By Pamela D. Winfield November 5, 2024 The professorship brings scholars from around the world to teach courses on Buddhism Winfield is currently serving as the 2024 Numata / Bukkyō Dendō Kyōkai visiting professor of Buddhist studies at McGill University in Montréal she is teaching a graduate level seminar on Zen and visual/material culture and pursuing research for her next book Winfield also delivered the keynote address for the annual Premodern Japanese Religions Conference at McGill University “Materiality as Method: How to Do Things with Zen” launched the conference SUNY Albany and High Point University shared their latest research at the intersection of Buddhist studies and visual/material culture Tagged: Elon University Professor of History Michael Matthews explores the romantic and sexual lives of Mexico City’s poor and working class during the rule of Porfirio Diaz The 6th annual Maker Takeover featured students’ experiments business and creative works to showcase the hard work taking place in Elon University’s Maker Hub Nia James says when doctors weren’t taking her pain seriously her athletic trainer and Elon alum John Lavender ’94 was her lifesaving advocate The graduate of Elon University’s human services studies program works on policy advocacy in Washington in a prestigious fellowship with the Religious Action Center of Reform Judaism Get more Elon news delivered to your inbox © 2025 Elon University | All Rights Reserved The SeaWolves are pleased to announce that applications are now being accepted for the 2025 Chace Numata Scholarship the SeaWolves Community Fund awarded 52 scholarships in memory of late SeaWolves catcher Chace Numata The SeaWolves Community Fund will once again award a minimum of $5,000 in scholarships to senior student-athletes participating in varsity baseball or softball Honorees will be selected based on their accomplishments applicants must meet the following criteria: The deadline to apply for the 2025 Chace Numata scholarship is February 15, 2025. Additional scholarship information, details on how to submit and past winners can be found here: https://www.milb.com/erie/community/numata-scholarship Those wishing to donate to the Chace Numata Scholarship can send checks payable to the SeaWolves Community Fund to: SeaWolves Community FundATTN: Numata Scholarship831 French StreetErie Additionally, you can support the scholarship by purchasing a Chace Numata T-shirt with the hashtag #LiveLikeNumi on the back. All proceeds from shirt sales benefit the Chace Numata Scholarship. T-shirts are available in both yellow and red and can be purchased here. ABOUT CHACE NUMATAChace Numata was a switch-hitting catcher with the Erie SeaWolves the Double-A affiliate of the Detroit Tigers He passed away during the 2019 baseball season at the age of 27 "Numi" played 10 professional seasons in the Phillies Yankees and Tigers organizations and was a leader on and off the field He was a talented baseball player and a world-class teammate It didn't matter if you were a professional athlete or a young child and his fun-loving personality could light up any environment The SeaWolves Community Fund will award a minimum of $5,000 in scholarships annually to senior student-athletes participating in varsity baseball or softball and their positive impact on the lives of others ABOUT THE SEAWOLVES COMMUNITY FUNDThe SeaWolves Community Fund is a 501(c)(3) non-profit organization committed to improving the lives of children and adults in the greater Erie region The SeaWolves Community Fund places a special emphasis on supporting youth programs focused on baseball and wellness by developing partnerships with those who share in our mission Each Chace Numata Scholarship is funded in part through individual and corporate donations to the SeaWolves Community Fund Yoshizawa Ryoichi is the third-generation owner of a 100-year-old finger joint shop Finger jointing is a traditional Japanese technique of assembling furniture and fixtures without nails or metal fittings Yoshizawa combines this finger jointing with lacquer work to create “lacquerware,” challenging himself to create new products that value human connections who founded “Yoshizawa Shibori Shop” in Numata City located in the northern part of Gunma Prefecture Yoshizawa sought to enhance the wood grain of his pieces by incorporating lacquer techniques he spent a year apprenticing at a lacquer shop in Tokyo’s Higashi-Mukojima district which involves applying raw lacquer to the wood and then wiping it off with cloth a method commonly used in woodworking to beautifully highlight the grain of the wood He applied lacquer to the woodworking pieces made by his father and himself As he was reflecting on his approach to his work and preparing to take over the family business as the third-generation owner This disaster made Yoshizawa realize the importance of human connections prompting him to reflect on his relationships with past customers and begin asking himself how he wanted to approach his work and what kind of craftsmanship he aspired to create moving forward “When my son was in his third year of high school ‘What do you plan to do in the future?’ He replied ‘I want to take over the family fingerboard shop.’ That one sentence made me think he was willing to work with me It made me want to create various things together who was considering ways to rebuild relationships with customers his son’s declaration to take over the family business inevitably changed his perspective on work With many of his regular customers growing older he also felt anxious about whether his son could continue doing good work in the future He wondered if it was better to continue making pieces for his regular customers in the same way he always had or if he should use the remaining years of his craftsmanship to create pieces freely Yoshizawa began searching for a way to build a future together with his son What he arrived at was a “process-oriented approach to craftsmanship” that values dialogue with those involved Yoshizawa says that he feels the greatest joy when creating works freely unbound by the traditional framework of finger joinery His shift from a craftsman focused on preserving tradition to a creator who enjoys expression has become a new charm of Yoshizawa Finger Joinery leading to the acquisition of new customers Yoshizawa revisited lacquer as a medium to create works freely unbound by the constraints of traditional woodworking techniques Lacquer has the role of organically connecting various materials I freely attach various materials using lacquer.” and even items like pottery and iron coated with lacquer he also began seriously experimenting with “colored lacquer,” which involves mixing pigments into lacquer to create various colors “Lacquerware crafts are often referred to as ‘lacquerware,’ but since ‘lacquerware’ refers to the work of lacquer artisans I prefer to call my work ‘lacquer craft.’ I value both woodworking and lacquer work equally so I refer to my work as ‘lacquer craft.’” The intersection of woodworking and lacquer connects various regional elements into works of art These connections then link people to one another Yoshizawa is very selective about who he works with he is increasingly involved in collaborative projects with chefs and architects from Japan and abroad where he makes proposals as a woodworker and works together with them to create something “Rather than being asked to make something specific I’m getting more jobs where I’m asked to join a project and come up with something interesting (laughs).” Yoshizawa’s approach to creating things involves having extensive conversations with clients to capture their desired image and then bringing it to life through creative ideas he is able to connect with people he truly wants to work with Who made that?’ rather than explaining techniques or skills I’m really enjoying being able to do that kind of work right now.” Yoshizawa’s encounter with a chef who inspired him to focus his work on specific targets was a pivotal moment in his career It all began when Chef Kotaro Noda of “bistrot64,” a restaurant in Italy that has earned two Michelin stars asked a friend to guide him around the Tone Numata area in search of ingredients and had accumulated knowledge over the years the conversation led to the idea of Chef Noda hosting a one-day dining event at a ski resort in Minakami Town Yoshizawa’s work was used as tableware and was highly praised by Chef Noda The conversation continued about creating something new together and when Chef Noda became the executive chef at Ginza Shiseido’s “FARO,” he commissioned Yoshizawa to create lacquerware boxes for serving dishes The creative world of a restaurant where Italian and Japanese cultures overlap Discussing how to design the tableware that would play a part in this world the time spent exploring a worldview that only the two of them could create was incredibly enjoyable Meeting Chef Noda inspired me to narrow my focus which has led to opportunities to create dishes that enhance various types of cuisine I now receive requests from chefs of different genres and also seek advice from them expanding my repertoire of dishes I can create “Creating and using tableware is about not overshadowing the dish itself and to highlight the chef’s creativity Finding that balance is the most important and challenging aspect of my work.” I don’t find much interest in pieces that overemphasize traditional craftsmanship making the “traditional” aspect too prominent “I should have done this a little differently.” When I can strike that middle ground I realize that my clients are very creative Whether they are Western or Japanese chefs they understand their own style and are seeking new ways to express themselves in exciting ways.” To narrow down the target audience while still meeting truly interesting people he holds an exhibition called “Autumn in the Sake Brewery” once a year gathering craftspeople from within and outside the prefecture in an old sake brewery she invites chefs to prepare daily changing lunches and dinners allowing participants to experience the interplay between food and tableware thereby gaining a deeper understanding of their use and feel Even chefs who have since earned Michelin stars continue to participate Minakami Town in Gunma Prefecture is known for its heavy snowfall Within this town lies the Fujiwara district where a traditional wooden tray called the “Fujiwara Bon” was once crafted This tray features a wooden surface carved with radial patterns using a chisel The craft began in the mid-Edo period as a way to utilize the abundant local timber and generate income during the winter months Some of the pieces were even presented to the imperial family and are now coveted by antique enthusiasts who had seen the Fujiwara Bons in his youth initially found them unappealing and thought they were outdated crafts that had fallen out of favor he began to see their beauty and launched a revival project as he approached his 60th birthday Yoshizawa feels that people who engage in craftsmanship tend to value the first-person perspective he creates opportunities to think in the second and third persons fostering a collective discussion about new challenges beyond the third-person perspective through interactions with others “I believe that without carefully observing old things and interpreting their beauty in your own way There are many techniques from the past that are no longer practiced today but can serve as references.” He incorporates old and traditional elements as one of his expressive methods blending them with images derived from conversations with clients rooted in traditional techniques and modern sensibilities harmoniously combines a sense of coolness that is both intuitive and timeless Yoshizawa has had more opportunities to think and talk about “food and agriculture.” New encounters have led to interesting interactions with people and connections with many stimulating individuals I want to have conversations that expand their world as someone who is older I feel a growing fear that the people who can broaden my world are gradually disappearing.” He emphasizes that interacting with people who can make the unknown world interesting and broaden one’s perspective is absolutely essential for someone who creates things When traditional techniques and inspiration from peers come together within Yoshizawa another fascinating work unlike anything seen before will undoubtedly emerge The Divinity School is pleased to announce that Matthew T Numata Visiting Professor Emeritus of Buddhist Studies has been awarded The 2024 Toshihide Numata Book Award for his recent book Tibetan Manuscripts and Early Printed Books 1: Elements (Cornell University Press The Toshihide Numata Book Award in Buddhism is presented on an annual basis to an outstanding book in the area of Buddhist studies The Award is administered by the Center for Buddhist Studies at the University of California Members of this year’s selection committee had high praise for the volume and depth of research by Kapstein and his team of collaborators and for the lavish production of the volume which includes copious high-quality illustrations One evaluator called it “a stunning contribution… Nothing even close has ever before been attempted making the success of this volume all the more impressive.” Another said it was “an essential reference work for the next generation of scholars of Tibetan Studies And a third concurred: “a must-read for anyone interested in ‘the history of the book’ whether in the Tibetan area or elsewhere.” Kapstein and an international team of specialists provide a comprehensive introduction to the material and aesthetic features of the wide range of Tibetan books described in detail and illustrated with copious full-color photographs and explore the major categories of traditional Tibetan books introducing their specific features and the main approaches to their study Kapstein is Professor Emeritus in the division of Religious Studies of the École Pratique des Hautes Études in Paris and Numata Visiting Professor Emeritus of Buddhist Studies at the University of Chicago Divinity School He was formerly director of the Tibetan Studies research team of the Center for the Study of East Asian Civilizations (Centre de recherche sur les civilisations de l’Asie orientale) His past publications include: The Tibetan Assimilation of Buddhism: Conversion Reason’s Traces: Identity and Interpretation in Indian and Tibetan Buddhist Thought (2001) The Rise of Wisdom Moon (Clay Sanskrit Library 2009) and the edited volumes The Presence of Light: Divine Radiance and Religious Experience (2004) Buddhism Between Tibet and China (2009) Japan-based biochemist Ali Malay has long questioned traditional theories about how spiders make their silk “The prevailing hypotheses seemed like magic you suddenly have these ordered structures appearing out of liquid precursors,” he says Known for its unmatched relative strength and flexibility spider silk has long eluded efficient synthetic replication in part because it has been challenging to replicate the complex transition from liquid precursors to highly structured fibres Malay is a scientist in the research group of Keiji Numata at the Japanese national research centre RIKEN in Wako near Tokyo whose work focuses on biopolymers such as spider silk The group has developed an alternative theory for how silk forms inside spiders This has enabled them to do something that had previously eluded scientists — create a system that mimics how spiders spin silk and is capable of mass-producing artificial spider silk fibres Central to this breakthrough is their partnership with Spiber Inc. a biotech startup based in Yamagata and specializing in nature-inspired structural proteins they are producing polymers from biomolecules instead of chemicals derived from fossil fuels a grant from the Japanese government sparked a pivotal collaboration between Spiber and Numata’s group Together they began investigating ways to replicate spider silk through a deeper understanding of the complex proteins called ‘spidroins’ that make up this material such as designing protein sequences to control physical properties,” says Hiroyuki Nakamura a researcher affiliated with Spiber and Keio University we aimed to understand how spiders naturally control these properties providing us with valuable insights for designing our artificial structural proteins.” By analysing spider silk from more than 1,000 species Keiji Numata’s team has a clearer idea about what gives the silk its unique properties.Credit: Daniela Duncan/Moment/Getty As part of these efforts, the RIKEN and Spiber teams were instrumental in elucidating the structures and physical properties of spider silk from more than 1,000 spider species across the world1 they analysed spider RNA to determine the amino-acid sequences of spider-silk proteins which is a particularly strong variety used for the radial lines in webs and also when spiders suspend themselves by a thread known for its toughness and mechanical usefulness allowed them to link properties such as tensile strength extensibility and toughness with specific sequences of amino acids This large-scale genomic and phenotypic mapping has resulted in the publicly accessible Spider Silkome Database (https://spider-silkome.org) a platform for understanding the evolutionary diversity and functional potential of spider silks The complexity among spider silks is fascinating “It prompts us to question why nature diversifies these proteins and it suggests that each variation has a unique function There’s a hidden language that we’re just beginning to tap into.” The researchers discovered that multiple components contribute to the mechanical properties of spider dragline silk2 Previous studies had identified two proteins which has short glycine-rich regions and stretches of multiple alanines that form strong crystalline domains vital for silk’s tensile strength which shares these motifs but also includes additional sequences that boost the silk’s extensibility and elasticity advances in proteomics and genomics have unveiled a more nuanced composition identifying three additional proteins: MaSp3 and spider-silk constituting element (SpiCE) Other molecular constituents found in spider silk also contribute to the overall functionality of silk But simply identifying spidroins is only part of the challenge of producing artificial spider silk The other lies in replicating the intricate transformation from a highly concentrated liquid into a fibre with a multilevel structure on the nanolevel As the silk dope travels along the long ducts within the spider it undergoes significant structural changes due to pH gradients and twisting geometric shifts that create shear forces and elongation The complexity of the process is compounded by dehydration and other factors all occurring simultaneously in a narrow space Using knowledge gained from the spider silkome database the team constructed a platform that creates new genetic material by combining DNA from different sources to investigate how different structural configurations affect fibre formation in dragline silk including versions with intact terminal and central repetitive domains as well as constructs lacking specific domains The researchers made a key discovery when phosphate ion additions to MaSp2 constructs initiated phase separation between two liquids This process concentrated the protein into dense phases crucial for its transformation from liquid to solid silk fibres MaSp2’s repetitive domain combines both water-attracting and water-repelling properties which is essential for this phase transition “These proteins naturally possess a hierarchical structure achieved through self-assembly encoded in their genetic and amino-acid sequences,” explains Malay This is not just a theoretical possibility — it’s a reality and an exciting step forward in biomaterials.” the real challenge lies in scaling up from benchtop to factory which is where Spiber’s expertise comes into play the company has developed various protein polymers including many with amino-acid sequences not found in nature Spiber refers to these innovative products as brewed protein materials Produced via a fermentation process using sugars and customized microbes brewed protein fibres offer a sustainable alternative to animal-derived materials such as wool The innovation has attracted significant attention in various industries including the apparel industry and secured over JPY 10 billion (US$64 million) in their latest funding to aid mass-production efforts Spiber’s unique approach combines advanced molecular design with an efficient way to feedback experimental measurements “This allows us to modify proteins or nucleotide sequences and immediately evaluate fibre productivity and quality,” he says The company also has a small industrial pilot plant which allows them to “quickly scale up candidates.” The researchers at RIKEN and Spiber are discussing ambitious plans to scale up their spider-silk production aiming to increase their production capacity from the few hundred milligrams currently made in the lab to much larger quantities silk produced by marine purple photosynthetic bacteria from airborne nitrogen and carbon dioxide for future sustainable materials in collaboration with Symbiobe Inc. we produced 100 tonnes of recombinant protein for artificial structural protein fibres at our plant in Thailand,” says Nakamura “We can quickly prototype products emerging from academic research on structural proteins This collaboration is propelling our entire industry forward.” Download references Volume 2 - 2022 | https://doi.org/10.3389/finsc.2022.1100671 Cooperative gating of localized ion channels ranges from fine-tuning excitation–contraction coupling in muscle cells to controlling pace-making activity in the heart Membrane deformation resulting from muscle contraction activates stretch-activated (SA) cation channels The subsequent Ca2+ influx activates spatially localized Ca2+-sensitive K+ channels to fine-tune spontaneous muscle contraction To characterize endogenously expressed intermediate conductance Ca2+-activated potassium (IK) channels and assess the functional relevance of the extracellular Ca2+ source leading to IK channel activity we performed patch-clamp techniques on cricket oviduct myocytes and recorded single-channel data we first investigated the identification of IK channels that could be distinguished from endogenously expressed large-conductance Ca2+-activated potassium (BK) channels by adding extracellular Ba2+ The single-channel conductance of the IK channel was 62 pS and its activity increased with increasing intracellular Ca2+ concentration but was not voltage-dependent These results indicated that IK channels are endogenously expressed in cricket oviduct myocytes the Ca2+ influx pathway that activates the IK channel was investigated The absence of extracellular Ca2+ or the presence of Gd3+ abolished the activity of IK channels we investigated the proximity between SA and IK channels administration of Ca2+ to the microscopic region in a pipette and application of membrane stretching stimulation increased SA channel activity Membrane stretch-induced SA and IK channel activity were positively correlated the emergence of IK channel activity and its increase in response to membrane mechanical stretch was not observed without Ca2+ in the pipette These results strongly suggest that IK channels are endogenously expressed in cricket oviduct myocytes and that IK channel activity is regulated by neighboring SA channel activity functional coupling between SA and IK channels may underlie the molecular basis of spontaneous rhythmic contractions Mammals and invertebrates use the combinatorial function of numerous ion channels expressed in muscle cells to control the electrical properties of membranes Elucidating their respective roles is essential to understanding the rhythmic contraction mechanisms produced by muscle tissue As a model system for the investigation of these problems, we used striated muscle cells from the oviduct of crickets, building on previous pioneering work in insects, such as fruit flies and locusts (1–16) the molecular physiological mechanisms still need to be fully