiomegaly and mild impairment of cardiac function, conditions that were sustained over 1 year without compensation during aging. In addition to these age-related changes, systemic stimulation with the α1-adrenergic receptor agonist phenylephrine, which induces sustained hypertension and hypertrophy development, induced expression of fetal cardiac genes that was more pronounced in adult Fhod3 -deleted mice than in the control mice, suggesting that Fhod3 modulates hypertrophic changes in the adult heart. We conclude that Fhod3 plays a crucial role in both postnatal cardiac development and functional maintenance of the adult heart., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Gene therapy refers to a rapidly growing field of medicine in which genes are introduced into the body to treat or prevent diseases. Although a variety of methods can be used to deliver the genetic materials into the target cells and tissues, modified viral vectors represent one of the more common delivery routes because of its transduction efficiency for therapeutic genes. Since the introduction of gene therapy concept in the 1970s, the field has advanced considerably with notable clinical successes being demonstrated in many clinical indications in which no standard treatment options are currently available. It is anticipated that the clinical success the field observed in recent years can drive requirements for more scalable, robust, cost effective, and regulatory-compliant manufacturing processes. This review provides a brief overview of the current manufacturing technologies for viral vectors production, drawing attention to the common upstream and downstream production process platform that is applicable across various classes of viral vectors and their unique manufacturing challenges as compared to other biologics. In addition, a case study of an industry-scale cGMP production of an AAV-based gene therapy product performed at 2,000 L-scale is presented. The experience and lessons learned from this largest viral gene therapy vector production run conducted to date as discussed and highlighted in this review should contribute to future development of commercial viable scalable processes for vial gene therapies.

When waiting for a traffic light or dancing to a musical beat, we unconsciously keep track of elapsed time and precisely predict the timing of forthcoming sensory events. Temporal monitoring and prediction are integral to our daily life, and are regulated by neuronal processes through multiple global networks involving the frontoparietal cortices, the basal ganglia and the cerebellum. These processes are also known to be influenced by a variety of internal state and neuromodulators. Here, we review recent advance of research in the field.

Predictive and tempo-flexible synchronization to an auditory beat is a fundamental component of human music. To date, only certain vocal learning species show this behaviour spontaneously. Prior research training macaques (vocal non-learners) to tap to an auditory or visual metronome found their movements to be largely reactive, not predictive. Does this reflect the lack of capacity for predictive synchronization in monkeys, or lack of motivation to exhibit this behaviour? To discriminate these possibilities, we trained monkeys to make synchronized eye movements to a visual metronome. We found that monkeys could generate predictive saccades synchronized to periodic visual stimuli when an immediate reward was given for every predictive movement. This behaviour generalized to novel tempi, and the monkeys could maintain the tempo internally. Furthermore, monkeys could flexibly switch from predictive to reactive saccades when a reward was given for each reactive response. In contrast, when humans were asked to make a sequence of reactive saccades to a visual metronome, they often unintentionally generated predictive movements. These results suggest that even vocal non-learners may have the capacity for predictive and tempo-flexible synchronization to a beat, but that only certain vocal learning species are intrinsically motivated to do it.

Fhod3 is a cardiac member of the formin family proteins that play pivotal roles in actin filament assembly in various cellular contexts. The targeted deletion of mouse Fhod3 gene leads to defects in cardiogenesis, particularly during myofibrillogenesis, followed by lethality at embryonic day (E) 11.5. However, it remains largely unknown how Fhod3 functions during myofibrillogenesis. In this study, to assess the mechanism whereby Fhod3 regulates myofibrillogenesis during embryonic cardiogenesis, we generated transgenic mice expressing Fhod3 selectively in embryonic cardiomyocytes under the control of the β-myosin heavy chain (MHC) promoter. Mice expressing wild-type Fhod3 in embryonic cardiomyocytes survive to adulthood and are fertile, whereas those expressing Fhod3 (I1127A) defective in binding to actin die by E11.5 with cardiac defects. This cardiac phenotype of the Fhod3 mutant embryos is almost identical to that observed in Fhod3 null embryos, suggesting that the actin-binding activity of Fhod3 is crucial for embryonic cardiogenesis. On the other hand, the β-MHC promoter-driven expression of wild-type Fhod3 sufficiently rescues cardiac defects of Fhod3-null embryos, indicating that the Fhod3 protein expressed in a transgenic manner can function properly to achieve myofibril maturation in embryonic cardiomyocytes. Using the transgenic mice, we further examined detailed localization of Fhod3 during myofibrillogenesis in situ and found that Fhod3 localizes to the specific central region of nascent sarcomeres prior to massive rearrangement of actin filaments and remains there throughout myofibrillogenesis. Taken together, the present findings suggest that, during embryonic cardiogenesis, Fhod3 functions as the essential reorganizer of actin filaments at the central region of maturating sarcomeres via the actin-binding activity of the FH2 domain.

