Kanazawa University NanoLSI Podcast

D. Carlero et al, ACS Nano 2024, 18, 30, 19518–19527Researchers from Kanazawa University's NanoLSI, IMDEA Nanoscience, and CNB-CSIC studied influenza A replication using high-speed atomic force microscopy. They observed that recombinant ribonucleoprotein complexes (rRNPs) can undergo multiple transcription cycles, with RNA structure stability influencing synthesis rates. Their findings offer new insights into viral replication mechanisms and RNA synthesis regulation, opening doors for further research on gene expression control. NanoLSI Podcast website

M.S. Alam et al, Small Methods 2024, 2400287Atomic force microscopy (AFM) was initially developed to visualize surfaces at nanoscale resolution. Researchers at WPI NanoLSI, Kanazawa University, have now extended AFM for 3D imaging, particularly for flexible nanostructures like carbon nanotubes. They demonstrated that dynamic mode AFM, which uses a vibrating tip, causes less friction and damage than static mode, making it ideal for imaging delicate biological systems like cells, organelles, and vesicles​. NanoLSI Podcast website

Madhu Biyani and colleagues at NanoLSI, Kanazawa University, have developed a sensitive and selective electrochemical biosensor for detecting the cancer biomarker ADAR1, using new aptamers. This cost-effective tool enables rapid ADAR1 detection in diluted samples, promising improved cancer prognosis and monitoring.NanoLSI Podcast website

Yanjun Zhang, Yuri Korchev, and colleagues used hopping probe scanning ion conductance microscopy to study hydrogen peroxide eustress on colorectal cancer cells, revealing varying cell stiffness and gradients. Their findings could lead to new cancer and inflammatory disease therapies. NanoLSI Podcast website

 Mikihiro Shibata and collaborators used high-speed atomic force microscopy to study nucleosome dynamics. They found that nucleosomes without histone tails, particularly H2B and H3, showed increased sliding and DNA unwrapping. These findings highlight the importance of histone tails in chromatin stability and structure. NanoLSI Podcast website

Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Japan, collaborating with Professor Sarikaya, Seattle, USA.The research described in this podcast was published in Small in February 2024Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/NanoLSI Podcast website

Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Holger Flechsig and Clemens Franz from WPI-NanoLSI, at Kanazawa University, in collaboration with Vincent Torre from the International School of Advanced Studies in Italy and former WPI-NanoLSI members Leonardo Puppulin and Arin Marchesi.The research described in this podcast was published in Nature Communications in January 2024Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Researchers observe the structural heterogeneity of a lipid scramblaseResearchers from Nano Life Science Institute (WPI-NanoLSI), at Kanazawa University report in Nature Communications that TMEM16F, a transmembrane protein that facilitates the passive movement of phospholipids and ions across membranes, explores a larger conformational landscape than previously thought to perform its unique functions. The finding refines our molecular understanding of crucial physiological processes such as blood coagulation and COVID-19 pathogenesis, and highlights the importance of probing membrane proteins in native-like environments.Lipid scramblases are proteins embedded in cell membranes that play a crucial role in shuffling phospholipids between the two lipid layers that form such cellular boundaries. TMEM16F, a member of the TMEM16 protein family, acts as both a calcium-activated ion channel and a lipid scramblase, meaning that it can facilitate the transfer of both, lipids and ions across the chemical environment outside and inside of the cell. These movements regulate several biological functions such as blood clotting, bone development, and viral entry and are therefore of great physiological and clinical interest. At the molecular level, the TMEM16F architecture has a double-barrelled shape in which two identical polypeptide chains (called subunits), each formed by ten transmembrane (TM) helices, stick together (a process known as dimerization) to form two separate and presumably independent ion and lipid pathways.Previously, it was thought that TMEM16F might work like a simple gate, with calcium ions serving as keys to unlock the two permeation pathways. Opening and closing the gate to different extents would let lipids and ions cross the plasma membrane alternately. However, structural investigations using cryo-electron microscopy (cryo-EM) -an in vitro technique that can reveal the 3D architecture of purified and frozen proteins at near-atomic resolution – have mostly captured TMEM16F snapshots in inactive conformations, with the ion and lipid gates presumably trapped in a closed state, raising questions about the validity of existing models.So how did the researchers set about shedding light on how TMEM16F works?To gain a better understanding of TMEM16F's structure and function relationship, Holger Flechsig and Clemens Franz from WPI-NanoLSI, Kanazawa University, in collaboration with Vincent Torre from the International School of Advanced Studies (Italy) and former WPI-NanoLSI members Leonardo Puppulin and Arin Marchesi, used advanced techniques such as single-molecule force spectroscopy (SMFS) and high-speed atomic force microscopy (HS-AFM) imaging. These methods allowed them to observe TMEM16F behaviour at the molecular level in physiological environments, providing insights into its structure, dynamics, and mechanical properties.The study uncovered that TMEM16F exhibits a wide range of structural conformations that have been overlooked so far. The research revealed unexpected changes in the dimerization interface and TMEM16F subunit arrangements, suggesting that TMEM16F operates in a more dynamic and versatile manner than previously thought. The authors propose thaNanoLSI Podcast website

Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Tareg Omer Mohammed, You-Rong Lin, and Clemens M. Franz at the Nano Life Science Institute (WPI-NanoLSI), at Kanazawa University.The research described in this podcast was published in the Journal of Cell Science in January 2024.Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/A novel role for S100A11 in focal adhesion regulationResearchers at Kanazawa University report in the Journal of Cell Science on a novel role of the small Ca2+ion-binding protein S100A11 [S one hundred A eleven] in focal adhesion disassembly.S100A11 is a small Ca2+ion-activatable protein with an established role in different cellular processes involving actin cytoskeleton remodeling, such as cell migration, membrane protrusion formation, and plasma membrane repair. It also displays F-actin binding activity and localizes to actin stress fibers, but its precise role in regulating these structures remained unclear.In their study, Tareg Omer Mohammed, You-Rong Lin, and Clemens M. Franz together with colleagues from the Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, in Japan, and Karlsruhe Institute of Technology, in Germany, report a novel localization of S100A11 to disassembling focal adhesions at the end of contractile stress fibers in HeLa and U2OS cells. Specifically, S100A11 transiently appears at the onset of focal adhesion disassembly, reliably marking the targeted adhesion sites for subsequent disassembly. Interestingly, S100A11 leaves focal adhesion sites before the completion of disassembly, indicating that S100A11 plays a specific role in the initiation of adhesion site disassembly, rather than the disassembly process itself.So what are focal adhesions anyway and what can we learn from them?Focal adhesions are integrin-containing cell/matrix adhesion sites enabling cells to adhere to the cellular environment and to apply cellular contraction forces during extracellular matrix remodeling. Directed cell migration requires the coordinated assembly of new adhesion sites at the front, and disassembly at the rear of the cell, and better understanding mechanisms regulating focal adhesion turnover is, therefore, an important goal in cell migration and invasion research. The newly discovered role of S100A11 in focal adhesion disassembly extends insights into the molecular mechanisms underlying focal adhesion site disassembly.The authors furthermore delineate a force-dependent recruitment mechanism for S100A11 to adhesion sites involving non-muscle myosin II-driven stress fiber contraction, activation of mechanosensitive, Ca2+ ion-permeable Piezo1 channels, and intracellular Ca2+ ion influx at mechanically stressed focal adhesions. In turn, locally elevated Ca2+ ion levels activates and recruits S100A11 to the adhesion sites targeted for disassembly. So how did they work this out?The force-dependent recruitment of S100A11 to stressed focal adhesions was confirmed using a micropipette pulling assay able to apply pulling forces onto individual focal adhesion sites. Even when myosin II-dependent intracellular contractility was inhibited, external pulling forces still recruited S100A11 to stretched focal adhesion sites, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ ion and Piezo1 function was still indispensable, indicating that myosin II-dependent contraction forces act upstream of Piezo1-mediated Ca2+ ion influx, in turn leading to S100A11 activation and focal adhesion recruitment.Lastly, the authors show impaired focal adhesion translocation and disassembly ratNanoLSI Podcast website