understood Drosophila melanogaster is an important model organism for genetics and molecular biology and has accumulated genetic information, including KCa channels, for more than 100 years. The KCa channel gene family consists of two groups, the BK (dSlo, SLO2) group and the SK group (dSK), which includes IK (33, 34) Mutant phenotypes of BK channels are often associated with negative regulation of ganglionic synapses, and dSlo mutants play a role in neuronal function, abnormal circadian activity, and locomotor disorders (35–37). In flight muscles, the mutant disrupts the homeostatic adjustments in neural circuits at the flight initiation (38, 39) The molecularphysiological importance of IK channels in oviduct muscle provides new insights into muscle contraction mechanisms in addition to flight and body wall muscles we characterized endogenously expressed IK channels in the cricket oviduct we investigated Ca2+-mediated functional coupling of SA and IK channels involved in forming rhythmic hyperpolarization Gryllus bimaculatus used in the experiments were sexually mature females purchased from a local pet store as food for pet reptiles genetic and environmental variability in the specimens were limited The crickets were housed in plastic containers with cardboard shelters until further analysis All crickets were bred at 27 ± 2°C Crickets were provided ad libitum access to feed and water for insects (I The isolated single lateral oviduct myocytes were maintained in fresh saline at room temperature (23-27°C) and used within 12 h the tips of the electrodes were first filled with the normal pipette solution then backfilled with the same solution containing a concentration of its inhibitors; a wait period of at least 10 min was ensured before data recording Gd3+ was dissolved in water to prepare stock solutions All inhibitor reagents used in the experiment were purchased from Sigma-Aldrich Corp All other reagents were purchased from Wako Pure Chemical Industries Figure 1 Ba2+-insensitive intermediate conductance single-K+ channel currents in isolated cricket oviduct cells (A) Two types of single K+ channel current recordings from cell-attached patches under extracellular K+ concentration of 140 mM are shown Representative single-channel current traces of large conductance Ca2+-activated potassium (BK) channels (left column) and intermediate conductance Ca2+-activated K+ channel (IK) channels are shown when held at +40 mV Notes at the BK channel currents are sensitive to extracellular (inside the pipette) 1 mM Ba2+ (1Ba) (B) Representative single-channel current traces at various membrane potentials (Vm) are shown in the figure Current activity does not exhibit voltage sensitivity (C) Average single-channel current (i)-Vm relationship (n = 6–12) Data points for Vm from −60 mV to +60 mV were fitted by linear regression yielding slope conductance values of 59.7 and 61.5 pS for the control and 1Ba Cells isolated from a total of 153 animals were used in the experiment and 259 tests were performed for data collection Figure 2 Single K+ channel I-V relationship at various extracellular K+ concentrations (A) Mean linear current-voltage (I-V) data at various extracellular K+ concentrations ([K+]o in mM): ● (B) Data show a semi-logarithmic plot of [K+]o against the difference between the reversal potential (⊿Erev) obtained at 140 mM and each K+ concentration Reversal potential (Erev) was obtained by fitting the i-Vm relationship for each K+ concentration in A by linear regression The slope of the regression line was 58.37 mV/decade Cells isolated from a total of 53 animals were used in the experiment and 121 tests were performed for data collection All data are expressed as means ± standard error of mean (SEM) We accumulated data for each condition from at least three independent experiments and trials used in each experiment are described in the figure legend Statistical significance was evaluated by Student’s t-test for comparisons between two mean values to assess statistical significance using Excel (Microsoft Corp. USA) or Origin 8 (OriginLab Corp.) software The data used for statistical analysis passed the Shapiro-Wilk normality test and the Levene equal variance test least-squares linear regression was performed using Excel (Microsoft Corp.) A P-value of <0.05 was considered significant The top trace in Figure 1A shows single-channel activity with large and medium conductance in the steady state of oviduct myocytes. Single-channel currents in the left column in K+-rich recording conditions are consistent with our previous observations (11) As shown in the upper traces of Figure 1A, both large (BK) and intermediate-sized single-K+ channel currents (IK) were observed either singly or multiples by trial when the membrane was held at +40 mV. During the course of this experiment, however, we newly found that the activity of this BK channel disappeared when 1 mM Ba2+ was added extracellularly. Thus, the extracellular addition of Ba2+ enabled selective recordings of the IK channels (Figure 1A this study performed recordings using Ba2+ in a pipette to eliminate BK channel activity IK currents recorded in patch membranes not coexisting with BK did not affect the slope conductance values (59.7 pS Figure 3 Intracellular Ca2+ concentration dependence of K+ channel activation (A) Representative traces of single K+ channel currents recorded under various intracellular Ca2+ concentration ([Ca2+]i) conditions at Vm = 60 mV from excised inside-out patches (B) Average mean open probability (NPO)-[Ca2+]i relationship of steady-state single-channel current The data fit the Hill equation with EC50 of 88.5 ± 7.8 μM and Hill coefficient of 0.9 Cells isolated from a total of 14 animals were used in the experiment and 41 tests were performed for data collection Activation of Ca2+-dependent IK channels in excitable cells is critical for feedback control of both Ca2+ influx and cell excitability (27) To investigate the effect of Ca2+ on IK channel activity we examined the effect of extracellular Ca2+ removal on IK channel activity We further investigated the proximity of IK and SA channels within microdomains by observing the effect of membrane stretch on IK channel activity Figure 4 Effect of Ca2+ removal from the bath solution and Gd3+ application to the bath solution on IK channel activity B) (left) Representative IKchannel currents before (control) and after Ca2+ removal from the bath solution (Ca2+-free) or after 30 mM Gd3+ application to the bathsolution The current was recorded from cell-attached patches at −140 mV B) (right) Average NPO of IK channel current *Significantly different (P < 0.05) from control values Cells isolated from a total of 15 animals were used in the experiment and 25 tests were performed fordata collection even if the suction was further increased to −60 cm H2O but IK channel activity was not observed (n = 12) Figure 5 Effect of membrane stretch on IK channel activity B) Representative stretch-activated (SA) and IK channel currents were recorded at −140 mV from cell-attached mode (n = 12) Recordings were obtained after changing to a Ca2+-free bath solution The pipette contains Ca2+ in A [Ca2+(+)] but does not contain Ca2+ in B [Ca2+(−)] 1–4 show enlargements in lower and upper traces Cells isolated from a total of 23 animals were used in the experiment and 43 tests were performed for data collection Figure 6 Activity relationship between SA and IK due to membrane stretch (A) Scatter plot of NPO (SA channel activity) was recorded at −140 mV versus membrane suction (n = 48) (B) Scatter plot of NPO (IK channel activity) recorded at −140 mV versus membrane suction (n = 40) B) The values for each relationship were obtained by fitting them with linear regression The slopes obtained from plots A and B revealed 0.026 and 0.019 cm H2O and analyses of the correlation function revealed values of 0.84 and 0.87 from the values of A and B (C) Scatterplots of mean NPO versus membrane suction (n = 7) for SA and IK The correlation coefficient between SA and IK was 0.99 Cells isolated from a total of 83 animals were used in the experiment and 108 tests were performed for data collection KCa is classified into three types according to the magnitude of conductance. Intermediate conductance calcium-activated potassium (IK) channels have a range of 20–85 pS between BK and SK channels (22–24) BK channels have activity defined by both voltage and intracellular Ca2+ whereas small and moderate KCa channels are sensitive to changes in intracellular Ca2+ and are activated in a voltage-independent manner the low intracellular Ca2+ sensitivity of IK channels presented in this study may be due to the loss of CaM performed in the inside-out mode This study performed simultaneous single current measurements of SA and IK channels with a small tip within sub-μm of a patch pipette in cell connection mode, directly demonstrating their proximity (Figures 5, 6). From these observations, IK channels were sufficiently characterized to construct microdomains in this study (27) that a time lag of ~30 s was observed between SA channel activity and increased IK channel activity This delay implies that the global Ca2+ rise in cells required for IK channel activity involves Ca2+ release from intracellular stores For KCa and voltage-gated calcium channel (CaV), the concept of 1:1 stoichiometry evaluates proximity due to physical interactions. However, it has been suggested that the stoichiometry of physical interaction and functional coupling is not congruent (27). Figure 6 demonstrates that the activation ratio of SA : IK channel was 3:2 when the strength of mechanical membrane stretching exceeded 20 cm H2O The results are the first to demonstrate functional measurements with two single-channel activities and provide quantitative activity ratios in function providing insights into computational science the characterization of single IK channels in oviduct myocytes enabled us to investigate their functional relevance to Ca2+ sources IK channels functionally coupled with rhythmic contraction-producing SA channels form a driving force for increased Ca2+ influx with periodic hyperpolarization We propose that cricket muscle cells are involved in spontaneous contraction via the IK and SA channel microdomains The original contributions presented in the study are included in the article/supplementary materials Further inquiries can be directed to the corresponding authors TN and MY: conceptualization and design of study TN and KS-N: performing experiments and analyses All authors contributed to the article and approved the submitted version This work was supported in part by Grants in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science and the Ministry of Education 21K06792 and JP20H05842 (Grant-in-Aid for Transformative Research Areas This work was also financially supported by the Naito Foundation Shu Fukuoka and Yoshino Lab members for their active support in cricket rearing The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher large-conductance Ca2+-activated potassium channel; CaK 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January 2023 Copyright © 2023 Numata, Sato-Numata and Yoshino. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Tomohiro Numata, bnVtYXRhQG1lZC5ha2l0YS11LmFjLmpw; Masami Yoshino, bXlvc2hpQHUtZ2FrdWdlaS5hYy5qcA== Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish New research published in npj Sustainable Agriculture reports that biomass made from the purple photosynthetic marine bacterium Rhodovulum sulfidophilum is an excellent nitrogen fertilizer Led by Keiji Numata from the RIKEN Center for Sustainable Resource Science (CSRS) and Kyoto University the study shows that this biomass is as effective as common inorganic synthetic fertilizers but avoids several side effects that harm the environment Increasing agricultural output using nitrogen-rich fertilizers is a common practice that is expected to increase in the coming decades as global food demands rise the ecologically harmful side effects of overusing common inorganic nitrogen fertilizers are also expected to skyrocket including substantial greenhouse gas emissions organic fertilizers such as compost and manure have much less nitrogen and must therefore be used in greater amounts for the same effect on plant growth as well as more carbon dioxide and nitrous oxide byproducts in the long run The Biomacromolecules Research Team at RIKEN CSRS has been searching for a natural source of nitrogen that can replace ammonia-based synthetic fertilizers and help prevent a future crisis Purple non-sulfur bacteria (PNSBs) are known to have enzymes that help them take nitrogen from the atmosphere and incorporate it into proteins no one has tested their effectiveness as fertilizers To create a PNSB fertilizer for the new study sulfidophilum and generated dried biomass from the released cellular material Analysis showed that the nitrogen content of the PNSB fertilizer was 11% by weight which is much higher than what is found in other organic fertilizers including biomass made from other microbes or microalgae The researchers compared how well the Japanese mustard spinach komatsuna (Brassica rapa var perviridis) grew when assisted by either inorganic fertilizers or the new PNSB biomass fertilizer The first major finding was that the mustard spinach could indeed absorb nitrogen from the dried biomass Further experiments showed that the biomass fertilizer boosted plant growth just as well as the nitrogen-rich inorganic fertilizers did even when the biomass fertilizer contained up to four times the amount of nitrogen similar to soil fertilized without any nitrogen The PNSB biomass fertilizer has a low carbon to nitrogen ratio and the nitrogen is released for plant use relatively slowly compared with inorganic fertilizers—about 60% in 30 days Although this means that twice as much biomass fertilizer will be needed for similar crop growth the big upside is that carbon dioxide and nitrous oxide emissions will be lower and less nitrogen will be dumped into the environment through leaching “In the long term this could revolutionize agriculture and mitigate its negative impact on the environment,” says Morey-Yagi Although the basic experiments prove the effectiveness of the biomass fertilizer the authors stress that results are preliminary and other factors will eventually need to be taken into consideration “A life-cycle assessment of this fertilizer will be essential for evaluating its environmental footprint across production how to scale-up the biomass production process must be considered The researchers are optimistic that these challenges will be met and that their discovery will help make fertilizers more eco-friendly also help prevent future problems with supply-chain distribution of inorganic fertilizers Because the biomass fertilizer is produced using carbon dioxide and nitrogen from the air they have named it Air Fertilizer® and have registered it for use as an organic fertilizer in Japan as a product marketed by Symbiobe Inc Adam Phillips RIKEN International Affairs Division Tel: +81-(0)48-462-1225 Email: adam.phillips [at] riken.jp Japanese mustards spinach grown in two inorganic nitrogen fertilizers (C1 and C2) Plants were grown at either 15°C – 25°C (Cool Fertilizer treatments were: no fertilizer (NF) and 4 represent an added amount of fertilizer corresponding to the amount of N in C1 Top Experience a stunning fireworks and music extravaganza up close Numata Fireworks offers an awe-inspiring showcase of state-of-the-art pyrotechnics blending seamlessly with musical rhythms to create an unforgettable sensory delight Featuring over 10,000 fireworks in a 45-minute spectacle Numata Fireworks promises a visual feast that will captivate onlookers The choreographed display dances across the night sky painting vibrant colors and mesmerizing patterns It’s an experience guaranteed to leave you with cherished memories of Japan Enjoy delicious Japanese street food from numerous stalls before the fireworks begin From purple sweet potatoes to wild horse sightings here’s how to make the most of Okinawa’s tropical escape Explore the best local brands shaping Tokyo’s fashion scene From vintage clothing to Hawaiian-themed goods here’s what’s worth checking out before it’s gone From buying and registering a bike to key rules of the road Collagen staples for beauty and health in Japan Our handpicked list of the best events going on this month and the untold tales of Japan’s queer community Everything you need to know before and during a natural disaster Stay up to date with Tokyo news and events This article was published more than 6 months ago standing) Sarah Lamb and Yumi Numata (seated) decided to buy a house together in 2022 to avoid renting or each buying a condo with limited space.Thomas Bollmann they’ve found a path to owning a home in today’s torrid housing market the three Toronto residents purchased a duplex in Toronto’s West End converting the two units into space for three people to live and Yumi and Sarah lived together as roommates (The three all became friends through a volunteer project they were working on.) “None of us individually had enough money to buy a house and none of us wanted to invest in little condos that we wouldn’t actually want to live in,” Ms “We each put in different amounts for the down payment and contribute equally to the monthly mortgage payments,” Ms Co-owning a home with people who are not directly or romantically related is still a niche but it’s growing in popularity as housing prices continue to stick at high levels Lesli Gaynor, a realtor and former social worker and restaurant owner, co-founded Husmates a digital platform that helps prospective co-owners find matches for house-hunting “We have sold approximately $100-million worth of co-ownership properties since 2018 That represents 67 homes and 182 different people who now live together in co-ownership situations,” he says “I became a co-owner myself when I [became] a single mom and bought a house with two other women,” Ms Gosai and their platform require prospective co-owners to go through an extensive workbook to make sure that they really want to co-own and help them find the most suitable partners Lamb says she considers the co-owning of her duplex to be relatively straightforward as long as the co-owners agree on how the financial and living situation will work “In some ways it’s not much different than the way many people may own properties with other people We simply added an agreement among ourselves on how we’re going to manage the property and live,” she says Banks and financial institutions are starting to catch up to the idea of co-ownership To make sure they are all on the same page – literally – the three women developed a mission statement for their home our home is an opportunity to enter the real estate market and begin building equity We will create a home that balances flexibility and independence with accountability and community; showing up for one another in support of day-to-day needs as well as longer-term goals that relate to our home and community,” she says “We see co-ownership as both a social and financial solution for a lot of people,” Mr but it’s also an attractive alternative for younger people with shared values who seek a household and for older people who are looking for a way to age in place rather than in an institution,” he says “Banks and financial institutions are starting to catch up to the idea of co-ownership by offering fractional mortgages where different co-owners have different terms,” he adds Chartrand and Numata developed a mission statement that describes their home as 'a collective rooted in trust friendship and shared responsibility' and 'an opportunity to enter the real estate market and begin building equity.'Thomas Bollmann Lamb says getting housing insurance was tricky “We had to go to a number of companies before our broker found one that fit,” she says While there’s no central database showing how many homes are co-owned, it’s now common enough that realtors and websites are recognizing the growing interest. In addition to Husmates, Collaborative Home Ownership in British Columbia offers advice and helps connect people to prospective matches Some governments recognize co-ownership’s increasing popularity and to some extent consider it a way to address the housing affordability crisis the Ontario government published a co-ownership guide explaining different co-ownership options and listing the benefits of these arrangements as “affordability more efficient use of housing stock and community.” “Fresh approaches will give people more options and access to housing that is affordable,” the Ontario guide says Gaynor agree that prospective co-owners should be aware that there are different types of co-ownership arrangements Having a rich uncle’s name on the deed is not the same as the co-ownership arrangement Ms There are also different ways to be a co-owner – for example if one person dies the survivors might inherit that share or it might go to the deceased person’s estate The trio has worked out other scenarios – important for any good contract – like looking at what happens if they no longer want to be co-owners together “We used some templates from Husmates and developed a contract that includes scenarios such as making sure there’s time when someone wants to leave for the others to figure out what that looks like for them as remaining co-owners,” she says instruction to family members and emergency contacts are all in place to reflect the agreement.” Keeping a happy home requires a bit of work “We’re strategic about how we want to live together and earlier this week we spent a few hours cleaning up.” “We try to intentionally work on the relational aspects of co-ownership – living together – with practical shared decision making,” adds Ms “And we revisit our agreement from time to time to ensure that our vision continues to be aligned.” Report an editorial error Report a technical issue Editorial code of conduct Authors and topics you follow will be added to your personal news feed in Following Welcome to The Globe and Mail’s comment community. This is a space where subscribers can engage with each other and Globe staff. Non-subscribers can read and sort comments but will not be able to engage with them in any way. Click here to subscribe If you would like to write a letter to the editor, please forward it to letters@globeandmail.com. Readers can also interact with The Globe on Facebook and Twitter Welcome to The Globe and Mail’s comment community This is a space where subscribers can engage with each other and Globe staff We aim to create a safe and valuable space for discussion and debate If you do not see your comment posted immediately it is being reviewed by the moderation team and may appear shortly We aim to have all comments reviewed in a timely manner Comments that violate our community guidelines will not be posted UPDATED: Read our community guidelines here We have closed comments on this story for legal reasons or for abuse. For more information on our commenting policies and how our community-based moderation works, please read our Community Guidelines and our Terms and Conditions defeated Australian Lionel Rose by unanimous decision in 15 rounds defending the WBC super featherweight title at the Prefectural Gymnasium in Hiroshima The Judges were Nobumitsu Inukai and Toru Kimura Privacy Policy / Cookie Policy AKRON, Ohio – Chace Numata, a catcher for the Detroit Tigers' Double-A affiliate, the Erie SeaWolves, died in an Erie hospital on Monday from injuries he sustained in a skateboarding accident native was selected in the 14th round of the 2010 MLB draft by the Philadelphia Phillies He signed a minor league deal with the Tigers in November and spent time between Double-A and Triple-A this season.  [ Opinion: Chace Numata's death reminds us to 'Live like Numi' ] Called by teammates as "the heart and soul" of the SeaWolves Numata was known to keep the clubhouse vibrant and teammates laughing during the season “He’s a giver,” SeaWolves outfielder Cam Gibson Me and him will go to Taco Bell at midnight and he forces himself in to pay for it always joking and making light of everything He’s a bright light in everybody’s life I can’t tell you the times he’s had heart-to-heart talks with me this year talking to me about what my future holds Regardless if I’m doing well or doing bad He has more heart and more soul than anybody in this entire organization.” The circumstances of Numata’s accident suggest he was riding his skateboard home in the downtown Erie area shortly after 2 a.m Surveillance video from an insurance business nearby showed Numata riding alone down East 4th Street when his skateboard suddenly stopped “It appears he just fell on the skateboard and landed headfirst on the ground,” Stucke said “It appears he was unconscious as soon as he fell A motorist saw Numata in the middle of the road “Chace was beloved by many from our Major League club through all levels of our player development system," the Tigers said in a statement Monday. "He had an engaging personality that quickly established him as a leader on and off the field and his presence will be forever remembered in our organization and beyond Our thoughts and prayers are with his family [ Erie SeaWolves pray for catcher: God, bring him back to us ] After the Tigers' 4-3 loss to the Minnesota Twins on Monday manager Ron Gardenhire called Numata a "gamer" who "loved to play."  "We saw him in spring training with my staff," Gardenhire said "It’s really tough out there in the clubhouse right now The SeaWolves' season ended on Monday in Akron the club honored Numata by writing his nickname his favorite sayings on a pair of cleats and they played some of Numata’s favorite songs in the clubhouse & the Sunshine Band’s “Give It Up.” “It’s surreal,” Erie second baseman Kody Clemens said Monday SeaWolves hitting coach Brian Harper said the club's typically rowdy dugout was "real somber and kind of quiet" over the weekend Numata hit .244 with four home runs and 28 RBIs in 77 games in the minors this season he spent a season in the Yankees farm system and eight seasons in the Phillies organization.  A GoFundMe account was established to help pay for Numata's medical expenses More than 240 people had donated more than $20,000 as of Monday afternoon including several players from the Tigers organization "Everyone who was around Chace knew there was never a stranger in the room when he was there," Numata's family said in a statement Monday "It didn’t matter if you were a professional athlete or young child he made sure to make everyone feel comfortable and welcomed while his personality could light up any environment he would be in Our family would like to say Mahalo nui loa (thank you very much) to everyone who prayed for and acknowledged Chace during this difficult time We heard you and we appreciate you all from the bottom of our hearts!" Contact Kirkland Crawford: kcrawford@freepress.com. Follow him on Twitter @HiKirkHere Your browser does not support JavaScript, or it is disabled．Please check the site policy for more information National Report a specialty made with fermented bean paste spread inside a baguette will soon be on the fast track to the capital aboard a bullet train from here when a Shinkansen starts transporting the local treat to Tokyo Station Prepared by the Friand bakery and pastry shop in Numata The regular Miso Pan (bread with miso paste) is priced at 302 yen ($1.90) while the Miso Butter (bread with miso paste and butter) will be sold for 324 yen Thirty pieces of each flavor will be delivered on a bullet train that leaves Takasaki Station after noon The breads will be offered from around 2 p.m at the Nomono Tokyo-eki Gransta Marunouchi store outside the ticket gate on the basement floor of the station on Tuesdays Gunma specialty yaki manju gets new spin as baumkuchen JR East to offer mobile order service to deliver Choshi cuisine Rare chance to see VIP room at Nikko Station on 111th anniversary Craft beer brewers become deep thinkers at Gunma station Information on the latest cherry blossom conditions Please right click to use your browser’s translation function.) A series based on diplomatic documents declassified by Japan’s Foreign Ministry Here is a collection of first-hand accounts by “hibakusha” atomic bomb survivors chefs and others involved in the field of food introduce their special recipes intertwined with their paths in life A series about Japanese-Americans and their memories of World War II In-house News and Messages No reproduction or republication without written permission Please view the main text area of the page by skipping the main menu. The page may not be displayed properly if the JavaScript is deactivated on your browser Japanese version Atsushi Numata is the owner and chef of Ni Japanese Deli in the Lower East Side Numata got a taste for cooking in the kitchens of his grandmother’s restaurant Numata came to New York City in the late ’80s to be closer to another passion: hip-hop music With experience as a line chef of large restaurants in NYC and Tokyo he soon founded Ni Deli with his wife and business partner Atsushi focuses on traditional recipes with seasonal local ingredients in his allergy-free He is also a DJ/music producer who has released an album of beats Chef Atsushi Numata discusses what led him to opening his own restaurant and how cooking Japanese food is a lot like making hip-hop music Can you talk about why you came to New York an exclusive reservation-only private dining restaurant my brothers and I would spend our summer breaks there One of the prep crew looked after us and got us to help with prepping in the kitchen I practically grew up in my grandmother’s restaurant Later I was producing music and beats for hip hop artists I came to New York in 1988 because of the music Occasionally my chef friends here needed help so I would sweat it out in the kitchen or come up with recipes Soon I was getting offers to consult for restaurants I worked in big restaurants as a line chef Slowly but surely I pulled away from music production How did you end up running a Japanese kitchen My wife and I used to live in the East Village We had a catering business and always wanted our own shop When she was pregnant with our first child she’d walk around the Lower East Side a lot She was a frequent at Essex Street Market and befriended a number of vendors there she was told there was going to be a stall available Because Essex Street Market was run by a government-subsidized company the rent was pretty reasonable we thought running our own business would provide a more flexible pace The word Ni に means “Nippon,” as in Japan; but also means New York which perfectly summed up the duality of our situation—we were both native Japanese living and working in New York City Our first child had so many allergies that she almost couldn’t eat any regular food Soy is one of the main ingredients in classic Japanese foods—I mean soy sauce and beans—and my daughter was allergic to all that We wanted to preserve our tradition and culture and so we had to come up with new allergy-free and gluten-free recipes We realized there was a wider audience for it there was interest in allergy-free macrobiotic foods but none that was with traditional Japanese meals That became the core concept of Ni: to provide traditional Japanese foods that are vegan and allergy-free but at the same time updating the usual ingredients with local and seasonal ones With the many Latino vendors in the market there was no shortage of South American ingredients and produce traditionally Japanese cooking is about sourcing local seasonal ingredients and I still use time-honored methods of preparing the meal Experimentation and seasonal ingredients became central to our menu What was one difference in working in a big kitchen versus running your own space Japanese food doesn’t require much firepower we only have one burner and very little equipment American kitchen systems are more compartmentalized with almost no crossovers which is a good thing for maintaining the quality I know some American chefs that don’t show up until after the prep It also has a lot to do with the Qi in that space—the energy I have created from scratch Somehow being in the space resets me to the beginning; I get really humble Can you talk us through your daily process We come in the morning around 8:30am after dropping the kids off at school Sometimes I would go to the Green Market in Union Square before to check out the new seasonal ingredients Sometimes I dream about what I can make with a certain ingredient the following day So with new ingredients fresh from the Green Market every day It’s really about experimenting on the spot—it has to work before lunch The lunch is busy with many regular customers; we tend to pause to chat and catch up New customers are usually curious and want to know about traditional recipes With just five different elements that make up a bento box meal What I mean is that even though the food looks simple it is often made with layers of preparation and marination The tastes must be different yet complementary And of course the colors and textures must please the eye as much as the tongue there are many elements working in harmony to be successful Your body works like nature—it responds to the seasons In the summer you enjoy cucumbers and during the winter they take me back to my childhood of helping my grandmother in her kitchen I know exactly what to do because of those days My mother’s and grandmother’s recipes always come back to me but now I’m remixing them with my New York twists New York City is great with its many different cultures I like to think about how other chefs use different ingredients What would your advice be for someone who wants to get into this You can always gain experience after you open the business Learn everything about the business and plan your vision Map out how everything is supposed to work Our first winter was a disaster: no one wanted cold food But mistakes and challenges make us stronger many New York restaurants give up too quickly There’s a restaurant I used to go to in the East Village make sure you have enough capital to sustain the business for at least a year We have been very fortunate to have the freedom to do what we believe in I remember at first our Latino neighbors didn’t want to try our food sushi?” With a little education they realized what we do is healthy They have also seen our kids grow up on this food but eventually you will have your own audience Building your own audience takes time sometimes From DJing to running a business to raising a family My kids play a major role in keeping me from burning out Be passionate about what you do and keep things interesting for yourself I get really excited going to the Green Market to check out the new produce and interact with the farmers Farmers love and appreciate the feedback you offer Then returning to my kitchen with my finds It’s like the old days of crate digging in dusty record shops then returning to the studio to make good vibes with samples and beats Dr Brian Weiss Any works by pre new wave Japanese bands, for example Tokyo Rockers Lonely Boy by Steve Jones Nikkatsu movies, especially with Akira Kobayashi “Big Kotta” by K Dub Shine Here's a space to search our entire website Try typing something like "creative blocks" "green" or "blue" and our snail will find what you're looking for Detroit Tigers and baseball families have suffered a tragic loss.Minor League catcher Chace Numata has died after being hospitalized for injuries suffered in a skateboarding accident in Erie that morning bleeding from the head after suffering a skateboarding accident in downtown Erie He was hospitalized at UPMC Hamot before dying three days later.Numata was a 14th-round pick by the Phillies in the 2010 Draft coming out of a Hawaii high school He stayed in the Philadelphia pipeline through the 2017 season He was a mid-season All-Star for Phillies affiliates in 2013 (Class A Lakewood) and 2016 (Class A Advanced Clearwater).The switch-hitting backstop signed a Minor League deal with the Yankees prior to the 2018 campaign and spent that season playing for four affiliates before signing as a free agent with the Tigers in November 2018 He spent the bulk of the 2019 season with Double-A Erie playing 71 games for the SeaWolves and hitting .239/.291/.354 while helping to guide a promising pitching staff that included top prospects Casey Mize He made his Triple-A debut with Toledo on April 28 and spent six games with the Mud Hens this season.Erie players -- and others across the Minors -- wore hats with the name "NUMI" on them over the weekend in support of the backstop The SeaWolves will be wearing “NUMI” on their caps in support of their teammate Chace Numata. pic.twitter.com/zH3u6fsShN Tigers prospect Logan Shore started a GoFundMe campaign over the weekend to raise money for the Numata family to help pay for travel expenses and hospital bills it had raised more than $21,000 with many across leaving memories of respect for Numata on the page's comment section."The Erie SeaWolves are heartbroken and deeply saddened by the passing of Chace Numata," the Eastern League club said in its own statement and his personality was positive and infectious and he had a very positive impact throughout the SeaWolves organization and the Erie community We extend our sympathies to the Numata Ohana and have them in our thoughts and prayers Sam Dykstra is a reporter for MiLB.com. Follow and interact with him on Twitter, @SamDykstraMiLB The catcher suffered a head injury following a skateboarding accident the family of Erie SeaWolves catcher Chace Numata announced the heartbreaking news that the 27-year-old minor-leaguer had passed away Numata had experienced head injuries following a fall while skateboarding on Friday and his teammates from the SeaWolves and all through the Tigers organization had rallied around him His family released the following statement regarding his tragic passing: “It is with deep sorrow that the Numata Ohana announces that Chace Kekoa Kenji Numata Hawaii that was blessed with the opportunity to professionally play the sport he loved for the Phillies and Tigers organizations for over ten seasons Everyone who was around Chace knew there was never a stranger in the room when he was there It didn’t matter if you were a professional athlete or young child Our family would like to say Mahalo nui loa to everyone who prayed for We heard you and we appreciate you all from the bottom of our hearts It is not believed that an automobile was involved in Chace’s accident as nearby security camera footage showed him falling from his board in the early morning hours The SeaWolves had just played their last home game on Thursday evening SeaWolves owner Fernando Aguirre released a statement on behalf of the Double-A club “The Erie SeaWolves are heartbroken and deeply saddened by the passing of Chace Numata who signed Numata to a minor league deal last November issued the following statement upon learning of his passing “The Detroit Tigers are deeply saddened to learn of the passing of Chace Numata who was a catcher for our Double A affiliate in Erie Though this was his first year with our organization Chace was beloved by many from our Major League club through all levels of our player development system He had an engaging personality that quickly established him as a leader on and off the field We at Bless You Boys echo these sentiments sending our thoughts and prayers to the Numata family There is a GoFundMe that was established to help with Chace’s medical bills, and the funds raised will go towards helping the Numata family during this difficult time. Consider donating here Japanese version Metrics details offer a relevant opportunity to investigate the role of natural selection in shaping genetic dominance: the high frequency of heterozygotes at these loci has been shown to enable the evolution of dominance among alleles mimetic wing color variations are controlled by an inversion polymorphism of a circa 2 Mb genomic region (supergene P) with strong dominance between sympatric alleles To test how differences in dominance observed on wing patterns correlate with variations in expression levels throughout the supergene region we sequenced the complete transcriptome of heterozygotes at the prepupal stage and compared it to corresponding homozygotes By defining dominance based on non-overlapping ranges of transcript expression between genotypes we found contrasting patterns of dominance between the supergene and the rest of the genome; the patterns of transcript expression in the heterozygotes were more similar to the expression observed in the dominant homozygotes in the supergene region Dominance also differed among the three subinversions of the supergene suggesting possible epistatic interactions among their gene contents underlying dominance evolution We found the expression pattern of the melanization gene cortex located in the P-region to predict wing pattern phenotype in the heterozygote We also identify new candidate genes that are potentially involved in mimetic color pattern variations highlighting the relevance of transcriptomic analyses in heterozygotes to pinpoint candidate genes in non-recombining regions Both levels of gene and allele expression can trigger dominance in heterozygotes and be submitted to selection in polymorphic loci where heterozygotes have a high frequency We thus aim at understanding the molecular mechanisms involved in dominance between supergene alleles which are likely shaped by natural selection through mimicry The rearrangement P1 involves an event of inversion that spans over 400 kb and is associated with the phenotype bicoloratus P2 and P3 involve two supplementary inversions of roughly 0.2 and 1.2 Mb respectively and are associated here with the phenotype tarapotensis Analysis of transcriptomic variation could overcome such difficulties because changes in expression levels among genes within the inversion could more likely associate with phenotypic differences than to their physical linkage we compared gene expression in the homozygotes (Pbic/Pbic and Ptar/Ptar) and heterozygotes (Pbic/Ptar) and specifically tested for departure from additivity We focused our analyses only on transcripts where the range of expression levels did not overlap between Pbic/Pbic and Ptar/Ptar genotypes By focusing on genes with contrasted levels of transcript expression in the homozygotes we were thus able to compare patterns of dominance between the supergene and the rest of the genome our experiment does not cover the whole timeframe of wing development so that we cannot rule out that other genes involved in color pattern variations located in the P-supergene would be differentially expressed at other developmental stages not studied here including 4 individuals with the genotypes Pbic/Pbic Of the 53719 transcripts of this reference transcriptome (obtained from all transcripts of the 24 afore-mentioned H 51181 and 49808 were expressed in the Pbic/Pbic homozygotes Pbic/Ptar heterozygotes and Ptar/Ptar homozygotes To test whether the expression at the supergene in heterozygotes Pbic/Ptar matches the expression observed in the Pbic/Pbic homozygotes we compared patterns of expression between Pbic/Ptar heterozygotes and the two corresponding homozygotes in terms of gene level of expression (D) along the genome Two categories of transcripts were considered depending on the levels of expression in the Pbic/Pbic and Ptar/Ptar homozygotes (category 1: when Min(NPbic/Pbic) > Max(NPtar/Ptar) and category 2 when Min(NPtar/Ptar) > Max(NPtbic/ Pbic) The dominance score was assessed depending on the minimum and maximum number of transcripts observed in the different replicates (represented by the transparent boxes) of the same homozygous (in red and blue) and heterozygous (in purple) genotypes (see Methods) A score ranging from −3 to 3 was then given to each transcript following its class in respect to the phenotypically-dominant Pbic/Pbic homozygous genotype D = 3 when overdominance of the bic allele is observed when Min (NPbic/Ptar) > Max (Nhigh) in category 1 or when Max (NPbic/Ptar) < Min (Nlow) in category 2 D = 2 when strict dominance of the bic allele is observed when Min (NPbic/Ptar) > [(Min (Nhigh) + Max (Nlow)]/2 and Min (NPbic/Ptar) ≤ Max (Nhigh) in category 1 or when Max (NPbic/Ptar) < [(Min (Nhigh) + Max (Nlow)]/2 and Max (NPbic/Ptar) ≥ Min (Nlow) in category 2 D = 1 when incomplete dominance of the bic allele is observed when Max (NPbic/Ptar) ≤ Min (Nhigh) and Min (NPbic/Ptar) > [(Min (Nhigh) + Max (Nlow)]/2 in category 1 or when Min (NPbic/Ptar) ≥ Max (Nlow) and Max (NPbic/Ptar) < [(Min (Nhigh) + Max (Nlow)]/2 in category 2 D = 0 when codominance of the two alleles is observed when the range of expression levels observed in the heterozygotes overlap the midpoint of the range of expression observed in both homozygotes when incomplete recessiveness of the bic allele is observed when Min (NPbic/Ptar) ≥ Max (Nlow) and Max (NPbic/Ptar) < [(Min (Nhigh) + Max (Nlow)]/2 or when Max (NPbic/Ptar) ≤ Min (Nhigh) and Min (NPbic/Ptar) > [(Min (Nhigh) + Max (Nlow)]/2 in category 2 when strict recessivity of the bic allele is observed when Max (NPbic/Ptar) < [(Min (Nhigh) + Max (Nlow)]/2 and Max (NPbic/Ptar) ≥ Min (Nlow) or when Min (NPbic/Ptar) > [(Min (Nhigh) + Max (Nlow)]/2 and Min (NPbic/Ptar) ≤ Max (Nhigh) in category 2 when underdominance of the bic allele is observed when Max (NPbic/Ptar) < Min (Nlow) in category 1 or when Min (NPbic/Ptar) > Max (Nhigh) in category 2 Non-parametric statistics (chi2-test) were then used to test whether frequencies of those categories differed (1) between genomic regions (supergene vs rest of the genome) and (2) between transcripts where the level of expression was higher in Pbic/Pbic (category 1) vs genes with non-overlapping expression levels are enriched in the P inversion relatively to the rest of the genome (Chi2 = 62.52 2798 and 3057 transcripts are upregulated in Pbic/Pbic and Ptar/Ptar homozygotes respectively indicating a slight deviation toward up-regulation in Ptar/Ptar homozygotes (Chi2 = 11.45 56 and 39 transcripts are overexpressed in Pbic/Pbic and Ptar/Ptar homozygotes respectively whereas differences in the P inversion does not deviate from parity (Chi2 = 3.08 it contrasts with genome-wide pattern (Chi2 = 4.90 A closer examination of the three subinversions P1 P2 and P3 revealed substantial differences overexpression of transcripts is more frequent in Pbic/Pbic than in Ptar/Ptar (30 vs where 4 and 13 transcripts are overexpressed in Pbic/Pbic and Ptar/Ptar homozygotes respectively (Chi2 = 4.90 For P3 differentially expressed transcripts no deviation from parity was observed with 22 and 19 up-expressed transcripts in Pbic/Pbic and Ptar/Ptar homozygotes respectively (Chi2 = 0.22 the difference in P2 is even larger than the genome-wide pattern which also indicates an overexpression in Ptar/Ptar homozygotes (Chi2 = 4.01 the most important deviation from genome-wide pattern is biased towards Pbic/Pbic in P1 and Ptar/Ptar in P2 Gray dots refer to transcripts with overlapping expression between the homozygous genotypes or between the homozygotes and the heterozygote Transcripts with non-overlapping expression levels between the homozygous genotypes are indicated in violet and turquoise colors according to whether the expression was higher in the Pbic/Pbic (category 1) or Ptar/Ptar homozygous genotype (category 2) Vertical lines indicate borders of the three subinversions according the Hmel2 reference genome coordinates of chromosome 15 11 belonged to intergenic regions whereas the remaining 14 transcripts belonged to 11 protein-coding genes namely HMEL000025 (Cortex) and HMEL000033 (Lmtk1 Both genes are on P1 and are overexpressed in Pbic/Pbic homozygotes Those multiple transcripts are most likely the result of alternative splicing Another evidence for alternative splicing comes from a partial overlap (~10 bp) between an intergenic transcript (comp38651_c0_seq1) that is overexpressed in Pbic/Pbic and shows a bic dominance in the heterozygote and the HMEL000021 transcript which is overexpressed in Ptar/Ptar and shows a tar dominance in the heterozygote When correcting for the P1 inversion coordinates these two overlapping transcripts with contrasting dominance patterns become adjacent to the P1/P2 breakpoint As indicated in the Methods above and Fig. 2 we