Attention may select objects or perceptual groups as fundamental units. Previous studies with event-related potentials (ERPs) have found that obligatory attention-spreading over spatial regions within stable objects is associated with intermediate feedforward visual processing, as reflected by the posterior N1 component of the ERP at a latency of 140-180 ms. The present study examined object-based spatial attention effects in response to individual objects, by recording lateralized ERP attention effects over the posterior scalp (i.e., contralateral versus ipsilateral to the attended visual fields). The stumuli were bilateral unfilled line objects with minimal figural enhancement, and their connectedness and the difficulty of the perceptual task were manipulated. The effects of spatial attention on successive ERP components (N1, P2, and N2) provided information on the timing of the different stages of processing that underlie the formation of perceptual objects.

Attention directed to a part of an object tends to obligatorily spread over all of the spatial regions that belong to the object, which may be critical for rapid object-recognition in cluttered visual scenes. Previous studies have generally used simple rectangles as objects and have shown that attention spreading is reflected by amplitude modulation in the posterior N1 component (150-200 ms poststimulus) of event-related potentials, while other interpretations (i.e., rectangular holes) may arise implicitly in early visual processing stages. By using modified Kanizsa-type stimuli that provided less ambiguity of depth ordering, the present study examined early event-related potential spatial-attention effects for connected and separated objects, both of which were perceived in front of (Experiment 1) and in back of (Experiment 2) the surroundings. Typical P1 (100-140 ms) and N1 (150-220 ms) attention effects of ERP in response to unilateral probes were observed in both experiments. Importantly, the P1 attention effect was decreased for connected objects compared to separated objects only in Experiment 1, and the typical object-based modulations of N1 were not observed in either experiment. These results suggest that spatial attention spreads over a figural object at earlier stages of processing than previously indicated, in three-dimensional visual scenes with multiple depth cues.

It has been suggested that uniform connectedness is the most fundamental factor in forming units of attentional selection, while there are evidences that attention can select a perceptual group that consists of separate elements with similar features. The present study examined the effects of connectedness and a boundary-feature similarity on early spatial-selection processes using a sustained-focal-attention paradigm of event-related potentials (ERPs). Bilateral stimuli were manipulated to have an orthogonal combination of connectedness (C-, C+) and a similarity in boundary feature (S-, S+). ERPs were recorded from 15 participants who were instructed to pay attention to the left or the right visual field and to respond to a target shape that appeared infrequently in the attended field. The ERP attention effect in the N1 latency range (125-185 ms) was decreased for stimuli with connectedness and/or boundary-feature similarity, and the effects of the two grouping factors were independent of each other. The present result suggests that multiple grouping factors, including connectedness, operate in parallel in early processes of object-based attention-spreading.