Researchers observe what ubiquitination hinges on Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Hiroki Konno and Holger Flechsig at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University.The research described in this podcast was published in Nano Letters in December 2023Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Researchers observe what ubiquitination hinges onResearchers at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University report in Nano Letters how the flexibility of a protein hinge plays a crucial role in the transfer of proteins in key cell processes.Ubiquitination – the addition of the protein ubiquitin – is a key stage in many cell processes, such as protein degradation, DNA repairs, and signal transduction. Using high-speed atomic force microscopy (AFM) and molecular modelling, researchers led by Hiroki Konno and Holger Flechsig at WPI-NanoLSI, Kanazawa University have identified how the mobility of a ubiquitination related enzyme hinge allows ubiquitination to take place.So what was known already about ubiquitination?Previous studies have identified a number of enzymes that facilitate ubiquitination, including an enzyme that activates ubiquitin (E1), an enzyme that conjugates it (E2), and an enzyme that catalyzes ubiquitin protein joining (that is, a ligase, E3) to a target protein. The HECT-type E3 ligase is characterized by a HECT domain that comprises an N lobe with the E2-binding site and a C lobe with a catalytic Cys residue, A flexible hinge connects the two lobes, leading to the hypothesis that ubiquitination is facilitated by the rearrangement of the protein around this hinge. Konno and their collaborators deployed their high-speed atomic force microscope to hunt for evidence that this was the case.So what did they find out?The researchers noted that when the HECT domain was crystallized with a type of E2 enzyme, it formed an L shape such that the distance between the catalytic Cys residue of the HECT domain and the catalytic Cys of the E2 enzyme was 41 Å – too far for the transfer of ubiquitin. However, in its catalytic conformation the HECT domain has a different shape where the distance between the two catalytic Cys residues is much closer – just 8 Å – so this is thought to be a “catalytic conformation”.Analysis of high-speed-AFM images of a wild-type HECT domain of E6AP revealed two conformations – one of which looked spherical and the other oval. Using AFM simulations they attributed the oval shapes to the L conformation and spherical shapes are either the catalytic conformation or the so called inverted T conformation, which had been observed in the another type of HECT domain where the distance between the Cys residues is 16 Å. To overcome the spatio-temporal resolution limitations of imaging, the experiments were complemented by molecular modelling to visualize HECT domain conformational motions at the atomistic level. Simulation AFM was used to generate a corresponding pseudo AFM movie, which clearly showed the change from spherical to the oval shaped topography.“Although experimental limitations do not allow us to resolve the intermediate conformations,” explain the researchers in their report of the work. “The performed modeling provides evidence that the transitions between spherical and oval HECT domain shapes observed under high-speed-AFM correspond to functional conformational motions under which the C-lobe rotates relative to the N-lobe, thereby allowing the change between catalytic and L-shape HECT conformations.”Further experiments with mutant HECT domains with less flexibility in the hNanoLSI Podcast website

Chromatin Accessibility: A new avenue for gene editingHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, led by Yusuke Miyanari.The research described in this podcast was published in Nature Genetics in February 2024Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Chromatin Accessibility: A new avenue for gene editingIn a study recently published in Nature Genetics, researchers from Nano Life Science Institute (WPI-NanoLSI) at Kanazawa University explore chromatin accessibility, that is, endogenous access pathways to the genomic DNA, and its use as a tool for gene editing.Our DNA is protected from unwanted external modifications by forming structures called nucleosomes that consist of threads of DNA wound around chunks of special proteins known as histones. This unique coiled shape prevents the access of undesirable molecules to a cell's DNA. However, for vital genetic functions—such as DNA repair—the right set of proteins require access to these DNA fragments. This phenomenon known as ‘chromatin accessibility' involves a privileged set of protein molecules, many of which are still unknown.Now, researchers from Nano Life Science Institute (WPI-NanoLSI) at Kanazawa University, led by Yusuke Miyanari, have used advanced genetic screening methods to unravel chromatin accessibility and its pathways.So how did they go about it?For the investigation the team used a combination of two technologies—CRISPR screening and ATAC-see. While the former is a method to suppress the function of a desired set of genes, the latter is a means to identify which ones are essential for chromatin accessibility. Thus, using this method all genes playing a crucial role in chromatin accessibility could be pinned down.With the help of these assays, novel pathways and individual players involved in chromatin accessibility were uncovered—some playing a positive role and some negative. Of these, one particular protein, TFDP1, showed a negative effect on chromatin accessibility. When it was suppressed, a significant increase in chromatin accessibility was observed, accompanied by nucleosome reduction. A deeper dive into the mechanism of TFDP1 revealed that it functions by regulating the genes responsible for production of certain histone proteins.The team then focused their study towards exploring biotechnological applications of their findings. After suppressing TFDP1, two different approaches were tried. The first approach involved gene editing using the CRISPR/Cas9 tool. This revealed that deletion of TFDP1 made the gene editing process easier. Now, most chromatin accessibility occurs in nucleosome-depleted regions or NDRs. However, by suppressing TFDP1 chromatin accessibility occurred not only in NDRs but across other regions as well. Secondly, the depletion of TFDP1 aided the process of converting regular cells into stem cells, a massive step forward in cellular transformation.This study identified new chromatin accessibility pathways and channels for exploring its potential even further. “Our study shows the significant potential to manipulate chromatin accessibility as a novel strategy to enhance DNA-templated biological applications, including genome editing and cellular reprogramming,” conclude the researchers.ReferenceSatoko Ishii, Taishi Kakizuka, Sung-Joon Park, Ayako Tagawa, Chiaki Sanbo, Hideyuki Tanabe, Yasuyuki Ohkawa, Mahito Nakanishi, Kenta Nakai, Yusuke Miyanari. Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility. Nature Genetics, FebNanoLSI Podcast website

Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Romain Amyot, Noriyuki Kodera, and Holger Flechsig at the Kanazawa University NanoLSI.The research described in this podcast was published in Frontiers in Molecular Biosciences  in November 2023Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Researchers predict protein placement on AFM substratesResearchers at Kanazawa University report in Frontiers in Molecular Biosciences a computational method to predict the placement of proteins on AFM substrates based on electrostatic interactions.The observation of biomolecular structures using atomic force microscopy (AFM) and the direct visualization of functional conformational dynamics in high-speed AFM (HS-AFM) experiments have significantly advanced the understanding of biological processes at the nanoscale. In experiments, a biological sample is deposited on a supporting surface (AFM substrate) and is scanned by a probing tip to detect the molecular shape and its dynamical changes. The observation of protein dynamics under HS-AFM is a delicate balance between immobilizing the structure on the supporting surface while at the same time preventing too strong perturbations by immobilization.The process of placing a biomolecular sample on the supporting surface and controlling its proper attachment is a challenge at the very start of every AFM observation. By the chemical composition of the buffer, interactions between the sample and substrate can be modified. Such surface modifications are often critical for successful AFM observations of protein structures and their functional motions. However, the molecular orientation of the sample is a priori unknown, and due to limitations in the spatial resolution of images, difficult to infer from a posteriori analysis.Romain Amyot, Noriyuki Kodera, and Holger Flechsig from Kanazawa University have now developed a physical model to predict the placement of biomolecular structures on AFM substrates based on electrostatic interactions. The method considers the substrates commonly used in AFM experiments (mica, APTES-mica, lipid bilayers) and takes into account buffer conditions. In computer simulations, a large number of possible molecular arrangements on the AFM substrate are sampled, and from evaluating the corresponding interaction energies, the most favorable placement is determined. Furthermore, the analysis allows predictions of the imaging stability under tip scanning.The authors provide several applications of the new method and obtain remarkable agreement of model predictions with previous experimental HS-AFM imaging of proteins. The findings can explain, for example, why HS-AFM observations of the Cas9 endonuclease, a protein playing a key role in genetic engineering applications, can reliably visualize functional relative motions of target DNA and Cas9 and capture DNA cleavage events at the single molecule level (see Fig. 1). Furthermore, as demonstrated for the ATP-powered chaperone machine ClpB, the model can explain how buffer conditions affect the stability of the sample-substrate complex and validate observations of previous HS-AFM experiments.In summary, the new method allows to employ the enormous amount of available structural data for biomolecules to make predictions of the sample placement on AFM substrates even prior to an actual experiment, and it can also be applied for post-experimental analysis of AFM imaging data. The developed method is implemented within the freely available BioAFMviewer software package, providing a convenient platform for applications by the broad BioAFM community.  Reference R. Amyot, K. Nakamoto, N. Kodera, H. FlechsiNanoLSI Podcast website