assigned signs to dominance estimates according to the overlap between the minimal counts of the heterozygotes relative to the intermediate expression between the two homozygous genotypes for a gene with a higher expression of the Pbic/Pbic homozygotes dominance will be positive if the heterozygous minimal counts will be higher than the intermediate expression level and negative if it is below this threshold The opposite trend follows for a gene with a higher expression in the Ptar/Ptar homozygotes We therefore investigated whether the dominance observed on the transcripts of the heterozygotes was caused by an overexpression of these transcripts in one out of the two homozygote genotypes We thus compared the number of dominant transcripts where an overexpression was found in the Pbic/Pbic individuals (category 1) vs we indeed found an excess of transcripts for which expression levels in heterozygotes tend to follow the homozygous genotype with the highest expression level (Chi2 = 206.82 A similar trend was also found for genes in the ancestral P1 inversion (Chi2 = 13.00 there was no deviation from parity (Chi2 = 0.06 P = 0.806) and the observed dominance patterns was thus not linked to an increased level of transcript expression in the Pbic/Pbic homozygous genotypes This final observation indicates that nearly half of dominant transcripts in the two derived subinversions may be due to the downregulation of transcripts by the bic allele of genes that are overexpressed in the phenotypically recessive Ptar/Ptar homozygotes Taken together our analyses of dominance of gene expression levels in Heliconius butterflies reveal that (1) expression levels of the transcripts in heterozygotes are more similar to the expression pattern observed in the dominant homozygous morph (2) this dominance pattern is enriched but not restricted to the wing-pattern controlling P inversion and (3) the most important deviation from homozygotes-based expectations is at the derived P2 subinversion If such a hypothesis turns out to be correct this would indicate that the evolution of complex dominance patterns in Heliconius may have involved a step-wise process with (1) a Haldane’s sieve-like evolution of a dominant derived allele at P1 where the over-expression of the dominant transcripts drive phenotypic development towards the bicoloratus phenotype and (2) the evolution of repression in the genes located in the younger sub-inversions P2 and P3 preventing the expression of the transcripts triggering the development of the recessive tarapotensis phenotypes This study thus represents a promising step towards the understanding of the evolution of dominance relationships in complex phenotypic traits Investigating the pattern of different heterozygous combinations at the P supergene would certainly shed light on the different molecular mechanisms causing the coordinated dominance observed at the phenotypic scale allowing to reconstruct the evolutionary history of dominance relationships a switch in developmental pathway can be triggered by variations in the levels of certain signaling proteins but can also be triggered by variations in the protein sequence We thus hope our study will stimulate larger-scale investigation of expression patterns allowing to refine our knowledge on the combinations of genetic elements involved in such major developmental switch The data can be found in the NCBI SRA and TSA repositories under the number PRJNA555830 (2016) Crossing fitness valleys: empirical estimation of a fitness landscape associated with polymorphic mimicry Llaurens V (2021) The integrative biology of genetic dominance Challi RJ, Kumar S, Dasmahapatra KK, Jiggins CD, Blaxter M (2016) Lepbase: the Lepidopteran genome database. bioRxiv https://www.biorxiv.org/content/10.1101/056994v1 Charlesworth D (1999) The genetic basis of inbreeding depression Willis JH (2009) The genetics of inbreeding depression Joron M (2016) Warning signals are under positive frequency-dependent selection in nature Hall MD (2018) Genetic constraints on adaptation: a theoretical primer for the genomics era (2014) Dominance hierarchy arising from the evolution of a complex small RNA regulatory network Nijhout HF (2001) Nonlinear developmental processes as sources of dominance (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis Haldane JBS (1956) The theory of selection for melanism in Lepidoptera (2018) Supergene evolution triggered by the introgression of a chromosomal inversion (2021) Mutation load at a mimicry supergene sheds new light on the evolution of inversion polymorphisms (2022) Association mapping of colour variation in a butterfly provides evidence that a supergene locks together a cluster of adaptive loci (2011) Chromosomal rearrangements maintain a polymorphic supergene controlling butterfly mimicry (2006) A conserved supergene locus controls colour pattern diversity in Heliconius butterflies Mallet J (1999) Variable selection and the coexistence of multiple mimetic forms of the butterfly Heliconius numata Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2 Joron M (2014) Evolution of dominance mechanisms at a butterfly mimicry supergene (2009) The Sequence Alignment/Map format and SAMtools Livraghi L, Hanly JJ, Loh LS, Ren A, Warren IA, Concha C et al. (2020) The gene cortex controls scale colour identity in Heliconius. bioRxiv https://www.biorxiv.org/content/10.1101/2020.05.26.116533v2.full Vekemans X (2009) Evolution of dominance in sporophytic self-incompatibility systems: I Genetic load and coevolution of levels of dominance in pollen and pistil Billiard S (2015) Molecular mechanisms of dominance evolution in Müllerian mimicry Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads Reed RD (2014) Wnt signaling underlies evolution and development of the butterfly wing pattern symmetry systems (2012) Diversification of complex butterfly wing patterns by repeated regulatory evolution of a Wnt ligand (2016) The gene cortex controls mimicry and crypsis in butterflies and moths Bourguet D (1999) Balanced polymorphisms and the evolution of dominance Llaurens V (2019) Unravelling the genes forming the wing pattern supergene in the polymorphic butterfly Heliconius numata Edwards R (2011) Quality control and preprocessing of metagenomic datasets Edwards R (2012) Identification and removal of ribosomal RNA sequences from metatranscriptomes Wilkie AO (1994) The molecular basis of genetic dominance Hakomori S (1990) Molecular genetic basis of the histo-blood group ABO system Pool JE (2016) Ancient balancing selection at tan underlies female colour dimorphism in Drosophila erecta Download references This study was funded by ANR JCJC DOMEVOL and the Emergence program of Paris City council to VL and by ANR SUPERGENE to MJ Université Paris-Saclay – Institut Diversité Université des Antilles) Muséum National d’Histoire Naturelle - CP50 Interactions Des Systèmes Amazoniens (LEEISA) HB designed and performed the dominance analyses on RNA-Seq data and wrote and edited the manuscript assembled the transcriptome and wrote and edited the manuscript performed the crosses and wrote and edited the manuscript MJ designed the experimental scheme and wrote and edited the manuscript supervised the project and wrote and edited the manuscript The authors declare no competing interests Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law Download citation DOI: https://doi.org/10.1038/s41437-022-00583-5 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article With shafts of natural light spilling into the cozy first floor of the C about 50 people recently gathered to celebrate the library’s Buddhist collections and the many contributions of the Rev The event also doubled as a reception for a new exhibit at the East Asian Library featuring some of the library’s most precious materials related to Chinese Filling the first floor are stunning prints of Buddhist temples and maps of pilgrimage routes; popular texts and treatises from Buddhist scholars; an excerpt of a 600-volume accordion-fold sutra; and more “Berkeley has been collecting materials in the field of East Asian Buddhism for close to 100 years,” said Peter Zhou “This exhibit will show how that collection has grown.” speakers discussed the closely intertwined history of Buddhist studies at Berkeley and the legacy of the Numata family Convinced that the teachings of Buddhism could inspire world peace started the philanthropic organization Bukkyō Dendō Kyōkai the organization has supported Buddhist studies at UC Berkeley from all sides and helping fund the library’s now 11-year-old home Top to bottom: Clockwise from left: The Rev Shoryu Katsura speaks at the event; Mitutoyo president Yoshiaki Numata presented a gift to the library; an item from the Buddhist collection now on display said it was an honor for the family to have contributed to the library’s construction vice chancellor for development and alumni relations through the campus’s Center for Buddhist Studies called Numata a “remarkable figure” whose contributions have been instrumental to the field of Buddhist studies at Berkeley where he studied mathematics and statistics After graduating from Berkeley with his master’s degree in 1928 now a global enterprise specializing in precision measuring tools our relationship with UC Berkeley will have spanned 100 years,” said Yoshiaki Numata “Our roots are deeply established in Berkeley Copyright © 2025 The Regents of the University of California. Except where otherwise noted, this work is subject to a Creative Commons Attribution-Noncommercial 4.0 License Statement from the Numata Family"It is with deep sorrow that the Numata Ohana announces that Chace Kekoa Kenji Numata It didn't matter if you were a professional athlete or young child #FORNUMI"Statement from the Detroit Tigers"The Detroit Tigers are deeply saddened to learn of the passing of Chace Numata teammates and coaches."Statement from Erie SeaWolves Owner Fernando Aguirre"The Erie SeaWolves are heartbroken and deeply saddened by the passing of Chace Numata Jake Rogers is used to answering questions Perhaps most impressive about the Detroit Tigers’ rookie catcher – even more so than his outstanding defense behind home plate – is that despite his batting average hovering near .100 Rogers stands at his locker and answers questions from reporters postgame Rogers was again in front of his locker at Comerica Park on Monday afternoon questions weren’t limited to reporters readying for an evening flight to Kansas City a Tigers media relations staffer asked Rogers if he was OK to talk “It’s tough,” Rogers said and he brought life to everybody else around him all I can do is keep his family and pray for him and think about him [ Erie SeaWolves prayed from Chace Numata: God, bring him back to us ] Numata’s death made for a somber clubhouse after the game the loss – in which the Tigers were bested by Max Kepler's two-run single in the eighth inning – played into the atmosphere But it's also because Numata made a lasting impact in his one season with the organization.  Baseball players – regardless of their paths professionally – are very much part of a fraternity And when something tragic like Numata’s death happens it reverberates through organizations across baseball “Things can happen so fast and it’s just was a .251 hitter in 10 minor-league seasons with the Phillies He was drafted by the Phillies in the 14th round of the 2010 MLB Draft not a lot of discussion,” Tigers manager Ron Gardenhire said “We saw him in spring training with my staff he was one of those gamer guys who really liked to play Really great personality and all those things and it’s really tough out in there in the clubhouse As players dressed and packed for their eight-game road trip to Kansas City and Oakland the loss to Minnesota seemed out of mind: Once again it was the product of an inexperienced team taking on a World Series contender Veteran right-hander Jordan Zimmermann pitched well allowing two runs in six innings for his second consecutive quality start but he could not win for the first time at home this season after the Tigers’ bullpen coughed up the lead in the top of the eighth the first two men of the eighth reached base shortstop Willi Castro could not corral a hard-hit ground ball up the middle With Gardenhire preferring to play the matchups “We had a chance to make a play and get out of the inning and it hit his glove,” Gardenhire said But Buck had a ton of pitches and we had a lefty up and that’s basically all we had.” The Tigers (40-95)  lost despite having enough offense to win: Miguel Cabrera hit a solo home run in the first; Travis Demeritte hit an RBI single and Victor Reyes hit a sacrifice fly in the fifth it wasn’t the biggest loss for the organization Rogers buttoned up his dress shirt in front of his locker where a Tigers hat with “NUMI” – Numata’s nickname written above the bill – was hanging Numata’s nickname will stay there for the remainder of the season he was an unbelievable baseball player and just having him back there made me try to work harder “I could feel him clipping on my heels He’s going to be there and I’ll support him and his family and he’s going to be with me.” Stockton University 101 Vera King Farris Drive Galloway, NJ 08205-9441 (609) 652-1776 Maps, Directions & Parking Accessibility Statement Additional Locations Metrics details The development of artificial spider silk with properties similar to native silk has been a challenging task in materials science we use a microfluidic device to create continuous fibers based on recombinant MaSp2 spidroin The strategy incorporates ion-induced liquid-liquid phase separation and shear-dependent induction of β-sheet formation We find that a threshold shear stress of approximately 72 Pa is required for fiber formation and that β-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence The MaSp2 fiber formed has a β-sheet content (29.2%) comparable to that of native dragline with a shear stress requirement of 111 Pa the polyalanine blocks have limited influence on the occurrence of liquid-liquid phase separation and hierarchical structure These results offer insights into the shear-induced crystallization and sequence-structure relationship of spider silk and have significant implications for the rational design of artificially spun fibers the confined dimensions and geometries of the spinning ducts can be approximated in the design of the miniaturized channels in the chip such quantitative analysis of the forces within microfluidic channels have not been carried out in rigorous detail to link the forces with the fiber formation and change of conformation recombinant spidroins in the native state are more responsive to naturally occurring triggers leading to self-assembly behavior under native-like conditions is of great interest and significance for better understanding of spider silk self-assembly mechanism Here we present a rationally designed microfluidic system that aims to emulate the functionality of the natural silk spinning apparatus by integrating recent insights regarding the in vivo self-assembly process By sequential administration of biomimetic chemical gradients that trigger phase separation and nanofibril formation we demonstrate the complete in situ assembly of hierarchically structured silk fibers from recombinant MaSp2 precursors with tunable β-sheet abundance and at near-instantaneous speeds consisting of three inlets and one outlet with a negative pressure controlling system The spidroin was first mixed with 50 mM CPB at pH 7 and then subjected to 1 M citrate–phosphate buffer (CPB) (pH 5) at the intersection The channel along which MaSp2 assembled was divided into sections A B and C for comparison in terms of the morphology and secondary structure The inset shows a picture of the actual microfluidic device b The different recombinant MaSp2 constructs used in this study representing various combinations of the N-terminal domain (NTD) the poly-Ala repeat normally found in the repetitive region was replaced by GSGSGSG c N-R12-C in the form of droplets and nanofibrils obtained by exposure to 0.5 M CPB at pH 7 and 5 Five independent experiments were performed with similar results we used a hybrid citrate-phosphate buffer (CPB) system as the general kosmotropic trigger in light of its improved buffering capacity throughout the relevant pH range (7.5–5.0) compared to the individual phosphate and citrate components a N-R12-C labeled with DyLight-488 was transferred to the microfluidic device for spinning under native-like conditions Three independent experiments were performed with similar results b 3D reconstruction of N-R12-C droplets distributed along the channel c Surface structure of N-R12-C droplets induced by LLPS inside the microfluidic device as visualized by SEM Two independent experiments were performed with similar results d N-R12-C fiber assembled inside the channel in section C3 with a hierarchical organization e 3D reconstruction of N-R12-C fiber aligned along the channel in section C3 Two independent experiments were performed f Oriented nanofibrils observed from the flexible continuous fiber in Milli-Q water after recovering it from the microfluidic chip g Nanofibrils split from the fiber to show hierarchical organization upon exposure to the Milli-Q water which provides further evidence of hierarchical organization While the above demonstrates a microfluidic platform capable of inducing rapid self-assembly of recombinant MaSp2 into hierarchically structured fibers in response to sequential physiological triggers another major concern is whether such a system can successfully induce the formation of β-sheet structures which are the main source of tensile strength in spider silk a Secondary structure of the MaSp2 fiber determined by Raman spectroscopy immediately after fiber assembly inside the channel b Negative control experiment of N-R12-C and N-R12-C(xA) outside the microfluidic device N-R12-C and N-R12-C(xA) were gently mixed with 1 M CPB at pH 5 and 7 on a cover slide at a 1:1 volume ratio A positive control was conducted by immersing N-R12-C nanofibrils in 90% methanol (MeOH) solution for 5 min after removal of 1 M CPB (pH 5) The dash line denotes the peak at 1654 cm−1 N-R12-C and N-R12-C(xA) in three sections along the channel Peak features were specifically evaluated within the amide I region d Raman results of N-R12-C fiber under various negative pressures The solid line denotes the peak at 1664 cm−1 e β-sheet contents of N-R12-C fiber assembled inside the microfluidic device under various negative pressures Peak fitting was carried out to calculate the β-sheet contents after deconvolution within the amide I region Data are presented as mean values ± SD; n = 3 N-R12-C and N-R12-C(xA) fibers formed by biomimetic spinning and manual drawing Source data are provided as a Source Data file the estimated β-sheet content increased from 12.5% in section A to 22.5% in section B and finally to a maximum of 29.2% in section C despite the assembly of morphologically similar fibers; we hypothesize a higher pressure threshold for triggering structural change in the 6- versus the 12-repeat construct with an applied pressure of −90 kPa (the upper limit for our system) being evidently insufficient to drive abundant β-sheet formation in N-R6-C a secondary structure transition was not observed in line with polyalanine forming the basis for the β-sheet crystalline component of the silk fibers a The viscosity-shear rate relationship of N-R12-C solution at various concentrations from 5 to 150 mg/ml b Shear-thinning behavior of N-R12-C solution at a concentration of 50 mg/ml with the result fitted by the Carreau model c Simulated shear stress distributed along the channel to show the difference in each section d Comparison of shear stress s in section C when changing the negative pressure from −60 to −90 kPa e The value of shear stress estimated in sections A B and C under different negative pressures f The β-sheet contents of N-R12-C fiber under specific shear stress estimated from the corresponding negative pressure The red line shows the fitting result by using the quadratic function −60 kPa was confirmed to be the threshold pressure for the formation of silk fiber which obtained an averaged β-sheet content of 17.8% the β-sheet content was gradually increased by 21.5% (at −70 kPa) It was assumed that more β-sheets could be induced within silk fibers if the pressure reached beyond −90 kPa that was limited by our experimental system This result indicates the potential for tuning fiber structure via subtle changes in the applied pressure the intensity at peak (010) suggested that the relative crystallinity of silk fibers prepared by manual drawing was positively related with the number of polyalanine blocks within the repetitive domain the crystallinity of N-R12-C(xA) fiber made by sufficient manual drawing was still lower than that of N-R12-C fiber made by microfluidic spinning these results suggest a further possibility of fine-tuning the properties of biomimetic silk by combining aqueous spinning methods with mechanical post-processing steps N-R12-C solution exhibited a typical shear-thinning behavior with decreased viscosity in response to an increase in shear rate the shear viscosity of native spidroins was in the order of 103 Pa s much higher than that of N-R12-C (all below 10 Pa s) and NT2RepCT (below 1 Pa s) This significant difference can be interpreted by the much higher concentration and molecular weight of native spidroins in comparison to these of recombinant spidroins To directly compare the shear viscosity between recombinant spidroins under the same concentration (such as 100 mg/ml) N-R12-C exhibited remarkably higher shear viscosity (~2 Pa s) more than 20 times higher than that  reported for NT2RepCT (0.05–0.1 Pa s) which could be attributed to the different number of repeat units (12 repeats vs 2 repeats) the alanine residues in the repetitive region were found to contribute significantly to the viscosity of spidroins as evidenced by the lower viscosity (apparent to the naked eyes) of N-R12-C(xA) when compared with N-R12-C In terms of physical forces generated within the device, as anticipated from the design, elongational flow effects within the device were minimized, with negligible values in the non-convergent channels of section A and C (Supplementary Fig. 9) there might be certain elongation stress applied to the material protein in the threshold region between sections A and B but we considered its effect trivial after confirming that the local shear stress was more than nine times higher than the elongation stress a Hierarchical structure of N-R12-C and N-R12-C(xA) fibers imaged by SEM Four independent experiments were performed with similar results b Schematic of spider silk self-assembly from LLPS to nanofibrillation to fiber formation triggered by native-like conditions Both chemical and physical triggers were needed and worked synergistically for fiber formation however the latter played a dominant role for the transition of secondary structure It is flexible to apply the microfluidic system to control the shear which was highlighted in this work to investigate its impact on crystallization it is promising to fabricate artificial silk fibers with subtly tuned β-sheet structure we report a biomimetic strategy in probing the β-sheet development under native-like assembly process from LLPS to nanofibrillization to hierarchically organized fiber This work provides a basis for the rational design of biomimetic spinning with regard to domain structure shear threshold and hierarchical reconstruction Future efforts will focus on investigating the silk assembly of combined MaSp1 and MaSp2 spidroins under biomimetic spinning conditions The heterodimerization formed by CTD and/or NTD will be comprehensively evaluated to better understand the interaction between spidroins in natural spinning process specific amino acid residues in the gene sequence will be edited/substituted to explore the underlying mechanism for LLPS Expression of recombinant MaSp2 was performed as previously described12 the pET15-derived constructs were transformed into E grown overnight as 100-mL preculture in LB medium with ampicillin (Amp; 100 µg/mL) at 37 °C with shaking at 180 rpm after which the