Superoxide production by Nox1, a member of the Nox family NAPDH oxidases, requires expression of its regulatory soluble proteins Noxo1 (Nox organizer 1) and Noxa1 (Nox activator 1) and is markedly enhanced upon cell stimulation with phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC). The mechanism underlying PMA-induced enhancement of Nox1 activity, however, remains to be elucidated. Here we show that, in response to PMA, Noxo1 undergoes phosphorylation at multiple sites, which is inhibited by the PKC inhibitor GF109203X. Among them, Thr341 in Noxo1 is directly phosphorylated by PKC in vitro, and alanine substitution for this residue reduces not only PMA-induced Noxo1 phosphorylation but also PMA-dependent enhancement of Nox1-catalyzed superoxide production. Phosphorylation of Thr341 allows Noxo1 to sufficiently interact with Noxa1, an interaction that participates in Nox1 activation. Thus phosphorylation of Noxo1 at Thr341 appears to play a crucial role in PMA-elicited activation of Nox1, providing a molecular link between PKC-mediated signal transduction and Nox1-catalyzed superoxide production. Furthermore, Ser154 in Noxo1 is phosphorylated in both resting and PMA-stimulated cells, and the phosphorylation probably participates in a PMA-independent constitutive activity of Nox1. Ser154 may also be involved in protein kinase A (PKA) mediated regulation of Nox1; this serine is the major residue that is phosphorylated by PKA in vitro. Thus phosphorylation of Noxo1 at Thr341 and at Ser154 appears to regulate Nox1 activity in different manners., Structured Digital Abstract: Noxo1 binds to p22phox by pull down (1, 2, 3) Noxo1 binds to Noxo1 by pull down (View interaction) Noxa1 binds to Noxo1 by pull down (1, 2, 3, 4, 5)., (© 2013 FEBS.)

Background: Dilated cardiomyopathy (DCM) is characterized by a dilated left ventricular cavity with systolic dysfunction manifested by heart failure. It has been revealed that mutations in genes for cytoskeleton or sarcomere proteins cause DCM. However, the disease-causing mutations can be found only in far less than half of patients with a family history, indicating that there should be other disease genes for DCM. Formin homology 2 domain containing 3 (FHOD3) is a sarcomeric protein expressed in the heart that plays an essential role in sarcomere organization during myofibrillogenesis. The purpose of this study was to explore a possible novel disease gene for DCM., Methods and Results: We analyzed 48 Japanese familial DCM patients for mutations in FHOD3, and a missense variant, Tyr1249Asn, which was predicted to modify the 3D structure and damage protein function, was found in a case with adult-onset DCM. Functional studies revealed that the DCM-associated mutation significantly reduced the ability to induce actin dynamics-dependent activation of serum response factor, although no remarkable change in the cellular localization was induced in neonatal rat cardiomyocytes transfected with a mutant construct of FHOD3., Conclusions: The DCM-associated FHOD3 variant may cause DCM by interfering with actin filament assembly.

Heart development requires organized integration of actin filaments into the sarcomere, the contractile unit of myofibrils, although it remains largely unknown how actin filaments are assembled during myofibrillogenesis. Here we show that Fhod3, a member of the formin family of proteins that play pivotal roles in actin filament assembly, is essential for myofibrillogenesis at an early stage of heart development. Fhod3(-/-) mice appear normal up to embryonic day (E) 8.5, when the developing heart, composed of premyofibrils, initiates spontaneous contraction. However, these premyofibrils fail to mature and myocardial development does not continue, leading to embryonic lethality by E11.5. Transgenic expression of wild-type Fhod3 in the heart restores myofibril maturation and cardiomyogenesis, which allow Fhod3(-/-) embryos to develop further. Moreover, cardiomyopathic changes with immature myofibrils are caused in mice overexpressing a mutant Fhod3, defective in binding to actin. These findings indicate that actin dynamics, regulated by Fhod3, participate in sarcomere organization during myofibrillogenesis and thus play a crucial role in heart development.