Sodium channel investigationHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Ayumi Sumino and Takashi Sumikama at the Kanazawa University NanoLSI.The research described in this podcast was published in Nature Communications in December 2023Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Sodium channel investigationResearchers at Kanazawa University report in Nature Communications a high-speed atomic force microscopy study of the structural dynamics of sodium ion channels in cell membranes.  The findings provide insights into the mechanism behind the generation of cell-membrane action potentials.The transport of ions to and from a cell is controlled by pore-forming proteins embedded in the cell membrane.  In particular, so-called voltage-gated sodium channels (VGSCs) govern the transfer of sodium (Na+) ions, and play an important role in the regulation of the membrane potential — the voltage difference between the cell's exterior and interior.  In electrically excitable cells such as neurons and muscle cells, VGSCs participate in the generation of action potentials; these are rapid changes in the membrane potential enabling the transmission of e.g. neural signals.  The precise structural changes occurring in VGSCs are not completely understood, however.  Now, Ayumi Sumino and Takashi Sumikama from Kanazawa University in collaboration with Katsumasa Irie from Wakayama Medical University and colleagues have succeeded in observing the structural dynamics of VGSC by means of high-speed atomic force microscopy (high speed-AFM), a method capable of imaging the nanostructure and subsecond dynamics of biomolecules.VGSCs can be in three different states: resting, inactive and active.  In the latter state, Na+ ions can pass through the channel; in the resting and inactive states, which are structurally different, ions cannot pass.  The basic structure of a VGSC consists of two modules: voltage sensor domains and pore domains.  These domains form a square arrangement, with the ion pore at its center.  An important open question is whether the voltage sensor domains dissociate from the pore domains when the channel closes.So how did they go about determining this?Sumino and colleagues performed experiments on three VGSCs.  One is the sodium channel of a particular bacterium (Arcobacter butzleri), the other two are mutants of it.  These three VGSCs have different voltage dependencies, with activation voltages starting at -120 mV, -50 mV and 0 mV, so that at the experimental conditions (0 mV), the VGSCs are in different states.In order to provide insights into the structural dynamics of these three VGSCs, the researchers applied high speed-AFM, a powerful technique for producing image sequences of biochemical compounds.  A single AFM image is generated by laterally moving a tip just above the sample's surface; during this xy-scanning motion, the tip's position in the direction perpendicular to the xy-plane (the z-coordinate) will follow the sample's height profile.  The variation of the z-coordinate of the tip then produces a height map — the image of the sample.  The generation of such AFM images in rapid succession then produces a video recording of the sample.The HS-AFM results revealed that for the mutant VGSC in the resting state, the voltage sensor domains are indeed dissociated from the pore domains.  Furthermore, the researchers found that the dissociated voltage sensor domains of neighboring channels connect to form pairs — this is referred to as dimerization.The observation of the dissociation of voltage sensor domains, as well as the dimerization between pore channels,NanoLSI Podcast website

Researchers fix the chirality of helical proteinsHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Naoki Ousaka, Mark J. MacLachlan and Shigehisa Akine at the Kanazawa University NanoLSI.The research described in this podcast was published in Nature Communications in October 2023 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Researchers fix the chirality of helical proteinsResearchers at Kanazawa University report in Nature Communications how they can control chirality inversion in α helical peptides.The function of a protein is determined by its structure – prompting great interest in how to manipulate these structures. The structure is defined not just by the sequence of amino acids that make it, but the shape these acids make – the secondary structure – as well as how that shape is then folded. The most common secondary protein structure is the α-helix, which can coil to the right or left. This coiling direction in turn determines how it engages with other chiral structures, which may be the form of a light beam or another molecule. Although molecular components and environmental factors can favor a particular coiling direction over the other, helical molecules tend to flip between the two coil directions. Now Naoki Ousaka, Mark J. MacLachlan and Shigehisa Akine at Kanazawa University in Japan have shown how they can control and fix the coil direction.Helical proteins are chiral molecules, which means that the molecule's shape cannot be fitted into its mirror image. In nature helical proteins often have other chiral components, such as sugars or amino acids, and these will determine which way the protein coils. However, there is a lot of interest in synthesizing artificial helical proteins that have different chemical components and hence functions not found in nature, and these may not have other chiral components. Nonetheless having both types or “enantiomers” of the chiral molecule can be hazardous because of the significant differences in behavior between the two chiral forms, one of which may be benign or even therapeutic while the other is toxic. Hence, there is demand for other ways of selecting and fixing the chirality.So how did they go about this?Ousaka, MacLachlan and Akine synthesized α helical molecules solely from achiral components. They included bulky segments so that the molecule tended towards the larger rings of the α helical structure, as well as side chains of piperidine – molecular components that are common in pharmaceuticals. These side chains can be cross linked to “staple” the molecule into either the righthanded or lefthanded coil, inhibiting flipping between the two – chiral inversion. Finally they added another molecular component, known as an ester  – the L-Val-OH residue. This would switch the direction of the coil in response to acidic or basic environments due to preferences in the interaction between oxygen atoms in the ester and the amino acid backbone.The researchers used a range of chiral characterization methods including circular dichroism, nuclear magnetic resonance and liquid chromatography. They found that with the molecule stapled just once, it would slow down the flipping between enantiomers by a factor of 106, although this still occurred over minutes. Changing the solution to acid or alkali also successfully determined which enantiomer was favoured. However, stapling the molecule twice slowed down the chirality inversion by a factor of 1012, so that the molecular chirality was stable for years. This increased energy barrier to chirality inversion could then be overcome by heating the sample to very high temperatures to switch betNanoLSI Podcast website

Genetic switches in tumor developmentHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Masanobu Oshima at the Kanazawa University NanoLSI.The research described in this podcast was published in Cancer Research in November 2023Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Genetic switches in tumor developmentResearchers at Kanazawa University report in Cancer Research how Kras and p53 mutations influence the tumor suppressor and promoter functions of a TGF- ß pathway. The findings may lead to a new approach for colorectal cancer therapy.Both the progression and the suppression of tumors are governed by biomolecular processes. Often, a particular process is involved in either cancer progression or suppression. Cancer treatment in the form of drugs then typically focuses on the respective deactivation or activation of the relevant biomolecular process. However, it has been established that a process known as transforming growth factor ß (TGF-ß) signaling*1 plays a role in both tumor suppression and progression. Now, Masanobu Oshima from Kanazawa University and colleagues have studied the precise genetic conditions underlying the outcome of TGF-ß signaling. Their findings may help the development of new therapeutic strategies for particular cancers.The suppressive effect of TGF-ß signaling happens through the stimulation of cell differentiation — the process through which dividing cells acquire their type or function. The malignant progression of cancers, on the other hand, comes from a process called epithelial-mesenchymal transition (EMT), in which an epithelial cell transforms into a mesenchymal cell type. The former is a ‘stationary' type of cell, found in epithelial tissue, whereas the latter is a more ‘migratory' type of cell found in development and cancer.So how did they investigate these processes and what did they found out?Oshima and colleagues performed experiments with tumor-derived organoids. They confirmed that TGF-ß family cytokine, activin plays a role in tumor suppression and progression dependent on the mutation types of driver genes. In certain cancer cells treated with activin, the researchers noted that the partial EMT is induced with tumor aggressiveness and development. On the other hand, certain mutated activin receptors were found to have cancer suppressor capabilities, which made the scientists conclude that genetic alterations underlie the dual function of activins.One of the two relevant genes is Kras which relays signals that regulate cell growth, division and differentiation. Oshima and colleagues found that a mutation of Kras blocks TGF-ß/activin-induced growth suppression. The other gene is known as Trp53, which encodes tumor protein 53, playing an important role in cancer regulation. A combination of Kras and Trp53 mutations at hot spots, known as gain-of-function mutation, was found to not just block tumor suppression but promote partial EMT and tumor proliferation.The experiments were done with mouse intestinal tumor-derived organoids with defined genetic backgrounds, which makes the results relevant for therapeutic strategies for human colorectal cancer. Quoting the scientists: “Based on these results, the control of TGF- ß/activin signaling appears to be an important preventive and therapeutic strategy against the malignant progression of colorectal cancer carrying […] mutations”.ReferenceDong Wang, Mizuho Nakayama, Chang Pyo Hong, Hiroko Oshima, and Masanobu Oshima. Gain-of-function p53 mutation acts as a genetic switch for TNanoLSI Podcast website