temperature was lowered to 20 °C and protein expression induced overnight with 0.4 mM isopropyl-beta-d-thiogalactopyranoside (IPTG) cell lysis was carried out by freeze-thawing the bacterial cell pellet that had been completely resuspended in 20 mM Tris-HCl pH 7.5 0.5 M NaCl supplemented with 1% Triton X-100 (Sigma-Aldrich) and cOmplete EDTA-free protease inhibitor tablet (Roche) The lysate was spun down and the supernatant fraction applied to a His-TRAP FF column (2x5ml in tandem) (Cytiva) The eluate was subjected to buffer exchange against 20 mM Tris-HCl pH 7.5 0.15 M NaCl using a VivaSpin spin concentrator with 10 kDa MWCO (Sartorius) The poly-histidine tag was removed by overnight thrombin digestion (Sigma-Aldrich) at 4 °C a final round of purification was carried out through LLPS was added until two stable liquid phases were formed followed by centrifugation and retrieval of the high-density phase The protein concentration was measured by absorbance at 280 nm using a NanoDrop instrument (Thermo Fisher Scientific) The protein concentration of 50 mg/ml was applied for the following experiments unless otherwise indicated SDS‒PAGE was performed on 4–20% mini-PROTEAN TGX precast gels (Bio-Rad) under denaturing conditions according to the manufacturer’s recommendations β-mercaptoethanol (βME) was omitted from the sample buffer and the samples were not heated prior to electrophoresis Gels were stained with 0.1% Coomassie R-250 in 40% ethanol and 10% acetic acid SU-8 (3050) was poured onto a clean wafer for spin coating for 30 s at 1650 rpm The wafer with uniformly dispersed SU-8 was transferred to the oven to prebake for 20 min at 95 °C and then slowly cooled for 10 min The photomask with specific channel patterns was closely aligned to the SU-8 wafer for photolithography followed by postbaking and developing to retain only the pattern on the silicon wafer The mold was successfully fabricated after stepwise high-temperature baking from 150 to 200 °C and silanization overnight by using trichloro(1H a PDMS slab containing channels was obtained by casting a silicone elastomer (SYLGARD 184 Dow Corning) with its curing agent on the mold Three inlets and one outlet with hole diameters of 1.5 mm and 4 mm the PDMS slab was attached to the coverslip to obtain the final microfluidic device the microfluidic device was operated under native pressure to enable a strong and stable interface between PDMS and the coverslip to avoid leaking risk The coverslip was selected rather than a relatively thick glass slide mainly because of the conditions required by Raman spectroscopy and CLSM The sequence of steps was as follows: (1) turn on the vacuum pump (with adjustable pressure output) and attach the connected tubing to the device outlet; (2) pipet 15 µl of 1 M CPB (pH 5) into inlet 3; (3) pipet 15 µl of 50 mM CPB (pH 7) into inlet 2; (4) pipet 1.5 µl of 50 mg/ml MaSp2 into inlet 1; and (5) disconnect the vacuum pump tubing The protocol was performed rapidly and was essentially complete upon pipetting of the protein solution recombinant spider silk was assembled inside the microfluidic channel The device was used to check the in situ MaSp2 morphology with various microscopes and characterize the secondary structure by Raman spectrometry The sample morphology was directly observed through the outlet of the microfluidic device by using an Olympus BX53 microscope To visualize the sample within the channel the microfluidic device was reversely placed on the glass slide before transferring to the optical microscope additional experiments were carried out to recover the freshly formed fibers from the microfluidic device and place them in a hypotonic environment (Milli-Q water) There were localized areas where the continuity of the fiber was broken to allow the visualization of high-aspect ratio nanofibrils High-resolution 3D imaging of biomimetic fibers within the microfluidic channels (using MaSp2 N-R12-C labeled with DyLight-488) was carried out using a TCS SP8 confocal microscope platform (Leica) under the Hyvolution2 regime Imaging was performed using a 20x/0.75 HC PL APO CS2 objective HyD 2 hybrid detector (Ex = 488 nm; Ex = 508–532 nm) Image deconvolution was achieved using Huygens Professional software (Scientific Volume Imaging) The surface structures of MaSp2 droplets and fibers were detected by using a JEOL-6000 instrument at an accelerating voltage of 15 kV To detect samples within the microfluidic device PDMS was gently detached from the device to allow part of the samples to be retained on the coverslip the coverslip was directly fixed to an aluminum stub with conductive carbon tape and then subjected to gold sputtering for 2 min It is noteworthy that the sample on the coverslip was covered with a thin layer of salts from the buffer solution Samples could be damaged or partly dissolved after rinsing with Milli-Q water no extra treatment was performed to remove the salt and the overall morphology of samples detected by SEM was quite similar to that detected by CLSM MaSp2 fibers prepared by manual drawing were immersed in liquid nitrogen for 1 min and then cut by a blade Raman measurements were carried out with a JASCO NRS-4100 confocal microscope with a 532 nm laser. Using a 100x oil objective, the laser beam was focused on the center of the MaSp2 samples through the coverslip side, as shown in Fig. 3a Spectra were recorded in a wavenumber range from 500 to 2000 cm−1 the grating was set as 1800 grooves mm−1 with a slit size of 10 × 8000 μm providing a resolution as high as 0.8 cm−1 The beam intensity of 16.9 mW and exposure time of 300 s were optimized to obtain a sufficient signal-to-noise ratio Under the protection of the buffer medium surrounding the MaSp2 samples no local heating effect was induced by the set laser power Consistent results were obtained by repeating the measurement at least five times under the same conditions to ensure data reliability MaSp2 was gently mixed with 0.5 M CPB (pH 7 or 5) in a customized chamber made from vinyl adhesive tape on a glass slide To create a similar background as inside the microfluidic channel native dragline silk from Trichonephila clavata and manually stretched MaSp2 fibers were immersed in 1 M CPB (pH 5) for Raman experiments The crystalline structure of MaSp2 fibers was determined by WAXS at SPring-8 synchrotron in Harima The BL05XU beamline was selected to have X-ray energy as high as 15 KeV at a wavelength of 0.08 nm The sample-to-detector distance was measured as 262 mm with an exposure time of 1 s set for each sample 1D profiles were converted using Fit2D software from 2D patterns PDMS was carefully peeled from the microfluidic device to better subtract the glass background in the following data analysis After either biomimetic spinning or manual drawing MaSp2 fibers were rinsed thoroughly with Milli-Q water to remove salts remaining on the fiber surface to avoid the adverse X-ray signal generated from salt crystals 7.1 µl of MaSp2 was pipetted onto the plate at various concentrations from 5 to 150 mg/ml When it comes to the set temperature (25 °C) the sample was subjected to the shear rate ramp from 0.1 to 5000 s−1 to measure the viscoelastic property of MaSp2 dope The interval time was set as 120 s under shear rate from 0.1 to 10 s−1 60 s from 11 to 100 s−1 and 25 s from 101 to 5000 s−1 It is worth noting that the ultrahigh shear rate region (above 104 s−1) is a region that cannot be measured by a rotational rheometer To account for the effect of MaSp2 concentration on viscosity we fit the Carreau model as a function of concentrations to the viscosity as below: Here c represents the dimensionless concentration of MaSp2 normalized with 50 mg/ml. Other parameters are summarized in Supplementary Table 3 constant diffusion coefficients in the entire calculation were utilized the diffusion constant of MaSp2 in 1 M CPB (pH 5) was determined by dynamic light scattering (DLS) using a Zetasizer Nano-ZS (Malvern Instruments) The viscosity of 1 M CPB (pH 5) was measured by a vibration-type viscometer (VM-1G Artificial spider silk fibers were prepared by manually drawing MaSp2 out of 1 M CPB at pH 5 A 0.5 μl MaSp2 solution was first pipetted onto the cell culture dish at a concentration of 100–130 mg/ml and then 4 μl CPB was immediately added over the MaSp2 solution A sharp tweezer was taken to pull out one end of condensed turbid proteins to some extent another tweezer was taken to uniaxially extend the fiber at the other end to the maximum length in the air Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article The data supporting the findings of this study are included in the published article and its supplementary information files. The simulation data generated in this study have been deposited in the GitHub repository (https://github.com/Dubbing173/Simulation-data.git). Source data are provided with this paper Multicomponent nature underlies the extraordinary mechanical properties of spider dragline silk strength and mechanical toughness of β-sheet crystals in silk Hierarchical spidroin micellar nanoparticles as the fundamental precursors of spider silks Self-assembly of spider silk proteins is controlled by a pH-sensitive relay A conserved spider silk domain acts as a molecular switch that controls fibre assembly Progress and trends in artificial silk spinning: a systematic review Impact of physio-chemical spinning conditions on the mechanical properties of biomimetic spider silk fibers Engineered spider silk proteins for biomimetic spinning of fibers with toughness equal to dragline silks Biological materials processing: time-tested tricks for sustainable fiber fabrication Biomolecular condensates undergo a generic shear-mediated liquid-to-solid transition Changes in element composition along the spinning duct in a Nephila spider Spider silk self-assembly via modular liquid-liquid phase separation and nanofibrillation Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains Liquid crystalline spinning of spider silk Spider silk: from soluble protein to extraordinary fiber Extensional flow behaviour and spinnability of native silk Mechanically strong proteinaceous fibers: engineered fabrication by microfluidics Modular assembly of a conserved repetitive sequence in the spider eggcase silk: from gene to fiber Recombinant spider silk from aqueous solutions via a bio-inspired microfluidic chip Assembly mechanism of recombinant spider silk proteins Interplay of different major ampullate spidroins during assembly and implications for fiber mechanics Two-in-one spider silk protein with combined mechanical features in all-aqueous spun fibers Flow analysis of regenerated silk fibroin/cellulose nanofiber suspensions via a bioinspired microfluidic chip Strong silk fibers containing cellulose nanofibers generated by a bioinspired microfluidic chip Biomimetic spinning of artificial spider silk from a chimeric minispidroin A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning Analysis of the pressure requirements for silk spinning reveals a pultrusion dominated process Structure of silk by Raman spectromicroscopy: from the spinning glands to the fibers In situ Raman spectroscopic study of al-infiltrated spider dragline silk under tensile deformation Inducing β-sheets formation in synthetic spider silk fibers by aqueous post-spin stretching Combination of amorphous silk fiber spinning and postspinning crystallization for tough regenerated silk fibers Native-like flow properties of an artificial spider silk dope Imaging the microscopic structure of shear thinning and thickening colloidal suspensions Divergent shear thinning and shear thickening behavior of supramolecular polymer networks in semidilute entangled polymer solutions Solvent removal during synthetic and Nephila fiber spinning Secondary structure transition and critical stress for a model of spider silk assembly Deciphering how naturally occurring sequence features impact the phase behaviours of disordered prion-like domains Liquid–liquid phase separation primes spider silk proteins for fiber formation via a conditional sticker domain Mechanism of silk processing in insects and spiders User-friendly semiautomated assembly of accurate image mosaics in microscopy Study on the temperature-induced sol–gel transition of cellulose/silk fibroin blends in 1-butyl-3-methylimidazolium chloride via rheological behavior Chen, J. et al. Replicating shear-mediated self-assembly of spider silk through microfluidics. GitHub, https://doi.org/10.5281/zenodo.10408439 (2023) Download references This work was financially supported by the Grant-in-Aid for Early-Career Scientists from JSPS (Grant No National Natural Science Foundation of China (Grant No Departmental General Research Fund (Grant No the Japan Science and Technology Agency Exploratory Research for Advanced Technology (JST-ERATO; Grant No and MEXT Data Creation and Utilization-type MaTerial R&D project (to K.N.) RIKEN Center for Sustainable Resource Science Research Institute for Intelligent Wearable Systems Research Centre of Textiles for Future Fashion Japan Synchrotron Radiation Research Institute prepared and characterized the recombinant spidroins designed and fabricated the microfluidic chip prepared the first draft of the manuscript and all authors contributed to the final draft reviewers for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-024-44733-1 Sign up for the Nature Briefing newsletter — what matters in science SENATOR SEAN RYAN SELECTS YUKI NUMATA RESNICK AS 2023 ‘WOMEN OF DISTINCTION’ HONOREE Accomplished Violinist Co-Founded Buffalo String Works to Promote Equitable Access to Youth Music Education in Western New York New York State Senator Sean Ryan recognized Yuki Numata Resnick as a member of the 2023 New York State Senate Women of Distinction class of honorees Resnick held the position of Assistant Professor of Music at the University at Buffalo from 2013-2016 a music program focused on leadership development and social change Resnick steadily grew the organization’s capacity to serve over 150 refugees Buffalo String Works employed 22 staff and faculty Resnick has performed on stages ranging from the Sydney Opera House to Carnegie Hall and has played with and recorded for bands including The National she recorded for Taylor Swift and can be heard on Swift’s Midnights She is also a featured musician on Ed Sheeran’s recently released record her debut solo record featuring brand new works by living composers Resnick is currently Director of Arts and Culture at the Community Foundation for Greater Buffalo Her role interacts closely with the Ralph C Foundation’s $100 million commitment to transforming the long-term viability of Western New York’s arts and culture community through the establishment of an endowment to support the sector now and into the future “It is my privilege to recognize Yuki Numata Resnick for the important work she has done for underserved communities in Buffalo she has used her gift to inspire children who too often miss out on the arts education available to their peers She is an incredible musician and an even better person and her commitment to ensuring children in Western New York have access to the arts makes this a well-deserved honor.” HAYATO KANNO: The person who proposed this project was originally from Gunma Prefecture. When it came to Takasaki, Maebashi, and Numata, the director [Koudai Kakimoto] did some location scouting in Gunma He decided to use Takasaki's Central Ginza Shopping Arcade That's how we decided to focus on the three locations of Takasaki What are the key characteristics or charm of Gunma Prefecture and how will they be represented in the series I think Gunma Prefecture is famous for its three mountains the teams aren't divided into red and white but based on these three mountains hot springs such as Kusatsu Onsen and Ikaho Onsen are attractive Hot springs and local specialties will appear in the series a type of karuta game unique to Gunma Prefecture that is a required subject for elementary school students It's also relatively easy to access Gunma from the Kanto region And I think the people of Gunma are very passionate It's one of the 100 Famous Mountains of Japan I think that's why the director and I were drawn to Gunma Will some of those locations appear in the series the story takes place in Takasaki City and Numata City so places like the front of Takasaki Station and although it is a little far from the station the Central Ginza Shopping Arcade in Takasaki will appear a slope called Dangozaka in Numata City appears the series is set to take place around Takasaki what other places within Gunma Prefecture can we expect to see but it's related to the story later in the series so I can't definitively say “here,” but other places will appear but onsen [hot springs] may appear in the story but do you have any recommendations for onsen or other interesting places for our readers It's the birthplace of the hot spring mark (♨️) in Japan Isobe has several places where you can enjoy free footbaths as well as places where you can enjoy rice crackers made with hot spring water but the place I visited and found interesting was Isobe Onsen There was an announcement on the Narenare -Cheer for you!- website announcing a tourism collaboration between the series and Takasaki Will we see some of those locations in the series Locations in the tourism collaboration visuals we released the other day won't appear in the series When creating collaborative visuals with the three cities we deliberately chose attractive destinations that won't appear in the series "Takasaki City's tourist destination is the Kannon statue." So and the waterfalls in Numata will not appear in the series I've heard the Takasaki/Maebashi area has a rivalry with Saitama Prefecture's Kumagaya City Will we see that rivalry appear in Narenare -Cheer for you!- In terms of rivalry and future episodes, I heard from someone from [Gunma] Prefecture when I was working on You Don't Know Gunma Yet that if I wanted to cover Takasaki City they would also like to see Maebashi City covered as well Maebashi High School in Maebashi City and Takasaki High School in Takasaki City have been holding a sports tournament once a year for decades Takasaki City and Maebashi City were also rivals and there may be a few episodes related to this in the series I think we tried to incorporate that characteristic of the region into our story I noticed in some of the promotional material there is a character using a wheelchair Can you tell us more about her that isn't in her character intro Megumi is a childhood friend of the main character they have been active in the cheerleading club together during the national middle school championships [Megumi] became ill and had to undergo surgery and recuperate and she is still working hard on her rehabilitation the first episode starts with Megumi working hard on her rehabilitation The theme of the series is “Cheering.” Is this competition cheering or will we see the characters cheering on people in Gunma Prefecture the theme is "cheering" and "cheering cheers people up" Cheerleading can be divided into two types: competitive cheering performed at competitions and cheering to support something the unit "Pom-Poms," including Kanata and the other members Tying into the [Japanese] title Nanare Hananare the “hananare” part refers to the glamorous aspect of competitive cheer which are competitive cheerleading performances the desire to cheer on someone is at its core I thought this was important and not just about the glamorous performances we used the title "Nanare Hananare" because it has both "hana" (flower) and "na" (stem) The theme of the work is about cherishing the feeling of cheering or wanting to cheer the story feels like a work that supports and pushes someone in the right direction In an interview with Jomo Newspaper, Rika Nakagawa (voice of Kanata Misora) spoke about the Gunma dialect What sort of research went into ensuring the characters sound like they are from Gunma Prefecture are there any phrases or slang you cued in on to give the series some color When we were writing the script and creating the story and it was decided Gunma would be the setting "What elements are typical of Gunma Prefecture?" There were two main things I said and we decided to include that in the story In my research on words when I worked on Omagun I came across word enders "ah-ne" and "nan-nan." Depending on the rising and falling intonation you get "ah-ne," which means "got it" or used as a question mark There are words whose meaning changes depending on the nuance of the ending intonation such as a strong "nan-nan," which means "What does that mean!?" or a falling intonation I carefully picked out those subtle nuances when we were recording the dubbing she'll point out nuances with "ah-ne" and "nan-nan" and suggest changing intonations accordingly But if we used the Gunma dialect throughout there was a chance [the dialogue] wouldn't be understood I picked a few words and phrases that left a strong impression and used them in the series Nakagawa spoke about how she wanted to portray the character Kanata Misora What was the deciding factor in her taking on the role and we got the impression she was straightforward and energetic We also felt the shadowy part of the conflict that Kanata was facing in the story I'm going to use the restroom." She then came out wearing a cheerleading uniform She was in a cheerleading club called Cheer Dance at university and did cheerleading so she came in wearing her uniform and auditioned and she performed a cheerleading routine on the spot it's easier for someone shorter to be in the top position we felt a connection between the two in that respect The staff unanimously decided Nakagawa was fit for the role Narenare -Cheer for you!- had a pre-screening event in Takasaki on April 18 Are there any other Narenare -Cheer for you!- events and dates anime fans might be interested in before the series begins in July fans who have already seen the pre-screenings know this but the last big promotional video with other members of the Pom-Poms will be released we're planning to hold several more pre-screenings of the series [with] announcements we have been uploading comments about once every two days called “One Line Pom-Poms” on the Official X account The posts use mini characters and illustrations and have gentle comments made by the girls to the audience and viewers that convey their own aura We publish interviews with each staff member called "How to Make Nare Nare" every Friday as you can hear from staff members who aren't the main staff who work on the anime if you've ever watched the ending credits and thought "I wonder what kind of work that is" or "What does the sound effects person do," we're currently interviewing everyone involved in making "Nare Nare" I'm sure they'll give us comments that we wouldn't normally hear We're even planning to interview Oyama and the others in charge of overseas productions later You said the Narenare -Cheer for you!- Official X account posted comments by each character Are the comments written by the voice actors Not exactly. With the help of P.A. Works we thought it was important for people to get to know the characters before the series aired we've been posting a little bit of encouragement with the characters so fans can have more opportunities to interact with them They are illustrations drawn by Takada Tomomi We also show Shion's usual mood when she's shopping We've been doing the One Line Pom-Poms for about three months now to expand on things like "I wonder if this character likes this kind of makeup" or "I wonder what this character is like?" They're a way to get to know the characters a little better and like them are there any things you'd like the audience to keep an eye out for while watching Narenare -Cheer for you!- There's one thing I'd like fans to see other than the story: the music. The opening and ending songs and some of the insert songs were written and composed by [Yujin] Kitagawa of Yuzu and I think they turned out to be excellent songs The other songs that appear in the series and the background music in the PV are also really appealing I would be very grateful if people would pay attention to the music the drama of the characters is quite central to the series when six high school girls get together and talk two are talking face to face while the others are also talking We're depicting this with a sense of realism There are quite a lot of overlapping lines These two are talking about something like a temple; one is talking about school But there are scenes [in Narenare -Cheer for you!