The isolated right half (RH) or left half (LH) of Xenopus embryos can undergo regulation so as to form well-proportioned larvae. To assess how the combined actions of maternal determinants and cell-cell interactions contribute to form the well-proportioned larvae, we quantitatively compared four-cell stage blastomere fate between normal larvae and regulated larvae from RH embryos. In normal larvae, the clones of the right dorsal blastomere (RD) and right ventral blastomere (RV) were located unilaterally. In contrast, in regulated larvae: (i) the RD clone exclusively occupied the anterior endomesoderm (AE) derivatives, coinciding no RV progeny in those derivatives of normal larvae. The clone bilaterally populated tissues along the dorsal midline, which characteristically included the medial regions of both somites adjoining the notochord, with higher percentages on the right and anterior sides. (ii) The RV clone extensively compensated for the missing left side at the expense of its right side contribution, and bilaterally occupied the ventroposterior and also dorsal regions excluding the AE derivatives. This clone considerably populated, with altered orientations, the derivatives of the left half gastrocoel roof plate (GRP), the left half GRP being essential for laterality determination. These results show that the high cell-autonomy in the AE constitutes a mechanism common to both normal and regulative development. In regulated larvae, cell-cell interactions shifted the midlines on the dorsal side slightly and the ventral side to a greater extent. The cell lineage difference in the left half GRP could result in a different utilization of maternal determinants in that area., (© 2012 The Authors Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.)

Attention selects objects/groups as the most fundamental units, and this may be achieved by an attention-spreading mechanism. Previous event-related potential (ERP) studies have found that attention-spreading is reflected by a decrease in the N1 spatial attention effect. The present study tested whether the electrophysiological attention effect is associated with the perception of object unity or amodal completion through the use of partly-occluded objects. ERPs were recorded in 14 participants who were required to pay attention to their left or right visual field and to press a button for a target shape in the attended field. Bilateral stimuli were presented rapidly, and were separated, connected, or connected behind an occluder. Behavioral performance in the connected and occluded conditions was worse than that in the separated condition, indicating that attention spread over perceptual object representations after amodal completion. Consistently, the late N1 spatial attention effect (180-220 ms post-stimulus) and the early phase (230-280 ms) of feature selection effects (target N2) at contralateral sites decreased, equally for the occluded and connected conditions, while the attention effect in the early N1 latency (140-180 ms) shifted most positively for the occluded condition. These results suggest that perceptual organization processes for object recognition transiently modulate spatial and feature selection processes in the visual cortex., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The formin family proteins play pivotal roles in actin filament assembly via the FH2 domain. The mammalian formin Fhod3 is highly expressed in the heart, and its mRNA in the adult heart contains exons 11, 12, and 25, which are absent from non-muscle Fhod3 isoforms. In cultured neonatal cardiomyocytes, Fhod3 localizes to the middle of the sarcomere and appears to function in its organization, although it is suggested that Fhod3 localizes differently in the adult heart. Here we show, using immunohistochemical analysis with three different antibodies, each recognizing distinct regions of Fhod3, that Fhod3 localizes as two closely spaced bands in middle of the sarcomere in both embryonic and adult hearts. The bands are adjacent to the M-line that crosslinks thick myosin filaments at the center of a sarcomere but distant from the Z-line that forms the boundary of the sarcomere, which localization is the same as that observed in cultured cardiomyocytes. Detailed immunohistochemical and immuno-electron microscopic analyses reveal that Fhod3 localizes not at the pointed ends of thin actin filaments but to a more peripheral zone, where thin filaments overlap with thick myosin filaments. We also demonstrate that the embryonic heart of mice specifically expresses the Fhod3 mRNA isoform harboring the three alternative exons, and that the characteristic localization of Fhod3 in the sarcomere does not require a region encoded by exon 25, in contrast to an essential role of exons 11 and 12. Furthermore, the exon 25-encoded region appears to be dispensable for actin-organizing activities both in vivo and in vitro, albeit it is inserted in the catalytic FH2 domain.

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91(phox) serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91(phox) requires the cytosolic proteins p67(phox), p47(phox), and Rac (a small GTPase). p67(phox), comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47(phox) and there interacts with Rac; these processes are prerequisite for gp91(phox) activation. Here we show that a region of p67(phox) (amino acids 190-200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67(phox) to support superoxide production by gp91(phox)-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91(phox)-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67(phox) interaction with the gp91(phox) NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67(phox) (amino acids 190-210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91(phox).