Researchers tune the speed of chirality switchingHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Shigehisa Akine at the Kanazawa University NanoLSI.The research described in this podcast was published in Science Advances in November 2023Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Researchers tune the speed of chirality switchingResearchers at Kanazawa University report in Science Advances how they can accelerate and decelerate chirality inversion in large cage molecules using alkali metal ion binding.Chiral molecules can have dramatically different functional properties while sharing identical chemical formulae and almost identical structures. The molecular structure of two types of a chiral molecule – so-called enantiomers – are mirror images of each other where one cannot be superposed on the other any more than your right hand can fit front-to-back on the left. While a lot of chiral molecules are traditionally considered fixed as left- or right-handed, chiral molecules based on helices are known to be able to switch in response to changes in their environment. Now researchers led by Shigehisa Akine at Kanazawa University have demonstrated how environmental changes can also accelerate or decelerate this chiral inversion process, providing “a novel time-programmable switchable system”.The researchers focused their study on “metallocryptand (R6)-LNi3”, an organic molecule featuring metal atoms in a cage-like molecular structure that can exist in one of two possible forms described as the P or M type (right- and left-handed, respectively). In its pure form (R6)-LNi3 has a preferred ratio of P type to M type of 12:88. Starting from a 50:50 ratio, the molecules will flip between one form and the other with a preference for flipping towards the M type to meet that ratio. The researchers measured this change in ratio using NMR and circular dichroic spectroscopy. However, add an alkali metal into the cage cavity and this preference can change.By adding alkali metal ions to the solution of the (R6)-LNi3 the researchers could confirm that the metal ions readily bound to the metallocryptand from the changes in the spectroscopic signatures of the molecules. In addition, the bound ion also shifted the preferred ratio by a margin and with a speed that depended on which alkali metal was used.So what is causing this effect?The researchers attribute the different rates and ratios to differences in binding constants not just between the metal ion and the two forms of the molecule but also a virtual binding constant for the molecule transitioning between the two. The binding between a caesium ion and the P type molecule was more than 20 times greater than that with the M type so the solution eventually switched to a higher proportion of the P type with a P:M ratio of 75:25 over the course of 21 hours. The final ratio with a rubidium ion was similarly bias to the P type reaching a slightly lower ratio of 72:28 but in just 100 minutes. With potassium ion the equilibrium ratio was lower again at 68:32 but reached within just a minute, three orders of magnitude faster than for the caesium ion. The researchers attribute this speed to the large virtual bonding constant with the transitioning molecule.With smaller ions – lithium and sodium ions – the preferred molecular type did not actually change but the final ratio was reached much faster. It is the first time researchers have demonstrated that such chiral inversion can be sped up and slowed down by tuning the molecules environment.“This research can provide a new insight into the development of an on-deNanoLSI Podcast website

Researchers identify the dynamic behavior of a key SARS-CoV-2 accessory protein Transcript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Richard Wong at Kanazawa University alongside Noritaka Nishida at Chiba University.The research described in this podcast was published in the Journal of Physical Chemistry Letters in September 2023 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Researchers identify the dynamic behavior of a key SARS-CoV-2 accessory protein Researchers at Kanazawa University report in the Journal of Physical Chemistry Letters high-speed atomic force microscopy studies that shed light on the possible role of the open reading frame 6 (or, ORF6) protein in COVID19 symptoms. While many countries across the world are experiencing a reprieve from the intense spread of SARS-CoV-2 infections that led to tragic levels of sickness and multiple national lockdowns at the start of the decade, cases of infection persist. A better understanding of the mechanisms that sustain the virus in the body could help find more effective treatments against sickness caused by the disease, as well as arming against future outbreaks of similar infections. With this in mind there has been a lot of interest in the accessory proteins that the virus produces to help it thrive in the body. “Similar to other viruses, SARS-CoV-2 expresses an array of accessory proteins to re-program the host environment to favor its replication and survival,” explain Richard Wong at Kanazawa University and Noritaka Nishida at Chiba University and their colleagues in this latest report. Among those accessory proteins is ORF6. Previous studies have suggested that ORF6 potently interferes with the function of interferon 1 (that is, IFN-I), a particular type of small protein used in the immune system, which may explain the instances of asymptomatic infection with SARS-CoV2. There is also evidence that ORF6 causes the retention of certain proteins in the cytoplasm while disrupting mRNA transport from the cell, which may be means for inhibiting IFN-I signalling. However, the mechanism for this protein retention and transport disruption was not clear.So how did they figure it out? Well, to shed light on these mechanisms the researchers first looked into what clues various software programs might give as to the structure of ORF6. These indicated the likely presence of several intrinsically disordered regions. Nuclear magnetic resonance measurements also confirmed the presence of a very flexible disordered segment. Although the machine learning algorithm AlphaFold2 has proved very useful for determining how proteins fold, the presence of these intrinsically disordered regions limits its use for establishing the structure of ORF6 so the researchers used high-speed atomic force microscopy (or AFM), which is able to identify structures by “feeling” the topography of samples like a record player needle feels the grooves in vinyl. Using high speed AFM the researchers established that ORF 6 is primarily in the form of ellipsoidal filaments of oligomers – strings of repeating molecular units but shorter than polymers. The length and circumference of these filaments was greatest at 37 °C and least at 4 °C, so the presence of fever could be beneficial for producing larger filaments. Substrates made of lipids – fatty compounds – also encouraged the formation of larger oligomers. Because high speed AFM captures images so quickly it was possible to grasp not just the structures but also some of the dynamics of the ORF6 behavior, including circular motion, protein assembly and flipping. INanoLSI Podcast website

Researchers define a nanopipette fabrication protocol for high resolution cell imagingTranscript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Yasufumi Takahashi at the Kanazawa University NanoLSI.The research described in this podcast was published in Analytical Chemistry in August 2023 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Researchers define a nanopipette fabrication protocol for high resolution cell imagingResearchers at Kanazawa University report in Analytical Chemistry how to produce nanopipettes that reliably provide nanoscale resolution scanning ion conductance microscopy images of living cells.A nanoscale view of living cells can provide valuable insights into cell structure and function. Over the years, various microscopy techniques have been enrolled to obtain a window into biological specimens at the nanoscale but all with their limitations and challenges. Although scanning ion conductance microscopy has demonstrated the capability to image living biological samples in solution with nanoscale resolution, it has been hampered by challenges in reliably producing nanopipettes with the optimum geometry for the job. Now researchers led by Yasufumi Takahashi at Kanazawa University's Nano LSI and Nagoya University have devised a protocol for reproducibly fabricating nanopipettes with the preferred geometry for high quality imaging. So what is scanning ion conductance microscopy and what kind of nanopipette does it need?Scanning ion conductance microscopy uses a nanopipette to control the distance between nanopipette and sample using an ion current as feedback signal. The shape of the nanopipette significantly influences the performance of the device. For instance, a wide aperture limits the possible resolution, a long shunt can lead to rectification effects that warp the ion current measurements, and if the glass of the nanopipette is too thick it can deform the sample before the proximity of the aperture has reached the point needed for constant ion current topographical mapping. As a result, the ideal nanopipette has a short shunt, small aperture and thin glass walls.The standard procedure for fabricating the nanopipette is to pull a capillary tube with a laser puller that heats the capillary tube it is manipulating. The capillary then narrows where it lengthens until it is finally drawn into two separate pieces. Although quartz can allow a little more control in the process of drawing the capillary tube into shape it is hydrophobic, which raises complications in actually filling the nanopipette with the aqueous solution needed for the ion current. For this reason, the researchers developed a protocol by which they could draw nanopipettes from borosilicate glass capillaries with the required control and reproducibility.Takahashi and his collaborators noted that ideally the starting capillary should have thick walls and a narrow inner diameter, however it is not easy to obtain capillary tubes to these requirements from commercial suppliers. Instead, they preheat the capillary for 5 s without pulling it, which causes the glass walls to the thicken and reduces the inner diameter. They also optimized the parameters for pulling the tube, such as the velocity.So did it work? Apparently soThe researchers demonstrated the performance of the nanopipettes they produced by imaging a cell undergoing a type of endocytosis, where it engulfs and absorbs some external material. They were able to image the microvilli – that is, tiny cellular membrane protrusions – found on the cell surface, as well as the endocytic pits that NanoLSI Podcast website