-] where the six girls get together so I think it would be interesting if people paid attention to the relationships and interactions between the characters Will people be able to make out all the overlapping conversations Two girls go to Takanosaki High School in Takasaki two go to Omaebashi High School in Maebashi and Megumi have a conversation like "Thank you for always taking care of us," the others are over here talking about what kind of video they want to make the conversations are very realistic when they get together as a group so I think it would be fun if fans could watch it Narenare -Cheer for you!- will premiere in Japan in July. Fans can follow the series' English-language X account at @narenare_eng The Erie SeaWolves continued to swing the bats well last week as they won their second series in a row for the first time since early June the SeaWolves swept a doubleheader Thursday and dominated Friday 9-3 and Saturday 14-1 Erie has outscored Altoona 44-5 over the past four games Through their red-hot play over the past two weeks the 'Wolves climbed within five games of a playoff spot and jumped over Altoona in the standings Here are five things we learned from this past week: Cheryl and Homey Numata traveled from Hawaii to be with the SeaWolves this weekend The parents of the late Chace Numata arrived on Friday to catch up with everyone at UPMC Park and were on hand Saturday to celebrate Chace Numata Saturday would have been the former Erie catcher's 29th birthday and Homey Numata threw out the first pitch The first pitch took on a whole new meaning with the ball that was used SeaWolves president Greg Coleman received a call from the Heberley family A young fan named Liam Heberley picked up a ball after a home run in the parking lot during the seventh inning of the SeaWolves' game at the Fisher Cats on Aug Liam Heberley and his dad were on their way home after watching six innings and interacting with several SeaWolves They checked the box score the next day and figured out it was a home run hit by Chace Numata It turned out to be the last home run Chace Numata hit before the accident that took his life one month later The young man decided the Numata family needed to have the ball He sent a letter and the ball to Coleman a few months ago Coleman handed the ball to Homey Numata to throw out the first pitch Cheryl and Homey Numata were speechless as the emotions were present on the field The Numatas also visited Reading and other places they had been during Chace Numata's career and they were also able to visit Baltimore to see Spenser Watkins The Numatas brought leis made from actual flowers from Hawaii to Erie for the entire team The SeaWolves wore the leis during introductions Every hitter in baseball wants to be as hot as Riley Greene right now 2 prospect for the Detroit Tigers homered in his fourth straight game on Saturday The last SeaWolves hitter to homer in four straight games was Dean Green on May 10-13 Greene needed a home run Sunday in the series finale to match Tyler Collins Greene is on an eight-game winning streak and is 16-of-34 (.471) with six home runs and 17 RBIs during that stretch On Saturday he blasted a three-run home run to spark an eight-run inning Chasing historyKerry Carpenter blasted his 10th home run Saturday a three-run shot during an eight-run inning for the SeaWolves He became the fifth player on the team to reach double digits in home runs The franchise record for double-digit home run hitters is six The 2021 SeaWolves with double-digit homers include Josh Lester (19) Drew Ward has eight homers and Andre Lipcius has six the SeaWolves' pitchers have allowed just five runs in 32 innings Joey Wentz started the dominant pitching by allowing one run in 4⅔ innings Thursday before Henry Martinez tossed 2⅓ scoreless innings to get the win Elvin Rodriguez followed in Game 2 by almost throwing a shutout He went 6⅓ scoreless innings and Billy Lescher got the final two outs The big-time pitching continued Friday as Beau Brieske allowed two runs in four innings Chavez Fernander tossed a scoreless inning Chance Kirby allowed one run in 2⅔ innings and Gerson Moreno threw 1⅓ scoreless innings Jesus Rodriguez allowed one run in six innings before Yaya Chentouf tossed two shutout innings and Ruben Garcia didn't allow a run in the ninth inning Clutch hittingIn the current four-game winning streak the SeaWolves have come through at the plate with runners in scoring position Erie went 7-of-11 and 1-of-5 with runners in scoring position during the doubleheader The 'Wolves then went 1-for-10 on Friday and 8-of-18 on Saturday Erie is 17-of-44 (.386) at the plate with runners in scoring position Contact Tom Reisenweber at treisenweber@timesnews.com his friends and teammates recalled their fondest memories If you buy something from an SB Nation link, Vox Media may earn a commission. See our ethics statement the 27-year-old catcher for the Erie SeaWolves had passed away due to a head injury he suffered during a skateboarding accident and personnel with the SeaWolves took to social media to remember the young man they had played alongside and showing the world what a bright light Chace had been to them It was clear that Chace was the kind of man who brought brightness and joy wherever he went and those who knew him are missing him a great deal today Chace... I write this with tears in my eyes. I was your teammate for only one year, but feel as if we have been friends for life. You were truly too good for this earth. Thank you for who you are to me. I love you dewd Rest In Peace my friend #FORNUMI Numi was kind enough to share with me a story about the day he caught Roy Halladay. I hear the passion, thoughtfulness, and humor that touched us all. It breaks mine and many hearts to see you join him. Now, go show ‘Mr. Doc’ all you’ve learned.Surf in paradise #ForNumi pic.twitter.com/RhviuLp8tV Numi is the most genuine, kind hearted, and loving person I have ever met. Please help donate to raise money for him as his family as they go through this tough time. Love you brother! Stay strong and keep fighting. #ForNumi Click to Donate: https://t.co/pYr3f9JPMs @Tigers Numata had only been in the Tigers organization since November of 2018 joining as a minor league free agent signing He was originally drafted by the Phillies in 2010 and spent the bulk of his career in the Phillies system before joining the Yankees minor league system in 2018 He then came to the Tigers in that offseason and spent 2019 between Double-A Erie and Triple-A Toledo It was obvious from the memorials posted online that no matter where he was playing he brought light and happiness to those around him His most recent teammates on the SeaWolves were effusive in their praise for his personality and friendship Numi, I love you brother. You are one of the best guys I’ve ever met. Your energy and love was felt by everyone. My thoughts and prayers go out to your family. You’ve changed my life forever and your presence in the clubhouse was a gift. Rest In Peace brother. #ForNumi pic.twitter.com/AqvilIHE3H I can’t explain how tough today has been. Numi not only were you the best teammate but the most selfless, positive, and down to earth person I have ever met. Thank you for being you A post shared by Alex Faedo (@alexfaedo) on Sep 2 We are deeply saddened by the passing of Chace Numata, a BlueClaw in 2013 & 2015. He had a constant smile and an infectious personality, making a lasting impact on the BlueClaws and our community. Our thoughts and prayers are with his family. #ForNumi https://t.co/Rz5nGlpdEJ pic.twitter.com/Jf1DA7XOl2 an organization which helps Minor League families and spouses posted this lovely image of Numata and his girlfriend Gabriella We are incredibly saddened to hear the news of Chace Numata’s passing. He was described by his teammates as the “heart and soul” of the SeaWolves and will be deeply missed by so many who loved him. Please continue to pray for his family & his girlfriend Gabriella.❤️ pic.twitter.com/FQWtdpzt85 By Ferd Lewis Editors' PicksObituaries Chace Numata was an All-State utility player and helped the Pearl City High School Chargers to the 2010 Oahu Interscholastic Association baseball championship from injuries suffered in a skateboarding accident The Reading Fightin Phils’ Alec Bohm slid into home plate ahead of a tag by Erie SeaWolves catcher Numata during a game Aug Chace Kekoa Kenji Numata played in more than 500 professional baseball games and had more than 2,000 plate appearances in a 10-year career but it was the infectious smile and buoyant personality that registered the biggest impact “If you took a poll of the guys — who is your favorite teammate — it would be Chace Numata 25-0,” Erie (Pa.) SeaWolves hitting coach Brian Harper told the Detroit News after Numata died Monday in Erie from injuries suffered in a skateboarding accident early Friday morning was a switch-hitting catcher for the Double-A farm team of the Detroit Tigers Numata was an All-State utility player and helped the Chargers to the 2010 Oahu Interscholastic Association baseball championship According to reports based on video camera footage Numata’s skateboard suddenly stopped as if it had hit something causing him to fall headfirst to the ground who found Numata unconscious and bleeding from the head ”He had an engaging personality that quickly established him as a leader on and off the field and his presence will be forever remembered in our organization and beyond,” the Tigers said in a statement “We had him up (with the major league team) in spring training,” Tigers manager Ron Gardenhire told the media “It is a tough one for the organization Numata also spent a year in the New York Yankees system and eight with the Philadelphia Phillies all of whom mourned the death of the one they called “Numi.” Pitcher Logan Shore of the Tigers organization tweeted kind-hearted and loving person I have ever met.” Words cannot describe how blessed I am to encounter a spirit like yours You were the heart and soul of our locker room.” paid tribute to their teammates by playing his favorite songs in the clubhouse and writing his name on their hats Numata hit a combined .244 this season in time with the Tigers’ Triple-A and Double-A teams and .251 for his pro career In high school the versatile Numata was at his best in helping to lead the Chargers to the 2010 OIA championship As a shortstop he drove in a run in the 4-1 quarterfinal victory over Moanalua Numata made four assists in the first three innings he went to the mound in relief of his brother with two men on base and one out and retired the side without giving up a run In the seventh inning he struck out the side on 11 pitches to preserve the victory In the 4-3 championship game victory over Campbell he turned a pivotal double play in the first inning and went to the mound in the seventh inning to bail the Chargers out of a bases-loaded situation and secure the victory Numata had planned to attend Central Arizona community college until the Phillies made him a 14th-round pick in the June 2010 MLB draft “You made the world a better place and you just made heaven an even better place to be,” Numata’s agent Detroit – One of the first things Tigers catcher Jake Rogers did when he heard the news was to call his former teammate at Double-A Erie Tigers minor-league catcher Chace Numata is in a hospital in Erie suffering what is believed to be a head injury Numata was found injured on a downtown street early Friday morning after what police called a skateboarding accident Surveillance cameras on the street showed Numata skating down East Fourth Street.  The Tigers released the following statement: “We’ve been made aware that early on Friday morning SeaWolves catcher Chace Numata was involved in an accident in Erie We have been in contact with hospital officials and SeaWolves leadership about his condition All of us with the Detroit Tigers ask for Chace to be kept in your thoughts and prayers at this time.” SeaWolves team president Greg Coleman confirmed the accident to Erie News but Numata’s condition was not available Friday afternoon More: Tigers' Niko Goodrum weighs health vs. possible return this season "Just keep him in your prayers," Tigers manager Ron Gardenhire said "It's a tough situation and hopefully the doctors are taking care of it Rogers began the season in Erie and played with Numata for more than a month "He was the life of the clubhouse," he said And was a really good presence behind the plate for those guys." officers were called to the first block of East Fourth Street on Friday at about 2:15 a.m on a report of a man down and found Numata on the ground A motorist told police that he was driving through the area when he saw something in the roadway and drove around it Numata was transported to the hospital by ambulance from the scene Become a Sponsor I am a member of a group of amateur musicians called the South Buffalo Fresh Air Music Club we were lucky enough to be able to recruit the talented violinist/singer/songwriter we learned that besides her work as an elementary school teacher and member of the celebrated Bel Canto string quartet Blair is also a Teaching Artist at Buffalo String Works (BSW) Blair enthusiastically invited us to one of the BSW concerts in which her students were performing and we were amazed at the work not only of the students We also got to meet their energetic and talented Executive Director I realized she is one of the best kept secrets of Buffalo music and one which all Buffalonians may be interested in learning about I hope that you will enjoy getting to know the indefatigable Yuki Numata Resnick I hear that you have been busier than ever during this awful pandemic about a week after our in-person activities were suspended in March I was in my office trying to work through our next steps when I received a text from a student’s mother asking but delighted that despite everything going on the children and parents were interested in forging on Since then we’ve had an intensive virtual program – we held a 6-week virtual summer camp in partnership with Say Yes Buffalo and our students are attending virtual classes 3 days a week Can you believe we have a 96% attendance rate? It’s quite incredible and we’re so grateful our families have stood by us as I update this interview to be even more current we just started 25 new students in our brand new Intro to BSW curriculum and we have more students in the pipeline We wanted to take this opportunity to give the community and Buffalo String Works (“BSW”) supporters the chance to learn more about you and your interesting life My father was a Japanese chef and my mom was a special education school teacher When people talk about the immigrant story My father came from Japan and my mother from Vietnam and Hong Kong and both believed that through their values of hard work they could make a better life for my sister Both of my parents worked incredibly hard but I think that I was particularly influenced by my mother who really advocated for her students often well beyond the end of the school day I noticed that she seemed to see possibilities in her students that others couldn’t and she became kind of a pioneer in integrating children with disabilities into traditional classrooms She often said that she thought that music seemed to play a role in bringing out the best in students and I’ve tried to bring some of her best practices into our work at Buffalo String Works my dad says that I was so bad at first that he wanted me to quit but my mom wouldn’t let him because she said then I would always quit if I think something is hard and the belief that with practice you get to enjoy the fruits of your labor but both of my parents appreciated classical music This support of my parents was so important to me and set me apart from some of the other kids at school I became actively involved in the music program at school and also continued my after-school lessons and by age 12 I earned a spot in a local youth symphony with musicians ranging in age from 12-25 I remember these experiences now when I see our students at Buffalo String Works spotlighted on stage at our performances after all their hard work Those experiences were empowering for me and I think they are for our students as well learning to play music teaches life-long skills After high school in Canada you headed to the University of Rochester’s prestigious Eastman School of Music music was still fun but I began to realize the stakes were even higher once I got to college It was a formative experience for me and I still have close personal and professional relationships from my undergraduate years a legendary professor known for his stern demeanor But I realized not long after that this was his way of encouraging me to fulfill my potential and he turned out to be like my musical grandfather in many ways you earned a graduate degree at the University of Michigan and began working as a musician My first music job was in the New World Symphony in Miami This program was established by Michael Tilson Thomas best known as the San Francisco Symphony’s longtime Music Director who envisioned a training orchestra for aspiring professional musicians Completing graduate school gave me more time to myself and I enjoyed that I learned to love just being in a room by myself with my violin and practicing During this time I was also lucky enough to spend my summers playing and training at the Tanglewood Music Center in Lenox While at Tanglewood I met some great friends and incredible musicians who were based in New York City so I moved to Washington Heights and got busy with chamber music and performing new This was my first experience as a musician without a safety net It was exhilarating and nerve-wracking at times but I have such good memories from that time in my life you left out something about your time in New York He and I were both playing at the New Yorker Music Festival with the band “The National.” Living in New York I had such unique opportunities to explore music making from working with living composers like John Zorn and Max Richter to recording on albums with bands like Passion Pit It was a thrill to straddle many worlds at once and it taught me to be more flexible and fluid in my definition of what music means to me it’s made all the more sweeter because I did meet my partner in life through these new experiences Well during my time at the Eastman School of Music I had developed a relationship with UB from playing at their “June in Buffalo Festival,” so while I was living in New York City I was offered an adjunct professor role at UB taught all day and then flew home at night I ended up making a good impression and eventually I taught undergraduate and graduate students at UB and found that I loved teaching As much as I enjoyed performing I always felt that there was more to music than I was allowing it to be I loved the idea of passing on the teachings from my own mentors and found that it completed something for me What have been some of your most memorable performances Probably at the top of the list was performing as a soloist at the Sydney Opera House in Australia overnight piece that the audience is meant to sleep to The performance is timed with sunrise so that the final notes are played as the sun comes up Another highlight was playing my entire “For Ko” album (for my son Ko) at the Big Ears Festival in Knoxville Big Ears is this amazing endeavor headed up by Ashley Capps (who also produces the music and art festival Bonnaroo) that brings together musicians of all genre from classical artists to indie rock musicians and everyone in between I read recently that the New York Times called the last Big Ears “A weekend of performances that soothed jolted and intrigued.” I highly recommend it Tell us about about your most recent collaborations in the world of pop music who is a professional trumpeter and sound engineer and I were invited to contribute to Taylor Swift’s new 2020 albums “Folklore” and “Evermore.” As a touring musician Kyle has been temporarily unemployed because of the pandemic so he industriously turned our attic into an ad hoc recording studio that allowed us to record my violin and viola parts and Kyle’s trumpet parts—all from home Kyle also got to do some engineering work on the recording which was a tremendous opportunity for him I immediately think of my parents who sacrificed so much to give my sister and me the best life possible They instilled terrific values that make me love the work that I do They also have become outstanding grandparents to our two children and Buffalo boosters—making it here for visits about three times a year is carefree like my husband and is the happiest kid Ko has been spending lots of time tinkering on our piano at home He also takes violin lessons – not from me though since I tend to be so disciplined and orderly and watching what some would call fluff TV including Schitt’s Creek starring my fellow Canadian-native Kyle and I devoured HBO’s “Succession” and oh We’re currently making our way through “Shtisel” I absolutely watched all of “Bridgerton” during the holidays Well quarantine has forced me to become a better cook I have developed some video editing and website design skills with the help of our BSW Operations Manager is it ever too late to learn to play a musical instrument In fact we have a couple of parents at BSW who enjoy playing with their children We love receiving videos of informal family performances and it makes me so happy to see them make music together sent us a video of her playing violin with her children and she was yelling out the same instructions that we use: “1 go!” “Ok and repeat!”  