Actin filament assembly in nonmuscle cells is regulated by the actin polymerization machinery, including the Arp2/3 complex and formins. However, little is known about the regulation of actin assembly in muscle cells, where straight actin filaments are organized into the contractile unit sarcomere. Here, we show that Fhod3, a myocardial formin that localizes to thin actin filaments in a striated pattern, regulates sarcomere organization in cardiomyocytes. RNA interference-mediated depletion of Fhod3 results in a marked reduction in filamentous actin and disruption of the sarcomeric structure. These defects are rescued by expression of wild-type Fhod3 but not by that of mutant proteins carrying amino acid substitution for conserved residues for actin assembly. These findings suggest that actin dynamics regulated by Fhod3 are critical for sarcomere organization in striated muscle cells.

The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox-p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production. p47phox translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for Ile152 in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr153 also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile155) of the p47phox homologue Noxo1 (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47phox, however, does not affect its interaction with p22phox or with p67phox. Consistent with this, a mutant p47phox (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of p67phox and Rac normally occurs in p47phox (I152A)-expressing cells. Thus the Ile152-containing region of p47phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.

Reactive oxygen species (ROS) have been attracting attention as mediators of various cell-signaling pathways. Nox-family NADPH oxidases have proven to be a major source of ROS production in various cell types and have crucial roles in various physiological and pathological processes. In this study, we show that Nox4, a member of Nox family, is prominently expressed in various neuroepithelial tumors by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical studies. We quantified Nox4 mRNA expression by real-time PCR in tumor specimens from 58 patients with astrocytomas and found that the expression levels of Nox4 mRNA in glioblastomas (WHO grade IV) were significantly higher than those in other astrocytomas (WHO grade II and III). In addition, we show that specific knockdown of Nox4 expression by RNA interference results in cell-growth inhibition and enhances induction of apoptosis by chemotherapeutic agents, such as cisplatin, in cultured glioma cell lines. Based on these observations, enhanced expression of Nox4 appears to be involved in cell proliferation and survival in glioma cells., ((c) 2008 Wiley-Liss, Inc.)

Formin-family proteins, in the active state, form actin-based structures such as stress fibres. Their activation mechanisms, however, are largely unknown except that mDia and its closely related formins can be activated by direct binding of the small GTPase Rho or Cdc42. Here we show that the Rho-dependent protein kinase ROCK phosphorylates the C-terminal residues Ser1131, Ser1137, and Thr1141 of formin homology domain protein 1 (FHOD1), a major endothelial formin that is normally autoinhibited by intramolecular interaction between the N- and C-terminal regions. Phosphorylation of FHOD1 at the three residues fully disrupts the autoinhibitory interaction, which culminates in formation of stress fibres. We also demonstrate that, in vascular endothelial cells, thrombin, a vasoactive substance leading to Rho activation, elicits both FHOD1 phosphorylation and stress fibre formation in a ROCK-dependent manner, and that FHOD1 depletion by RNA interference impairs thrombin-induced stress fibre formation. Based on these findings we propose a novel mechanism for activation of formin-family proteins: ROCK, activated by G protein-coupled receptor ligands such as thrombin, directly phosphorylates FHOD1 at the C-terminal region, which renders this formin in the active form, leading to stress fibre formation.

The superoxide-producing phagocyte NADPH oxidase is activated during phagocytosis to destroy ingested microbes. The adaptor protein p40phox associates via the PB1 domain with the essential oxidase activator p67phox, and is considered to function by recruiting p67phox to phagosomes; in this process, the PX domain of p40phox binds to phosphatidylinositol 3-phosphate [PtdIns(3)P], a lipid abundant in the phagosomal membrane. Here we show that the PtdIns(3)P-binding activity of p40phox is normally inhibited by the PB1 domain both in vivo and in vitro. The crystal structure of the full-length p40phox reveals that the inhibition is mediated via intramolecular interaction between the PB1 and PX domains. The interface of the p40phox PB1 domain for the PX domain localizes on the opposite side of that for the p67phox PB1 domain, and thus the PB1-mediated PX regulation occurs without preventing the PB1-PB1 association with p67phox.