Hydration matters: The interaction patterns of water and oxide crystals revealedTranscript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Keisuke Miyazawa at the Kanazawa University NanoLSI.The research described in this podcast was published in Nanoscale in July 2023 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Hydration matters: The interaction patterns of water and oxide crystals revealed.https://nanolsi.kanazawa-u.ac.jp/en/highlights/28428/ In a study recently published in the journal Nanoscale, researchers from Kanazawa University and AGC Inc. use three-dimensional atomic force microscopy to study the hydrated form and structure of commonly occurring oxide crystals. While sapphire and quartz are oxide crystals used in a wide range of industrial applications, the atomic-scale structures of these materials are not well understood. The major chemical components of sapphire and quartz are aluminum oxide and silicon dioxide, respectively. These components have a high affinity for water, which affects the chemical reactivity of the crystals. Thus, a thorough knowledge of the water-binding properties of these oxides is important for further innovative applications. To date, traditional microscopic methods have only provided insights into the two-dimensional topography of their surfaces. Now, a research team led by Keisuke Miyazawa from the NanoLSI at Kanazawa University has developed three-dimensional (3D) microscopy technique for a detailed study of the interaction of the surfaces of these materials with water.So how did they do it?The team started by looking at the surface structures and its hydration structures of sapphire and α-quartz in water. For this, they used an advanced form of microscopy known as 3D atomic force microscopy (3D-AFM). Oxide crystals usually have hydroxyl (OH) groups, which are the main “water-binding” molecules, closely linked with the oxides. Hence, the team studied the OH groups and its hydration structures on both crystals when immersed in water. They found that the hydration layer on sapphire was not uniform because of the nonuniform local distributions of the surface OH groups. On the other hand, the hydration layer on α-quartz was uniform because of the atomically flat distributions of the surface OH groups. When the interaction force of these oxides with water was subsequently measured, it was found that a greater force was required to break the water-crystal bonds in sapphire than in α-quartz. Lastly, it was also discovered that this affinity was much higher in regions where the oxides were in close proximity to the OH groups. This study showed that the hydration structures of oxides are dependent on the location and density of OH groups, in addition to the strength of the OH groups' hydrogen bonding (the chemical bond used to bind to water). What's more, it was successfully shown here that 3D-AFM can be used in unraveling the interaction of water with several surfaces, a potential avenue for understanding solid-liquid interactions better. “This study contributes to the application of 3D-AFM in exploring atomic scale hydration structures on various surfaces, and hence, to a wide range of solid–liquid interfacial research fields,” conclude the researchers.   ReferenceSho Nagai, Shingo Urata, Kent Suga, Takeshi Fukuma, Yasuo Hayashi and Keisuke Miyazawa. Three-dimensional ordering of water molecules reflecting hydroxyl groups on sapphire (001) and α-quartz (100) surfaces   NanoLSI Podcast website

Ion channel block unraveled Transcript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Takashi Sumikama at the Kanazawa University NanoLSI in collaboration with Katsumasa Irie from Wakayama Medical University and colleagues.The research described in this podcast was published in Nature Communications in July 2023 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Ion channel block unraveledResearchers at Kanazawa University report in Nature Communications how calcium ions can block sodium ion channels located in cell membranes. Structural analysis and computer simulations made it possible to identify where and why calcium ions get stuck. Ion channels are structures within cell membranes that enable specific ions to travel to and from the cell. Such transfer is essential for a variety of physiological processes like muscle cell contraction and nerve excitation. In so-called tetrameric cation channels, the ion selectivity results from the unique structural and chemical environment of the part referred to as the selectivity filter, which is located between two intertwined helical structures. Tetrameric ion channels are prone to ‘divalent cation block', the blocking of the channel by ions like calcium (as in Ca2+). Such blocking regulates the ionic current, which is involved in various neural activities such as memory formation. How divalent cation block happens exactly is still unclear at the moment — in particular, a direct observation of the cation actually blocking the ion pathway has not been reported yet. Now, Takashi Sumikama from Kanazawa University in collaboration with Katsumasa Irie from Wakayama Medical University and colleagues has discovered the mechanism behind divalent cation block in NavAb, a well-known tetrameric sodium (Na) channel. Through structural analysis and computer simulations, the researchers were able to reveal the relevant structural features and molecular processes at play.So how did they go about this structural analysis?NavAb is a sodium channel cloned from a bacterium (Arcobacter butzleri) and has a well-known structure. Sumikama and Irie's colleagues performed experiments with NavAb and three mutants. The structures of the mutants were determined for environments with and without calcium. The scientists focused on the differences in electron densities for the different structures, as these provide insights into the locations of the calcium ions. They found that for the mutants displaying calcium blocking, one or two calcium ions are located at the bottom of the selectivity filter. They also discovered that two other divalent cations — magnesium (as in Mg2+) and strontium (Sr2+) ions — blocked the calcium-blocking mutant sodium channels.The researchers then performed computer simulations to obtain a detailed understanding of the interaction between the calcium ions and the mutated NavAb channels. The simulations reproduce the dynamics of ions passing — or not passing — the channel. In the absence of calcium ions, sodium ions were observed to penetrate the channel. In the presence of calcium ions, penetration significantly decreased in the calcium-blocking mutants. The simulations also confirmed that the blocking calcium ions are ‘stuck' at the bottom of the selectivity filter, and revealed that this ‘sticking' is related to the increased hydrophilicity (affinity to water) of relevant structural parts of the mutated channels.The results of Sumikama and Irie's colleagues provide an important step forward towards a full understanding of the mechanism of divalent cation block in NavAb, an important and representaNanoLSI Podcast website

Brain cancer linked to nuclear pore alterations  Transcript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Masaharu Hazawa and Richard Wong at the Kanazawa University NanoLSI, alongside Mitsutoshi Nakada and colleagues at Kanazawa University.The research described in this podcast was published in Cell Reports in August 2023 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Brain cancer linked to nuclear pore alterations Researchers at Kanazawa University report in Cell Reports how alterations in nuclear pores lead to the degradation of anti-tumor proteins. Several types of cancer are believed to be linked to alterations of macromolecular structures known as nuclear pore complexes.  These structures are embedded in the nuclear envelope, a membrane barrier that separates the nucleus of a cell from the cytoplasm (the liquid filling the rest of the cell).  They consist of proteins called nucleoporins, which regulate the transport of molecules across the nuclear envelope, including enzymes that enable the synthesis of DNA.  Whether nuclear pore complex alterations play a role in glioblastoma, the most common type of cancer originating in the brain, is unclear at the moment.  Now, Masaharu Hazawa, Mitsutoshi Nakada and Richard Wong from Kanazawa University and colleagues have found a link between the functioning of nuclear pore complexes and glioblastoma — specifically, they demonstrated the inactivation of a tumor-suppressing protein called p53. The protein p53 is crucial in cancer prevention.  The corresponding gene TP53 encodes proteins that prevent mutations of the genome and is the most frequently mutated gene in human cancers.  Gaining insights into how p53 inactivation happens is crucial for understanding tumorigenesis in general and glioblastoma in particular.So how did the researchers go about it?Mitsutoshi Nakada and Richard Wong and colleagues first checked whether any nuclear pore complex proteins were amplified (that is ‘overexpressed') in glioblastoma.  They found that one such protein, called NUP107, showed overexpression.  Further investigations revealed that NUP107 is a potential oncoprotein in glioblastoma; its overexpression degrades the function of the cancer-suppressing p53 protein.  They also found that MDM2, another protein, is overexpressed simultaneously with NUP107.  MDM2 is also known to mediate p53 protein degradation. Further studies will be necessary to uncover the full molecular pathways at play, but the scientists speculate that the increased amount of NUP107 proteins in the nuclear pore complexes of glioblastoma cells results in nuclear pore complex structures that regulate the transport of molecules from the nucleus to the cytoplasm in a way that promotes p53 degradation.  This scenario is referred to as nuclear transport surveillance.  Experiments in which NUP107 proteins were depleted re-activated p53, consistent with NUP107 providing the structural stability of glioblastoma NPCs. The findings of Mitsutoshi Nakada and Richard Wong and colleagues confirm that alterations of nuclear pore complexes contribute to the pathogenesis of glioblastoma.  As Mitsutoshi Nakada and Richard Wong put it : “Together, our findings establish roles of nuclear pore complexes in transport surveillance and provide insights into p53 inactivation in glioblastoma.”  ReferenceDini Kurnia Ikliptikawati, Nozomi Hirai, Kei Makiyama, Hemragul Sabit, Masashi Kinoshita, Koki Matsumoto, Keesiang Lim, Makiko Meguro-Horike, Shin-ichi Horike, Masaharu Hazawa, Mitsutoshi Nakada, and Richard&NanoLSI Podcast website