She could be our next violin Teaching Artist Click here for Part II Website: buffalostringworks.org It’s not unusual for authors to come and go Guest authors range from collegiate interns to writers who will be contributing for a short stint of time Guest authors might also have a series in mind Authors are encouraged to submit their ideas to BRO (Buffalo Rising Online) upon which time we will work with the writer towards a productive end As one of the region’s most read publications since its launch in 2004 Buffalo Rising’s diverse editorial team delivers a wide range of relevant content to our readers as it unfolds in real time An independently owned and operated digital publication Buffalo Rising provides editorial and critical reviews on all topics that relate to Buffalo Click Here to Share Your Story 2023 at 4:47 pm ETA service for Masashi Numata will be held on June 3 (Courtesy of Marie Mockers Numata )(Courtesy of Gabe Tenaglia)PRINCETON Numata was in hospice care at the time of his death “The time of his death translates to 05/04/2023 in Japan which represents death departure and renewal,” his family said in a statement Numata was a 7th Degree Black Belt Master (SHIHAN) in the traditional Japanese Martial Art of Aikido Numata was born in Himeji Japan and raised in Shioanji Temple as an orphan from age 6 He began studying Aikido in his early 20s under Bansen Tanaka Sensei Numata moved to Princeton with his wife Marie Mockers Numata in 2001 to establish Princeton Aikikai under the leadership of Yamada Sensei teaching youth and adult classes until the COVID-19 pandemic Katzenbach School for the Deaf for over 10 years "He has touched the lives of many Aikido practitioners through his dedication to teaching Aikido His humble unwavering commitment to his students both in the USA and Japan has had a profound and positive impact on students’ lives,” his family said Numata’s final wish was for his body to be cremated and his ashes to rest under a tree A funeral service will be held the All Saints Church in Princeton on June 3 Marie and his three children - Taketeru Steve Numata Kiyoka Arielle Numata and Ayaka Aurelie Numata His family has thanked the following people for their support during this difficult time: Rev Alphonsus Catholic Church; the staff of Holy Redeemer Health Hospice Care Team; staff at Penn Medicine Princeton Medical Center; Princeton Dog Training Center; Princeton community members; Robbinsville High School staff; Princeton Aikikai students and friends and family Get more local news delivered straight to your inbox. Sign up for free Patch newsletters and alerts. Former President and CEO of ELM Design Co. After working at a major electronics manufacturer he joined Yamaha Motor design subsidiary IDL (Integrated Design Laboratory) in 1982 Working initially on designing 50cc scooters for the Japanese domestic market a subsidiary created by the merging of YAC Co. which had been handling concept model design work Numata was in charge of designing the Majesty maxi-scooter for the European and Japanese markets and later designing the Mio scooter and other models for ASEAN markets It was at an evaluation meeting held in the summer of 2011 Yamaha Motor was creating a new in-house design division and we were debating whether or not ELM Design should be absorbed into it mulling over and discussing this issue for quite some time and at the end of it all it was decided that ELM Design would be absorbed But I was the only one who voted against it I was proud of the designers that made up ELM Design and the company I’d nurtured to try to take our product design capabilities to the highest level Considering design excellence to be the end-all be-all we implemented all kinds of measures to push our limits Yamaha Motor had always been a company that believed product design should be left to professionals and that’s how the company actually operated Yamaha’s stance of keeping design work separated from the rest of the company to avoid the influence of in-house theorizing was a stance also widely known throughout the design world Even many of the designers at the boat design office—Yamaha’s only remaining in-house design group at the time—moved to join ELM Design in 1995 Without connecting product design to brand strategy you can’t profess to be a company that truly values design If we were to honestly admit our devotion to the importance of design we wouldn’t have hesitated to accept this change because we ourselves had decided to expand our fields of activity and aim for greater heights one aspect of both IDL and later ELM Design was that they were group companies that had given birth to new vectors of value in areas besides motorcycles What also set them apart was that they both had a large number of designers with backgrounds in home appliances The mindset that we should always create things that were innovative and stylish was something strongly engrained in our DNA there are always moments of doubt when creating something new What’s scary is saying something is “new” when it’s merely a form of self-indulgence or overeagerness Although it may not be comparable to Darwin’s theory of evolution it’s very important to stop and ask yourself if you would have made a different choice at the moment some new evolution took place The reason for a product’s form that you had believed in unquestioningly until then may already be irrelevant because of new advances in manufacturing technology and pioneering new and previously unknown fields is great fun High school players played in the Chace Numata Senior All-Star Game on Saturday at Joey DeSa Field By Andrew Lee / Special to the Star-Advertiser Hawaii PhotosPrep sports Ohio — They miss Numi’s voice and how he would scream: “Frick yeah!” And they miss his energy and encouragement Chace (Numi) Numata, the "heart and soul" of the Erie SeaWolves, the Detroit Tigers Double-A Affiliate, suffered a serious head injury on Friday morning in a skateboarding accident in Erie. trying to get into the Eastern League playoffs — although they were eliminated Sunday night — Numata has remained in the hospital “There is no manual for how you deal with this,” SeaWolves manager Mike Rabelo said They are playing with heavy hearts.” The approximately two-hour bus drive from Erie to Akron on Friday was emotional “Everybody was checking their phones trying to figure out what just happened.” The Tigers have not specifically announced his condition although it is obviously a serious situation Rabelo gave his players phone numbers to call for counseling at a team meeting on Friday and Brian Harper “I’m a Christian and I believe God answers prayers and I believe that’s our biggest vehicle to help Chace “I know Chase is a believer and believes in God I’m going to pray for Chace – we can pray as a team for him,’” he said touch Chace and bring him back to us.’” Several players have written “Numi” on their caps bought a pair of cleats and they wrote his favorite sayings in black marker He lives in an apartment in Erie with Cam Gibson and Derek Hill “He’s a giver,” Gibson said I can’t tell you the times he’s had heart to heart talks with me this year talking to me about what my future holds “It’s almost like you feel bad if you get a little happy,” Gibson said “It doesn’t feel right to be happy when someone that you love is suffering But here is the crazy thing: This would be driving Numi crazy “If we were in the dugout and it was somber Numi would say something or make a fool of himself to make sure everybody snaps out of that trance they are in,” Gibson said “He’s the most joyful kid I know,” Hill said I knew I wanted to be his friend,” Hill said “You want to gravitate toward him because of his personality He was one of the big leaders on this team – emotionally even if we wanted to talk something.” It’s been such a strange season for the SeaWolves From the Alex Faedo and Drew Carlton combined no-hitter, Erie’s first in almost 10 years To the magical night that Casey Mize threw a no-hitter in his Double-A debut To the injuries and shutdowns – Faedo was out for nearly a month with a back strain and Mize has been shut down in the middle of a playoff push to rest his arm And while the SeaWolves have fought – they were eliminated from playoff contension on the second-to-last day of the season – everything is out of whack without their catcher “It’s just different,” pitcher Tarik Skubal said For Numi is the thing that is going around When the Tigers put Numata on the injured list an undrafted free agent who was playing in High-A Lakeland The Florida State League ended its season early because of Hurricane Dorian and he was flying home to Canada “I didn’t have my catcher gear with me," MacLaren said "So the (clubhouse manager) in Erie sent me some catcher gear for me I brought my bat and my gloves and spikes and stuff A GoFundMe account was formed to pay for medical expenses More: Here's how to help pay for medical bills the SeaWolves keep playing – the season finale is Monday afternoon – but nothing is the same Contact Jeff Seidel: jseidel@freepress.com. Follow him on Twitter @seideljeff. To read his recent columns, go to freep.com/sports/jeff-seidel Metrics details Animal cells can regulate their volume after swelling by the regulatory volume decrease (RVD) mechanism RVD is attained through KCl release mediated via volume-sensitive outwardly rectifying Cl− channels (VSOR) and Ca2+-activated K+ channels Swelling-induced activation of TRPM7 cation channels leads to Ca2+ influx we examined whether TRPM7 plays any role in VSOR activation When TRPM7 was knocked down in human HeLa cells or knocked out in chicken DT40 cells not only TRPM7 activity and RVD efficacy but also VSOR activity were suppressed Heterologous expression of TRPM7 in TRPM7-deficient DT40 cells rescued both VSOR activity and RVD accompanied by an increase in the expression of LRRC8A TRPM7 exerts the facilitating action on VSOR activity first by enhancing molecular expression of LRRC8A mRNA through the mediation of steady-state Ca2+ influx and second by stabilizing the plasmalemmal expression of LRRC8A protein through the interaction between LRRC8A and the C-terminal domain of TRPM7 TRPM7 functions as an essential regulator of VSOR activity and LRRC8A expression While it is thus clear that there is a functional coupling between TRPM7 and Ca2+-activated K+ channels the possibility of functional coupling between TRPM7 and the volume-sensitive Cl− channel has not been studied it is intriguing to examine whether TRPM7 exhibits any molecular interaction with LRRC8A we used electrophysiological and genetic approaches to investigate functional and molecular interactions between VSOR/LRRC8A and TRPM7 in human epithelial cell-derived HeLa cells and chicken B cell-derived DT40 cells We demonstrate that TRPM7 expression is essential for LRRC8A expression and VSOR activity and thereby playing a crucial prerequisite role in the RVD process a Representative whole-cell current traces elicited by step pulses in isotonic and hypotonic conditions in Mock-transfected (Mock) and siRNA-TRPM7-treated (siRNA) cells b Mean current (I)–voltage (V) relationships for swelling-activated whole-cell currents observed in Mock and siRNA cells (n = 12–18) Each data point represents the mean ± SEM (vertical bar) of n samples respectively) <0.005 compared to the Mock cell data by t-test c Effects of TRPM7 knockdown on expression of TRPM7 and LRRC8A mRNAs Left panel shows the PCR products from Mock or siRNA cells for TRPM7 and the constitutively transcribed control The nucleotide sequences of the PCR products obtained with TRPM7- and LRRC8A-specific primers were completely identical to the corresponding sequences for TRPM7 (human: NM_017672) and LRRC8A (human: NM_001127244) Right panels show the bar graph representation of the relative expression values of the optical densities in pixels of the PCR bands of TRPM7 (left bars: 45.3 ± 5.1%) and LRRC8A (right bars: 59.4 ± 3.4%) in siRNA cells compared to those in Mock cells The values were calculated from five independent PCR amplifications after normalization to the corresponding band of GAPDH control *P (=0.00013 and 0.00010 for TRPM7 and LRRC8A respectively) <0.0005 compared to Mock cell data by t-test The downregulation of LRRC8A expression in TRPM7-silenced HeLa cells supports the existence of a molecular relationship between TRPM7 and LRRC8A that be responsible for the observed functional coupling between TRPM7 and VSOR activity in HeLa cells failed to affect the VSOR whole-cell currents (a b) and the endogenous expression of hLRRC8A (c) in HeLa cells a Representative I–V relationships of currents in response to ramp pulses (50 ms duration) from −100 to +100 mV before (control) and after 2-day treatment with 30 µM NS8593 (NS8593) b Mean peak current densities at +100 mV in the absence (black column) and presence of NS8593 (red column) (n = 6) * indicates a P (=0.000058) value of <0.0001 compared to the corresponding control (left column) by t-test c Cytosolic Ca2+ levels were determined by fura-2 ratio imaging in the cells before (control) and after treatment with 30 µM NS8593 for 2 days (NS8593) and TRPM7-siRNA for 2–3 days (siRNA) (n = 24) respectively) <0.0001 compared to the corresponding control data by one-way ANOVA followed by the post hoc Tukey’s test d Representative PCR products of human TRPM7 and GAPDH in the cells before (control) and after 2-day treatment with 30 µM NS8593 (NS8593) or 2 mM EGTA (EGTA) from three independent experiments The expression of TRPM7 and LRRC8A mRNAs was greatly decreased in NS8593- or EGTA-treated cells while that of GAPDH mRNA stayed almost constant The nucleotide sequences of the PCR products obtained with TRPM7- and LRRC8A-specific primers were completely identical to the corresponding sequences in TRPM7 (human: NM_017672) and LRRC8A (human: NM_001127244) e Swelling-induced peak VSOR current densities recorded at +40 mV before (control) and after treatment with NS8593 (NS8593) Each column represents the mean ± SEM (vertical bar) of n samples respectively) <0.05 compared to corresponding control data by one-way ANOVA followed by the post hoc Tukey’s test a Representative recording showing the time course of whole-cell currents recorded at +100 and −100 mV during hypotonic stimulation in the cells before and after extracellular application of 1 mM Mg2+ (Mg2+) Currents were elicited by ramp-clamp (every 5 s at 4 mV/ms) Bars above the recording indicate the time during applications of hypotonic solution (Hypo) and Mg2+ b Representative I–V relationships of currents in response to ramp pulses from −100 to +100 mV before (at i in a) and during application of Mg2+ (at ii in a) c Peak current densities at +100 (blank columns) and –100 mV (filled columns) before (0Mg2+) and after application of 1 mM Mg2+ (1Mg2+) (n = 13) respectively) <0.05 compared to corresponding data in 0Mg2+ group by t-test a Whole-cell current response to hypotonic stimulation Upper left panel shows representative recording exhibiting the time courses of whole-cell currents observed at +30 mV (open circles) and –30 mV (filled circles) which correspond to the equilibrium potentials for cations and anions under the ionic conditions employed for this recording Lower left panel shows the reversal potentials (Erev: open triangles) after application of hypotonic solution (Hypo) in WT DT40 cells Currents were elicited by ramp-clamp (every 10 s at 4 mV/ms) The value of Erev was evaluated by analyzing the measured reversal potentials of the all current traces in response to ramp pulses Right panels show representative I–V relationships of currents in response to ramp pulses from –100 to +100 mV applied at (i) Insets represent I–V curves expanded in the y-axis direction c Relationships between the mean peak current densities recorded at +30 mV (VSOR activity) and those at –30 mV (M7 activity) (n = 7) in WT DT40 cells (b) and in gTRPM7-deficient DT40 cells complemented with hTRPM7 (c) a PCR products of human or chicken TRPM7 gTRPM7-KO DT40 cells (KO) and hTRPM7-expressing KO cells (+hM7) The expression of chicken LRRC8A mRNA was greatly decreased in KO cells and increased in +hM7 cells The nucleotide sequences of the PCR products obtained with TRPM7- and LRRC8A-specific primers were completely identical to the corresponding sequences in TRPM7 (human: NM_017672; chicken: NM_001177555) and LRRC8A (chicken: XM_015279569.2) b The bar graphs represent the relative expression values of the optical densities in pixels of the PCR bands of gLRRC8A compared to the corresponding band of ACTB control These values were calculated from six independent PCR amplifications respectively) <0.05 compared to control data by one-way ANOVA followed by the post hoc Tukey’s test d Time courses of changes in the mean cell volume of WT DT40 cells after a hypotonic challenge (Hypo: 78% osmolarily) The RVD event was inhibited by DCPIB (10 µM) (c) and gTRPM7-KO (KO) whereas it was facilitated in hTRPM7-expressing KO cells (+hM7) (d) e Summarized data showing the percent volume recovery (RVD) at 30 min after application of hypotonic solution (n = 6–20) respectively) <0.05 compared to corresponding data in control data by one-way ANOVA followed by the post hoc Tukey’s test these experiments show that TRPM7 expression upregulates the molecular expression of LRRC8A thereby increasing VSOR activity and enhancing RVD efficacy a Representative fluorescence micrographs of gTRPM7-KO DT40 cells immunostained with anti-HA antibody before (KO) and after complementary expression with WT hTRPM7 or its mutant (Alexa-488: green) as well as their bright field micrographs b The percentage of TRPM7 localization to the plasma membrane is significantly lower in the C-terminal defects (+Δ-kinase: *P (=1.1 × 10−233) <0.0001 compared to +WT data by χ2 test) Observations and analyses were performed from 53–110 individual cells The data are representative of three independent experiments d Representative whole-cell VSOR currents elicited by step pulses in isotonic conditions (Iso) and in hypotonic conditions (Hypo) in gTRPM7-knockout (KO) DT40 cells and those complemented with hTRPM7 (+WT) e Peak VSOR current densities recorded at +100 mV (n = 8–26) *P (=0.000000000069 and 0.0000000029 for KO and +Δ-kinase respectively) <0.0001 compared to corresponding +WT data by one-way ANOVA followed by the post hoc Tukey’s test f Representative I–V relationships of whole-cell TRPM7 currents in KO and +K1648R DT40 cells exposed to hypotonic (86% osmolarity) solution The I–V curves were recorded under ramp-clamp (50-ms duration applied every 5 s at 4 mV/ms) from −100 to +100 mV in the absence (i g Peak current densities recorded at +100 mV (blank columns) and –100 mV (filled columns) in hypotonic conditions (n = 6–19) *P (=0.0000029 and 0.0012 at –100 mV and 0.00000000020 and 0.0000015 at +100 mV for KO and +Δ-kinase respectively) <0.005 compared to corresponding data in +WT cells by one-way ANOVA followed by the post hoc Tukey’s test These results suggest that TRPM7-mediated regulation of VSOR activity is independent of kinase activity of TRPM7 but is dependent on the presence of its cytosolic α-kinase domain a Confocal images of DAPI-stained HeLa cells before (DAPI) and after immunostaining with anti-GFP antibody for WT hTRPM7 or its mutant (TRPM7-EGFP: green) and those with anti-mCherry antibody for LRRC8A (LRRC8A-mCherry: red) during exposure to hypotonic solution (78% osmolarily) b The percentage of LRRC8A localization to the plasma membrane (PM) is significantly lower for the TRPM7 C-terminal defect (+Δ-kinase hypo: *P (=0.00060) <0.001 compared to corresponding WT data by χ2 test) Observations and analyses were performed from 14 to 121 individual cells c Co-immunoprecipitation of hTRPM7-EGFP (WT) or -K1648R-EGFP with LRRC8A-mCherry in HEK293T cells co-transfected with hLRRC8A-mCherry and EGFP-tagged hTRPM7 or its mutant Immunoprecipitations (IP) with a GFP-specific antibody were subjected to western blot with an antibody to mCherry aliquots of sample are loaded on a separate gel it is concluded that the enzyme activity of α-kinase domain is not involved in the regulatory role of TRPM7 on VSOR expression and activity The present study adds to this growing theme of functional and molecular interactions between TRP cation channels and anion channels in regulating important aspects of cell physiology such as RVD The latter occurs when swelling-induced TRPM7 activation augments both functional activities of VSOR Cl− channels and molecular expression of the core component of VSOR that TRPM7 dually exerts an augmenting action on VSOR activity: first by molecularly increasing expression of LRRC8A mRNA through the mediation of steady-state Ca2+ influx and second by stabilizing the plasmalemmal expression of LRRC8A protein through the molecular interaction with the C-terminal α-kinase domain in a manner independent of its enzyme activity the molecular and functional link between TRPM7 and VSOR could an efficient target for therapeutic development and clinical treatments Human cervical HeLa cells or human embryonic kidney 293T (HEK293T) cells were grown as a monolayer in modified Eagle’s medium or Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 100 μg/ml streptomycin under 95% air–5% CO2 atmosphere at 37 °C the cells were detached from the plastic substrate and cultured in suspension with stirring for 15–300 min HeLa cells were provided after culturing for 2 days in the presence of 30 μM NS8593 or 2 mM EGTA Chicken B cell-derived DT40 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 μg/ml streptomycin in a humidified 95% air the pcDNATM4/TO vector was prepared to contain a sequence in which two tetracycline operator sequences (TetO2) are inserted between the TATA box of the CMV promoter and the transcription initiation site The hTRPM7-constructed pcDNATM4/TO vectors (Invitrogen) were transfected into a gTRPM7-deficient DT40 cell line and subjected to a drug selectivity test supplemented with blasticidin and zeocin the end of full-length human TRPM7 cDNA and human TRPM4 cDNA was cleaved with an appropriate restriction enzyme and inserted into the multicloning site of pIRES2‐EGFP Cells were dissociated by mechanical agitation and lodged onto coverslips placed in tissue culture dishes Membrane currents of these cells were recorded at room temperature (22–27 °C) using the whole-cell mode of the patch-clamp technique with an Axopatch 200B (Axon Instruments/Molecular Devices patch electrodes prepared from borosilicate glass capillaries had an input resistance of 3–7 MΩ Current signals were filtered at 5 kHz with a four-pole Bessel filter and digitized at 20 kHz pCLAMP software (version 10.5.1.0; Axon Instruments/Molecular Devices) was used for command pulse control Data were also analyzed using Origin software (OriginLab Corp. the series resistance was compensated (to 70–80%) to minimize voltage errors and the liquid junction potential emerged between the extracellular solution and the pipette solution was evaluated by using Clampex software (Axon Instruments/Molecular Devices) and compensated before each experiment cell swelling was induced by adjusting the intracellular osmolality to 360 mosmol/kg H2O with D-mannitol and recorded the amplitude of whole-cell currents To evaluate the Pasp/PCl value for VSOR currents in DT40 cells CsCl was replaced with Cs-aspartate in the pipette solution The bath solution contained (in mM): 100 Na-aspartate (or Cs-aspartate) and 10 HEPES (pH adjusted to 7.4 with NaOH (or CsOH) and osmolality adjusted to 320 mosmol/kg H2O with D-mannitol) The liquid junction potential emerged between these solutions was evaluated to be +7.9 mV To estimate the PNa/PCs value for TRPM7 currents in DT40 cells Cs-aspartate was replaced with Na-aspartate in the pipette solution In the case of simultaneous measurements of cationic and anionic macroscopic currents for Fig. 6 the pipette solution was replaced with (in mM) 28.5 CsCl and 0.3 GTP (pH 7.4 adjusted with CsOH; and osmolality adjusted to 300 mosmol/kg H2O with D-mannitol; ionic strength ~130 mM) The external solution containing (in mM) 110 CsCl and osmolality adjusted to 320 mosmol/kg H2O in isotonic solution or 275 mosmol/kg H2O in hypotonic solution with D-mannitol) The liquid junction potential emerged between these solutions was evaluated to be +0.6 mV where VCtl and VA are the mean cell volumes before and after hypotonic challenge A negative control siRNA conjugated with Alexa Fluor 488 (AllStars; Qiagen Cells were used in experiments 48–72 h after siRNA transfection gTRPM7-deficient DT40 cells were transfected with a recombinant plasmid pIRES2‐EGFP vector (Clontech DNA and lipofectamine 2000 were mixed in Opti-MEMTM medium (Thermo Fisher Scientific) to form a complex The formed complex was added to a 6-cm non-coated dish containing DT40 cells and allowed to stand in a CO2 incubator at 37 °C for 6 h the cells were transferred to and incubated in fresh medium for an additional 24–48 h Transfection of LRRC8A-mCherry with pEGFP-N or TRPM7-EGFP into HEK293T or HeLa cells were also performed by using Lipofectamine 2000 for 36–48 h gTRPM7-deficient DT40 cells stably expressing hTRPM7-WT, -K1648R, or -∆-kinase were grown on 6-cm dishes. For the experiments of Fig. 9 or ∆-kinase-EGFP and hLRRC8A-mCherry were co-transfected in HeLa cells Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 The cells were incubated with Anti-HA (1:1000 dilution USA) primary antibody followed by subsequent incubation with the anti-rat Alexa-488-conjugated (1:4000 dilution anti-rabbit Alexa-488-conjugated (1:4000 dilution or anti-mouse Alexa-647-conjugated (1:4000 dilution A-21236; Thermo Fisher Scientific) secondary antibody Fluorescence images were acquired with a confocal laser-scanning microscope (Zeiss LSM710 and line analyses were made with ZEN software (Carl Zeiss) The intracellular Ca2+ concentration was measured by ratio imaging of fura-2 fluorescence using SPARK10M plate reader (Tecan Group Ltd. After culturing 1 × 104 HeLa cells in 96-wells Fura-2 acetoxymethyl (2.5 μM: Dojindo Laboratories Japan) in DMEM medium was added to each well of a 96-well plate and the plate was incubated at 37 °C for 30 min and then washed twice with Tyrode solution containing (in mM) 140 NaCl and 10 D-glucose (pH adjusted to 7.4 with NaOH and osmolality adjusted to 300 mosmol/kg H2O with D-mannitol) Plates were cultured at 37 °C for 1 h to stabilize cells in Tyrode solution By exciting fura-2 fluorescence at 340/380 nm intracellular Ca2+ fluorescence signals were measured using a 510-nm filter Fluorescence intensity was