Researchers define a protocol for narrow nanoneedle fabrication and high-resolution imaging of living cells using AFM  Transcript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Takehiko Ichikawa and Takeshi Fukuma at the Kanazawa University NanoLSI.The research described in this podcast was published in STAR Protocols in September 2023 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Researchers define a protocol for narrow nanoneedle fabrication and high-resolution imaging of living cells using AFM Researchers at Kanazawa University report in STAR Protocols procedural details and tips for nanoendoscopy-AFM, for capturing images of nanoscale structures inside living cells. Images of nanoscale structures inside living cells are in increasing demand for the insights into cellular structure and function they can reveal. So far, the tools for capturing such images have been limited in various ways, but researchers led by Takeshi Fukuma and Takehiko Ichikawa at Kanazawa University have now devised and reported a full protocol for using atomic force microscopy (AFM) to image inside living cells. AFM was first developed in the 1980s and uses the changes in the forces between a sample surface and a nanoscale tip attached to a cantilever to “feel” surfaces and produce images of the topography with nanoscale resolution. The technique has grown increasingly sophisticated for extracting information about samples and at speeds sufficient for the tool to capture moving images of dynamics at the nanoscale. However, so far, it has been limited to surfaces. Other techniques exist that can provide a view of the inside of a cell but with limitations. For instance, there is electron microscopy, which is capable of resolving details at the nanoscale and smaller, but the required operating conditions are not compatible with living cells. Alternatively, fluorescence microscopy is regularly used on living cells, but while fluorescence techniques exist to increase the resolution, there are practical challenges that inhibit fluorescence imaging at the nanoscale. AFM suffers from neither limitation and by embellishing the tool with a nanoneedle to penetrate cells, Fukuma, Ichikawa and their collaborators have recently demonstrated the capability to image inside cells at the nanoscale, which they describe as nanoendoscopy-AFM.So how does it work?In their protocol, the researchers break down the method for nanoendoscopy-AFM into 4 stages. The first few steps involve cell preparation and staining with a fluorescent dye and checking the fluorescence, which is used to identify the imaging area quickly. Next is the fabrication of the nanoneedles themselves, for which there are two options – either etching away a nanoneedle structure with a focused ion beam or building one up with electron beam deposition. Then comes the nanoendoscopy stage itself, and in the report, the researchers describe the approach for both 2D  and 3D nanoendoscopy. There are even details outlined to describe the best way to clean up after the nanoendoscopy images are captured before finally outlining the data processing needed to visualize the measured data. The method is replete with tips for successfully accomplishing each stage, as well as a guide for troubleshooting when things are not quite working out. This technique should be suitable for the observation of intact intracellular structures, including mitochondria, focal adhesions, endoplasmic reticulum, lysosomes, Golgi apparatus, organelle connections, and liquid-liquid phase-separated structures. They conclude, “This protocol can NanoLSI Podcast website

High-speed atomic force microscopy takes on intrinsically disordered proteins Transcript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Toshio Ando at the Kanazawa University NanoLSI, alongside Sonia Longhi at Aix-Marseille University and CNRS in France.The research described in this podcast was published in Nature Nanotechnology in November 2020 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/High-speed atomic force microscopy takes on intrinsically disordered proteinsKanazawa University's pioneering high-speed atomic force microscope technology has now shed light on the structure and dynamics of some of life's most ubiquitous and inscrutable molecules – intrinsically disordered proteins. The study is reported in Nature Nanotechnology.Our understanding of biological proteins does not always correlate with how common or important they are. Half of all proteins, molecules that play an integral role in cell processes, are intrinsically disordered, which means many of the standard techniques for probing biomolecules don't work on them. Now researchers at Kanazawa University in Japan have shown that their home-grown high-speed atomic force microscopy technology can provide information not just on the structures of these proteins but also their dynamics.Understanding how a protein is put together provides valuable clues to its functions. The development of protein crystallography in the 1930s and 1950s brought several protein structures into view for the first time, but it gradually became apparent that a large fraction of proteins lack a single set structure making them intractable to xray crystallography. As they are too thin for electron microscopy, the only viable alternatives for many of these intrinsically disordered proteins are nuclear magnetic resonance imaging and small angle xray scattering. Data collected from these techniques are averaged over ensembles and so give no clear indication of individual protein conformations or how often they occur. Atomic force microscopy on the other hand is capable of nanoscale resolution biological imaging at high-speed, so it can capture dynamics as well as protein structures.So what kind of insights can high-speed AFM offer for these proteins? In this latest work researchers at Kanazawa University alongside collaborators in Japan, France and Italy applied the technique to study several intrinsically disordered proteins. They identified parameters defining the shape, size and chain length of protein regions, as well as a power law relating the protein size to the protein length. Not only that but they got a quantitative description of the effect of the mica surface on protein dimensions. The dynamics of the protein conformations captured thanks to the high-speed capabilities of the technique revealed globules that appear and disappear, and transformations between fully unstructured and loosely folded conformations in segments up to 160 amino acids long.Studies of the measles virus nucleoprotein in particular helped identify not just the shape and dimensions but also characteristics of the order-disorder transitions in the region responsible for molecular recognition, which allows viruses to identify host factors so that they can reproduce. They could also determine larger scale structures of the virus's phosphoprotein that are not accessible to nuclear magnetic resonance (which can only give an indication of distances between amino acids separated by less than 2 nm). The researchers suggest that the formation of certain compact shapes observed may explain the resistance to proteolysis – protein breakNanoLSI Podcast website

Kanazawa University NanoLSI Podcast:Heat and manipulate, one cell at a time Transcript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Satoshi Arai at the Kanazawa University NanoLSI.The research described in this podcast was published in ACS Nano in June 2022Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Heat and manipulate, one cell at a timeResearchers at Kanazawa University report in ACS Nano the development of a nanoparticle that acts as a heater and a thermometer.  Inserting the nanoparticle in living cells results in a heat spot that, by switching it on and off, enables the controlled modulation of local cellular activities.Being able to heat nano-sized regions in biological tissues is key to several biomedical applications.  Indeed, many biological processes are temperature-sensitive, and the ability to locally modify temperature provides a way to manipulate cellular activity.  A notable purpose is the destruction of cancer cells by heating them.  Beside the need for an in-tissue local heating mechanism, it also important to be able to instantaneously measure the generated temperature.  Satoshi Arai from Kanazawa University and colleagues have now engineered a nanoparticle that is both a nanoheater and a nanothermometer at the same time.  They successfully showed that the insertion of a single, controllable heat spot in tissue can be very effective in modifying cellular function.The nanoparticle, called “nanoHT” by the scientists — an abbreviation of “nanoheater-thermometer” — is essentially a polymer matrix embedding a dye molecule (called EuDT) used for sensing temperature, and another dye molecule (called V-Nc) for releasing heat.  The latter happens through the conversion of light into thermal energy (the photothermal effect, which is also exploited in solar cells): shining a near-infrared laser (with a wavelength of 808 nanometer) onto V-Nc results in fast heating, with a stronger increase in temperature for higher laser power.The temperature sensing is based on the thermal fluorescence effect of EuDT.  When irradiated with light of one wavelength, the molecule emits light at another wavelength — that is, it fluoresces.  The higher the temperature, the less intense the fluorescence becomes.  This inverse relationship can be used to measure temperature.  Arai and colleagues tested the performance of nanoHT as a thermometer, and established that it can determine temperatures with a resolution of 0.8 °C and less.So what could you use this nanoHT for?The researchers then performed experiments with a type of human cells called HeLa cells.  They looked at the effect of heating through nanoHT, and found that at a temperature increment of about 11.4 °C, the heated HeLa cells died after only a few seconds.  This finding suggests that nanoHT could be used to induce cell death in cancer cells.Arai and colleagues also studied how nanoHT can be used to affect the behavior of muscles.  They introduced the nanoparticle into a myotube, a type of fiber present in muscle tissue.  Upon heating the myotube by approximately 10.5 °C, the muscle tissue contracted.  The procedure worked reversibly; letting the myotube cool again led to muscle relaxation.The work of Arai and colleagues shows that local heating at a subcellular scale by means of nanoHT enables the controlled manipulation of a single cell's activity.  Regarding applications, the scientists believe that – to quote them from their paper -  “the targeted application of nanoHT has a diverse and versatile range of capabilities to regulate cellular activities that wNanoLSI Podcast website

Xiabing Lyu: Exosomes to regulate the human immune system (Kanazawa/ Recorded in June 2023)  Xiabing Lyu is an Assistant Professor at the Nano Life Science Institute (WPI-NanoLSI), Kanazawa University. Here, she describes her research on engineering exosomes that regulate anti-viral and anti-tumor immune system responses.  Details here: https://nanolsi.kanazawa-u.ac.jp/en/about/members/life-science/ The Kanazawa University NanoLSI Podcast offers updates of the latest news and research at the WPI-NanoLSI Kanazawa University. The Nano Life Science Institute (NanoLSI) at Kanazawa University was established in 2017 as part of the World Premier International (WPI) Research Center Initiative of the Ministry of Education, Culture, Sports, Science and Technology (MEXT). Researchers at the NanoLSI are combining their cutting-edge expertise in scanning probe microscopy to establish ‘nano-endoscopic techniques' to directly image, analyze, and manipulate biomolecules for insights into mechanisms governing life phenomena such as diseases. Further information WPI-NanoLSI Kanazawa University website https://nanolsi.kanazawa-u.ac.jp/en/NanoLSI Podcast website

Endoscopy of a living cell on the nanoscale Transcript of this podcast Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Takeshi Fukuma at the Kanazawa University NanoLSI. The research described in this podcast was published in Science Advances in December 2021 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Endoscopy of a living cell on the nanoscaleResearchers at Kanazawa University report in Science Advances a new technique for visualizing the inside of a biological cell. The method is an extension of atomic force microscopy and offers the promise of studying nanoscale inner cell dynamics at high resolution in a non-destructive way.In order to advance our understanding of how biological cells function, visualizing the dynamics of intra-cellular components on the nanoscale is of key importance.  Current techniques for imaging such dynamics are not optimal — for example, fluorescence microscopy can visualize ‘labeled' molecules but not the target components themselves.  Now Takeshi Fukuma from Kanazawa University and his colleagues have developed a label-free, non-destructive nanoimaging method, which they call nanoendoscopy-AFM – it's a version of atomic-force microscopy that can be deployed within a living cell.  The research was carried out as a collaboration between Kanazawa University and the National Institute of Advanced Industrial Science and Technology (AIST), with Marcos Penedo, the lead author of the publication reporting the new method, recently moving from Kanazawa University's Nano Life Science Institute (WPI-NanoLSI) to the École Polytechnique Fédérale de Lausanne, Switzerland.So what is AFM in the first place?The principle of AFM – or atomic force microscopy ti give its full title - is to have a very small tip move over the surface of a sample.  During this ‘xy' scanning motion, the tip, attached to a small cantilever, will follow the sample's height, that is, the (‘z') dimension or profile, producing a measurable force on the cantilever.  The magnitude of the force can be back-converted into a height value; the resulting height map provides structural information about the sample's surface.The researchers designed a novel AFM setup where the needle-like tip is brought in and out of the interior of a cell.  The process is reminiscent of an endoscopy — the procedure of looking at an organ from the inside, by inserting a small camera attached to a thin tube into the body — which is why Fukuma and colleagues call their technique nanoendoscopy-AFM.  Letting the nanoneedle travel in an ‘xyz' trajectory, and going in and out of the cell results in a 3D map of its structure.  They tested the technique on a cell from the so-called HeLa cell line commonly used in medical research, and could clearly identify internal granular structures in a scanned volume of 10 x 10 x 6 µm3.But how does the cell fare under this kind of interrogation?During a scan, the nanoneedle penetrates the cell membrane (and the nuclear membrane) many times.  The scientists checked whether this repeated penetration causes any damage to the cell.  They performed a viability test on HeLa cells by using two fluorescent marker molecules.  One molecule emits green fluorescence from a living cell, the other red fluorescence from (the nucleus of) a dead cell.  The researchers found that when using nanoprobes smaller than 200 nm, nanoendoscopy-AFM does not severely damage cells.The method is also particularly useful for probing surfaces within the cell, for example the inner side of the cell membrane or the surface of the cell nucleus.  Fukuma and colleagues call this application 2D NanoLSI Podcast website

Kanazawa University NanoLSI Podcast: Enhancing carbon dioxide reductionTranscript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Yasafumi Takahashi at the Kanazawa University NanoLSI and Yoshikazu Ito and Yuta Hori at the University of Tsukuba.The research described in this podcast was published in ACS Nano in June 2023Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Enhancing carbon dioxide reductionResearchers at Kanazawa University report in ACS Nano how ultrathin layers of tin disulfide can be used to accelerate the chemical reduction of carbon dioxide — a finding that is highly relevant for our quest towards a carbon-neutral society.Recycling carbon dioxide released by industrial processes is a must in humanity's urgent quest for a sustainable, carbon-neutral society.  For this purpose, electrocatalysts that can efficiently convert carbon dioxide into other, less impactful chemical products are widely researched today.  A category of materials known as two-dimensional (2D) metal dichalcogenides are candidate electrocatalysts for carbon dioxide conversion, but these materials also typically facilitate competing reactions, which compromises their efficiency.  Yasufumi Takahashi from Nano Life Science Institute (WPI-NanoLSI), at Kanazawa University and colleagues have now identified a 2D metal dichalcogenide that can efficiently reduce carbon dioxide to formic acid, a compound that not only occurs naturally but is also an intermediate product in chemical synthesis.Takahashi and colleagues compared the catalytic performance of 2D sheets of molybdenum disulfide and tin disulfide.  Both are 2D metal dichalcogenides, with the latter of particular interest because pure tin is a known catalyst for the production of formic acid.  Electrochemical tests of these compounds revealed that with molybdenum disulfide, instead of carbon dioxide conversion, hydrogen evolution reactions were promoted.  Hydrogen evolution reactions refer to reactions yielding hydrogen, which can be useful when the production of hydrogen gas fuel is intended, but in the context of carbon dioxide reduction it is an unwanted competing process.  Tin disulphide, on the other hand, showed good carbon dioxide reduction activity and suppressed hydrogen evolution reactions.  The researchers also carried out electrochemical measurements for bulk tin dioxide powder, which was found to have less catalytic carbon dioxide reduction activity.So how is tin disulphide facilitating carbon dioxide reduction?To understand where the catalytically active sites are in tin disulphide, and why the 2D material performs better than the bulk compound, the scientists applied a method called scanning electrochemical cell microscopy (SECCM).  SECCM is used as a nanopipette to form the meniscus shape nanoscale electrochemical cell for the surface reactivity sensing probe on the sample.  The measurements revealed that the whole surface of the tin disulphide sheet is catalytically active, not only ‘terrace' or ‘edge' features in the structure.  This also explains why 2D tin disulphide has enhanced activity compared to bulk tin disulphide.Calculations provided further insights into the chemical reactions at play.  Specifically, the formation of formic acid was confirmed as an energetically favorable reaction pathway when using 2D tin disulphide as catalyst.The results of Takahashi and colleagues signify an important step forward towards the use of 2D electrocatalysts in electrochemical carbon dioxide reduction applications.  Quoting the scientists: “These findings will provide a better understanding and desigNanoLSI Podcast website