calculated as the F340/F380 ratio and shown as changes in the intracellular free Ca2+ concentration gTRPM7-deficient DT40 cells and those stably expressing hTRPM7-WT Expression of hTRPM7 and its mutants was induced by adding 1 µg/ml tetracycline to the medium and culturing for 24–48 h HEK293T cells were cultured for 48 h under each condition DT40 or HEK293T cells were lysed in radioimmunoprecipitation assay buffer (pH 8.0) containing 0.1% SDS and then centrifuged at 17,400 g for 15 min Whole-cell lysates were fractionated by 7.5% SDS-PAGE and electrophoresed on a poly-vinylidene fluoride (PVDF) membrane The blots were incubated with primary antibody anti-HA antibody (1:1000 dilution: 06340; Nacalai Tesque Japan) or anti-α-tubulin (1:2000 dilution: T6074; Sigma-Aldrich) as internal standard and then incubated with secondary antibody of mouse IgG (1:3000 dilution Amersham enhanced chemiluminescence (ECL): Amersham UK) and HRP-linked whole sheep Ab (1:3000 dilution: NA931; Merck Co-immunoprecipitation was performed by using the Capturem™ IP & Co-IP Kit (Takara-Bio) and according to the manufacturer’s instructions HEK293T cells were solubilized in Lysis/Equilibration Buffer containing Protease Inhibitor Cocktail then centrifuged at 17,400 g for 20 min to remove cell debris the cell lysate was incubated with anti-GFP polyclonal antibody (1:1000 dilution: 50430-2-AP; Proteintech) for 1 h Then immunoprecipitated proteins were fractionated by 7.5% SDS-PAGE and electrotransferred onto a PVDF membrane The blots were incubated with anti-mCherry monoclonal antibody (1:1000 dilution: GTX630189; GeneTex Inc.) or anti-α-tubulin and visualized using the ECL system (Thermo Fisher Scientific) The chemiluminescence of the membrane was detected by the LAS system (LAS-3000; Fujifilm Japan) and analyzed by the Image Gauge software (Fujifilm) the χ2 test was made to evaluate the association between two categorical variables with a value of P < 0.05 considered to be significant Further information on research design is available in the Nature Research Reporting Summary linked to this article All the source raw data used for generating main Figures are provided as Supplementary data files and are available from the corresponding author on reasonable request Ion channels and transporters involved in cell volume regulation and sensor mechanisms Physiology of cell volume regulation in vertebrates Ca2+ sensitivity of volume-regulatory K+ and Cl− channels in cultured human epithelial cells Activation of K+ and Cl− channels in MDCK cells during volume regulation in hypotonic media Hypotonically activated chloride current in HSG cells Stretch-induced activation of Ca2(+)-permeable ion channels is involved in the volume regulation of hypotonically swollen epithelial cells Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels Cell swelling activates K+ and Cl− channels as well as nonselective stretch-activated cation channels in Ehrlich ascites tumor cells TRPM7 is a stretch- and swelling-activated cation channel involved in volume regulation in human epithelial cells Role of potassium and chloride channels in volume regulation by T lymphocytes Volume expansion-sensing outward-rectifier Cl− channel: fresh start to the molecular identity and volume sensor Properties of volume-regulated anion channels in mammalian cells Cellular and molecular physiology of volume-sensitive anion channels is an essential component of volume-regulated anion channel Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC Selective transport of neurotransmitters and modulators by distinct volume-regulated LRRC8 anion channels Distinct contributions of LRRC8A and its paralogs to the VSOR anion channel from those of the ASOR anion channel Molecular identities and ATP release activities of two types of volume-regulatory anion channels Cell volume-activated and volume-correlated anion channels in mammalian cells: their biophysical The leucine-rich repeat as a protein recognition motif Distinct pharmacological and molecular properties of the acid-sensitive outwardly rectifying (ASOR) anion channel from those of the volume-sensitive outwardly rectifying (VSOR) anion channel TRPM7 is involved in acid-induced necrotic cell death in a manner sensitive to progesterone in human cervical cancer cells Direct mechano-stress sensitivity of TRPM7 channel Specific and essential but not sufficient roles of LRRC8A in the activity of volume-sensitive outwardly rectifying anion channel (VSOR) LRRC8A is essential for swelling-activated chloride current and for regulatory volume decrease in astrocytes Molecular composition and heterogeneity of the LRRC8-containing swelling-activated osmolyte channels in primary rat astrocytes Transient receptor potential channels in mechanosensing and cell volume regulation Natural and synthetic modulators of SK (K(ca)2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7 The chicken B cell line DT40: a novel tool for gene disruption experiments DCPIB is a novel selective blocker of I(Cl,swell) and prevents swelling-induced shortening of guinea-pig atrial action potential duration LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability Polyvalent cations as permeant probes of MIC and TRPM7 pores The modulation of TRPM7 currents by nafamostat mesilate depends directly upon extracellular concentrations of divalent cations Biophysics and physiology of the volume-regulated anion channel (VRAC)/volume-sensitive outwardly rectifying anion channel (VSOR) a bifunctional protein with kinase and ion channel activities Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7 Crystal structure of the atypical protein kinase domain of a TRP channel with phosphotransferase activity Volume-sensitive organic osmolyte/anion channels in cancer: novel approaches to studying channel modulation employing proteomics technologies Transient receptor potential cation channels in disease Molecular determinants of sensitivity and conductivity of human TRPM7 to Mg2+ and Ca2+ Functional coupling of TRPC2 cation channels and the calcium-activated anion channels in rat thyroid cells: implications for iodide homeostasis A functional tandem between transient receptor potential canonical channels 6 and calcium-dependent chloride channels in human epithelial cells Coupling of TRPV6 and TMEM16A in epithelial principal cells of the rat epididymis TRPV4 heats up ANO1-dependent exocrine gland fluid secretion Modulation of water efflux through functional interaction between TRPV4 and TMEM16A/anoctamin 1 Pain-enhancing mechanism through interaction between TRPV1 and anoctamin 1 in sensory neurons Transient receptor potential vanilloid 1 is required for intrinsic osmoreception in organum vasculosum lamina terminalis neurons and for normal thirst responses to systemic hyperosmolality An N-terminal variant of Trpv1 channel is required for osmosensory transduction TRPV2 is a component of osmotically sensitive cation channels in murine aortic myocytes Hypotonicity induces TRPV4-mediated nociception in rat Functional TRPV4 channels are expressed in human airway smooth muscle cells A TRPV family ion channel required for hearing in Drosophila Vanilloid receptor-related osmotically activated channel (VR-OAC) Hypotonic shocks activate rat TRPV4 in yeast in the absence of polyunsaturated fatty acids Differential activation of the volume-sensitive cation channel TRP12 (OTRPC4) and volume-regulated anion currents in HEK-293 cells The mechanosensitive nature of TRPV channels a nonselective cation channel that confers sensitivity to extracellular osmolarity Dual role of the TRPV4 channel as a sensor of flow and osmolality in renal epithelial cells Evidence that TRPC1 (transient receptor potential canonical 1) forms a Ca(2+)-permeable channel linked to the regulation of cell volume in liver cells obtained using small interfering RNA targeted against TRPC1 TRPC1 forms the stretch-activated cation channel in vertebrate cells Hypoosmotic- and pressure-induced membrane stretch activate TRPC5 channels Synergistic activation of vascular TRPC6 channel by receptor and mechanical stimulation via phospholipase C/diacylglycerol and phospholipase A2/omega-hydroxylase/20-HETE pathways A common mechanism underlies stretch activation and receptor activation of TRPC6 channels Hypotonicity-induced cell swelling activates TRPA1 Regulation of the murine TRPP3 channel by voltage Molecular and functional characterization of the melastatin-related cation channel TRPM3 Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia TRPV4 exhibits a functional role in cell-volume regulation Dependence of regulatory volume decrease on transient receptor potential vanilloid 4 (TRPV4) expression in human corneal epithelial cells On the role of TRPC1 in control of Ca2+ influx Involvement of Ca2(+)-induced Ca2+ release in the volume regulation of human epithelial cells exposed to a hypotonic medium A necessary role for cell shrinkage in apoptosis Cryo-EM structure of the volume-regulated anion channel LRRC8D isoform identifies features important for substrate permeation Uncovering the arrhythmogenic potential of TRPM4 activation in atrial-derived HL-1 cells using novel recording and numerical approaches Reduction of ionic strength activates single volume-regulated anion channels (VRAC) in endothelial cells Activation of volume-regulated chloride currents by reduction of intracellular ionic strength in bovine endothelial cells Intracellular ionic strength regulates the volume sensitivity of a swelling-activated anion channel Reduced intracellular ionic strength as the initial trigger for activation of endothelial volume-regulated anion channels LRRC8 proteins form volume-regulated anion channels that sense ionic strength Studies of the mechanism of activation of the volume-regulated anion channel in rat pancreatic beta-cells Endoglin selectively modulates transient receptor potential channel expression in left and right heart failure Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells Download references Perraud for providing DT40 cells and those expressing TRPM7 mutants and for advice on the culture technique of these cells This work was supported in part by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science and the Ministry of Education JP20H05842 (Grant-in-Aid for Transformative Research Areas and Central Research Institute of Fukuoka University (No Japan Society for the Promotion of Science Department of Synthetic Chemistry and Biological Chemistry National Institute for Physiological Sciences and approved the final version of manuscript Download citation DOI: https://doi.org/10.1038/s42003-021-02127-9 The Journal of Physiological Sciences (2024) Pflügers Archiv - European Journal of Physiology (2024) The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown 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For all open access content, the relevant licensing terms apply. but were a big part of what made many classic games so special In a time when memory limited what games could do working cohesively with the graphics and story could weave together a cinematic narrative that conveyed a whole lot of emotion the ex-Sega composer behind some of the most memorable soundtracks of the 8- and 16-bit era She began by adapting existing music for Sega Master System / Game Gear conversions like Psychic World and Sega Genesis arcade ports like Ghouls ‘n Ghosts and Forgotten Worlds Then she began composing original music for games like Phantasy Star III Phantasy Star IV: The End of the Millennium the SMS/GG Land of Illusion Starring Mickey Mouse IPPO explained how her musical roots go back to when she was a student at a technical college “There were very few female students and even fewer students who could play keyboard instruments like the piano I belonged to five bands and was very busy at the time of the school festival an experience which proved very useful in my later years.” She learned it was looking for new hires in the music department and she was interested in applying and she had never composed anything before “I didn’t have any tools when I was a student “The only thing I had was an analog synthesizer (POLY800 I bought this with my savings to use in the bands Sega required me to bring a cassette tape with a tune I composed to the job interview I only had a cassette recorder which could play/record two cassette tapes at the same time So I played the bass guitar and recorded the sound then played the next part to the sound of the cassette tape and recorded the combined sound I played the next part to the previous sound and repeated this classic method to compose It took a very long time because if I made one slight mistake those two player/recorder’s speeds were slightly different and as I played the pitch shifted gradually So I had to tune the instrument every time I played the new part ‘How could I do such hard work!’ I composed three tunes and brought the tape to the job interview.” they liked the compositions and she got the job One of the first assignments she received was converting the music from the side-scrolling arcade platformer to the Mega Drive (the Japanese counterpart of the Sega Genesis) It was difficult because the two versions had very different sound data “Capcom had given me the sound data of the arcade version and I was supposed to transfer the data to the console My boss told me not to change the sound data but to change the sound driver and I wasn’t used to that kind of programming I managed to do the job asking questions to my colleagues and the programmers ‘You should do this,’ or ‘You can do it this way IPPO found that when she changed the sound driver without changing the actual data the music and sound effects played but sounded very different She had to edit each tone to make the sound similar but it was hard because the timber changed Compounding the issue was that to know how the music should sound IPPO had to play the arcade game many times “I was not good at playing games at the time.” It didn’t help that Ghouls ‘n Ghosts was part of a series notorious for its difficulty After the Ghouls ‘n Ghosts conversion was complete Sega put her in charge of another arcade-to-Mega Drive port but instead wrote a new tool (with the help of a programmer) which converted Capcom’s sound data to a more Mega Drive-friendly format I worked quicker and more easily using the converter I’m telling this secret for the first time in my life,” she said The first game she composed original music for was Phantasy Star III The third part in Sega’s epic science-fiction RPG series it also changed up the Phantasy Star formula taking bold leaps with the narrative and gameplay from the haunting rhythms of the dungeon track to the dynamic overworld theme that increases in complexity depending on the number of members in your party I was surprised to learn IPPO only had a month to do all the tracks but recalled that they didn’t have much time and were in a great rush she composed all the tunes in that month (with a little extra time for revisions and adjustments) It was also her first time using Sega’s proprietary program for creating the music The usual workflow involved her composing the score in Prelude then asking the planner/director to listen to the track But you can’t use data from Prelude (SMF) directly on the Mega Drive You have to convert the SMF data into Mega Drive sound data I had to try and optimize the sound to utilize the least amount of data possible.” Her sound room and the development team were on separate floors she had to go on a walk to deliver a floppy disk to the developers The dynamic music system that changed with the flow of battle and the number of party members took some trial and error he is credited as S2.) When he told me that he’d like the music to change I wondered how I should do it and deal with the sound data so he had a clear idea how he wanted the music of Phantasy Star III to be and explained it to me I was asked to have the music change depending on who was in the party like there were too many types of characters who can join the party and if all the characters have their own unique music the game wouldn’t work in the later stages as it took up too much memory “I think I could’ve done more to link the music in better ways and I could have used more musical patterns If I were to do Phantasy Star III music now and it would take so much more time than I did back then!” composing the music involved lots of research She would generally start by reading documents that gave a synopsis of the game and details on characters and settings she had the director explain what they hoped for “The sound work is harder if the graphics are not made at least to some extent (because you make sound effects watching the graphics) so I usually start working much later than the programmers and the artists,” she said “In this way it’s much easier to integrate the director’s idea of the music with mine When I have to compose for the scenes whose graphics are not drawn yet I try to discuss with the director to know what they want Some directors just say ‘Any music will do,’ or ‘Do whatever you want to.’ In such instances “If a director ordered me to write music in the woods Then suddenly music starts in my head and I catch it and start composing In 1993 IPPO went to work on the fourth game in the series The game is considered by many to not just be the best JRPG on the Sega Genesis alongside SNES JRPG epics like Chrono Trigger and Final Fantasy VI covering the plight of the Algol Star System as a new cast of characters fights a generational battle to vanquish evil once and for all The manga-style cutscenes with panels were a big part of what made the story so impactful As she worked on a separate floor from the rest of the development team at Sega she asked to be in the same room with the other staff “You might be surprised and say ‘What?!’ at my saying this,” IPPO said and the planner (the director) were all working in the same room and if they weren’t directly linked to the sound aspects the sound designer just goes on without knowing about those revisions if they think the sound designer must know I couldn’t always check the graphics of the game if I wanted to see how the combat sequences with weapons were animated I had to go to the room where the consumer software development took place.” she moved all her gear to the floor where the other Phantasy Star IV were “I could ask any programmer to show me any scene but I hadn’t been able to do these ‘normal’ things The chance to have bonding moments like chatting with the staff and having snacks with them may seem irrelevant in the job Working together in the same room made it so much easier for me to compose or make sound effects I had little difficulty seeing the images I needed I remember thinking ‘I knew working in the same room would be good She composed many of the tracks for the cutscenes in Phantasy Star IV before she saw the graphics because they weren’t completed until almost the end of the project But she had seen the hand-drawn storyboards and had a general idea of what kind of scenes they were and what the actual dialogue was “I looked at the storyboards over and over again “Unlike battle sequences and walking on the overworld the script is important in drama sequences so I composed trying not to make my scores too dramatic Only with the ending sequence did I wait for the graphics to be completed before I composed This is because I had to adjust the timing of the graphics and the score to be in unison with each other I measured the timing of every scene and wrote them on paper I wrote down the dialogue and explanations of each scene I thought it would be very hard to compose scores for that long ending sequence with perfect timing I knew the players of Phantasy Star IV must have spent a long time to reach the ending so I composed the score to be peaceful and happy Though saying goodbye to the characters within the game must be sad I wanted the players to have bright feelings as the saga came to an end.” Phantasy Star IV also had one of what she considered her best decisions when it came to the usage of a score After the final cutscene and the credits start to play she used the same score as the opening cutscene though with slight adjustments to the tempo and speed She was originally supposed to compose a totally new score for the end but when the programmer showed her the ending and the staff roll “It must be very cool to end this game using the same score again.” the game in many ways wrapping up the series that had begun with the first Phantasy Star “Some accused me of cutting corners,” IPPO stated Nobody wants to cut corners in working on the important scenes it’s rare that the same score is used for the title and the staff roll.” IPPO’s role wasn’t just limited to composing music Creating sound effects was an important part of her work as well different people make sound effects and music “There are games in which sound effects are very important to make the game exciting One is ‘the imaginary sound,’ the others are ‘realistic sounds.’ ‘The imaginary sound’ is the sound you use when a wizard makes magic but it’s also fun to invent a sound which doesn’t exist.” “When you use ‘real sounds,’ you don’t just record the sound and use it Many times you have to look for a ‘similar sound’ and record that instead what would you do if the developers ordered the sound of a sword and a shield crashing Do you have a genuine sword and shield in your house I begin by hitting the things in my household with a metal stick searching for a ‘similar’ sound Once they ordered me to make a ‘wall-hitting sound,’ so I hit the wall and recorded the sound but they said ‘No.’ I tried everything and eventually gave them a sound of me shutting the door of the refrigerator and the client said this is it!’ The ‘real’ sounds in a person’s head aren’t always the real sounds which exist but are strongly influenced by prejudices and images I create sound effects that are completely different from the one ordered I once processed a piano sound by using the spring of a music box to create a roar of a monster Her instincts have led her to make choices that are as bold as they are effective as she believes silence can sometimes be more dramatic than music ’This scene should be left without any music.’ But when I suggested that the developers used to disagree since they were worried players might mistake it for an audio bug or that players might think we’re cutting corners And in the case of Starship Damrey (a 3DS survival horror game) I didn’t feel ‘This short scene should be without music,’ but ‘This entire game should be without music “Since Damrey was an adventure game,” she said “the decision to make it free of music was tough But the story writer said ‘I feel the same way!’ and the game was completed without BGM except for some event sequences I still think it was the best decision for that game Creative staff think and think before making the final decision Any creator must work that way to make the best decisions and choices.” IPPO left Sega shortly after wrapping on Phantasy Star IV but can’t say anything at this stage in development she revealed how iterative her composing process is and how with every soundtrack she writes many more tracks than are actually used especially when I feel it isn’t what I imagined or if I just don’t like them I repeat this process ‘til I get what I imagined ‘This is okay.’ Sometimes I find the tune which I thought okay the previous day isn’t okay at all the next day and discard it It’s like a Japanese ceramic artist who breaks what they have just made one after another.” part of her philosophy to composing shined through as she described the visceral nature of making music “It’s important to listen to all kinds of music,” she said “It is better if you don’t just ‘listen to’ the music but to ‘feel’ the music with your body if possible If they want you to compose a classical style music you should go to a classical music concert You should have experiences by which your heart is shaken with the live performances and there are many ways to let people listen to what you’ve composed I’m sure those of you who are interested in composing are already doing this so I don’t think you need advice from me.” This interview was translated by Daisuke Onitsuka