Kanazawa University NanoLSI Podcast: Experiments provide insights into the molecular mechanism for memory and learning Transcript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Mikihiro Shibata at the Kanazawa University NanoLSI alongside Hideji Murakoshi at The Graduate University for Advanced Studies and the National Institute for Physiological Sciences, and their colleagues.The research described in this podcast was published in Science Advances in June 2023Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Experiments provide insights into the molecular mechanism for memory and learning Researchers at Kanazawa University report in Science Advances  high-speed atomic force microscopy experiments that show the structural and chemical changes in an enzyme thought to play a vital role in modulating the strength of neural connections.Synapses connect neurons allowing the transmission of signals around the neural network. The strength of these connections varies – for instance strengthening or weakening depending on the signals received and how. This synaptic “plasticity” underlies learning and memory and the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to play a key role. Previous studies have provided some clues to the mechanisms of CaMKII protein activity in these functions but no-one had seen these proteins in action. Now Hideji Murakoshi at the Graduate University of Advanced Studies and the National Institute for Physiological Sciences and Mikihiro Shibata at Kanazawa University and their colleagues have used high speed atomic force microscopy (HS-AFM) to observe the structural dynamics of these proteins for the first time, not only in various states but in three different species.CaMKII is common to a vast range of species from mammals like rats to older, non-mammalian species like the roundworms (C. elegans) and hydra. In particular, certain structural features of the protein are particularly well preserved, including the kinase domain, the regulatory segment that inhibits the activity of the kinase domain and the hub domain. In addition, the protein has binding sites, phosphorylation sites and linker regions – however, the linker region shows a little more variability across species suggesting that its function and activation mechanisms are more bespoke for the different species.So what did we know about how this protein works? (3min)Previous studies had suggested that the regulatory segment's inhibition of the kinase domain is released when Ca2+/calmodulin binds to the regulatory segment. The activated kinase domains then phosphorylate each other, activity that persists even after the Ca2+/calmodulin becomes dissociated, which has been “hypothesized to be a form of molecular memory”, as the researchers describe in their report.Murakoshi, Shibata and their colleagues studied the protein using atomic force microscopy, which feels topologies using a nanoscale tip like a needle reading a vinyl record, raster scanning the image plane to build up a picture of the sample structure. With HS-AFM, these images are collected quickly enough to record movies of how these structures change. The researchers noted various measures of the proteins size and motion – the gyrus of rotation – as well as reactions such as kinase domain oligomerization (that is, where there is a limited level of polymerization to join molecules into chains) and phosphorylation – the addition of a phosphoryl group (PO3), which can activate enzymes like kinase.They found that the kinase domain was quite mobile, although this decreased with Ca2+/calmodulin binding. ThNanoLSI Podcast website

Kanazawa University NanoLSI Podcast:Zooming in on neurotoxic aggregatesTranscript of this podcast Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research led by Kenjiro Ono  at the Kanazawa University NanoLSI. The research described in this podcast was published in Nano Letters in May 2023 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/ Zooming in on neurotoxic aggregatesResearchers at Kanazawa University report in Nano Letters how high-speed atomic force microscopy leads to insights into processes relevant to Alzheimer's disease.  Moreover, the technique is shown to be an excellent tool for studying the effect of drugs against the disease.According to the amyloid hypothesis, Alzheimer's disease — the most common type of dementia — is caused by flaws in the production, accumulation, and disposal of amyloid-beta in the brain.  Amyloid-beta refers to a group of peptides (protein fragments) that over time form plaques in the brain of a person with Alzheimer's disease.  Drugs aiming to reduce the aggregation of amyloid-beta have been developed, but recent findings show that different types of amyloid-beta aggregates have different contributions to the development of Alzheimer's disease.  In particular, intermediate aggregates such as protofibrils are more toxic than the actual final fibrils, the main component of amyloid-beta plaques.  A precise understanding of the complex aggregation pathways is therefore necessary for the further development of efficient drugs against Alzheimer's disease.  Kenjiro Ono from Kanazawa University and colleagues have now succeeded in visualizing the structural dynamics of protofibrils, as well as the effect of a recently developed drug based on anti-amyloiide-beta antibodies.So how did they go about it?The scientists looked at the formation and the structure of amyloid-beta protofibrils by means of high-speed atomic force microscopy (AFM).  The latter method has in recent years emerged as a powerful nanoimaging tool for studying biomolecules and their dynamics at high spatiotemporal resolution.  High-speed AFM observations showed that protofibrils have a nodal structure, with stable structural features — specifically, the binding angle between nodes — across several samples.  Importantly, this nodal structure is distinct from proper, mature fibrils, which have a helical structure.Ono and colleagues then investigated the dissociation of protofibrils.  They found that the length of protofibrils depends on their concentration, suggesting that aggregates can dissociate spontaneously.Now onto the interaction with drugsTo obtain detailed insights into the functioning of anti-amyloid-beta antibody drugs, the researchers examined the binding between amyloid-beta protofibrils and a new drug known as lecanemab.  They found that the binding ability – termed the affinity - of lecanemab for protofibrils is almost independent of the size of the protofibrils — in other words, the affinity does not substantially vary throughout the aggregation process.  High-speed AFM observations further revealed that lecanemab covers the surface of small, pre-protofibril aggregates.  In doing so, the drug inhibits the further aggregation into protofibrils, which in turn prevents the formation of proper amyloid-beta fibrils and plaques.The results of Ono and colleagues provide direct evidence of a mechanism through which an antibody drug interferes with the amyloid-beta aggregation process.  More generally, the work confirms the versatility of high-speed-AFM as a tool for studying biochemical pathways.  NanoLSI Podcast website

Kanazawa University NanoLSI Podcast:Experiments reveal chilli-sensitive molecular structure fluctuation changes in TRPV1Transcript of this podcast Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Ayumi Sumino at the Kanazawa University NanoLSI alongside Motoyuki Hattori at Fudan University in China, and their colleagues. The research described in this podcast was published in the journal Proceedings of the National Academy of Science in May 2023 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Experiments reveal chilli-sensitive molecular structure fluctuation changes in TRPV1Researchers at Kanazawa University report high-speed atomic force microscopy experiments that show how ligands associated with stimulating and suppressing activation of the TRPV1 protein increase and decrease the molecule's structural variations. The observations provide insights into how these heat- and chilli-sensing proteins function.The skin senses heat – both from increased temperature and molecules like capsaicin in chillies – through the activation of protein receptors called Transient receptor potential vanilloid member 1 or TRPV1. However, the mechanisms behind the function of TRPV1 have not been clear. Now Ayumi Sumino at Kanazawa University in Japan and Motoyuki Hattori at the Fudan University in China and their colleagues provide important insights into this mechanism. Using high-speed atomic force microscopy to compare the protein with and without stimulating or suppressing molecules – ligands – bound to it, they obtain what they describe as “the first experimental evidence showing the correlation between molecular fluctuation and the gating state (ligand binding)”.So what was already known about this mechanism?Well once activated, the TRPV1 channel opens, allowing ions to permeate and signalling to the nervous system that a noxious stimulant is present. And in 2011 researchers at the Howard Hughes Medical Institute in the US put forward a theoretical basis for the activation of the receptor derived from thermodynamics, a theoretical framework that has since been corroborated by experiment. The idea was that the molecule would respond to heat with a change in heat capacity, which is related to the fluctuations in the molecule's conformation. Structures for the TRPV1 protein were known from previous cryo electron microscopy studies but these did not clarify how the fluctuations in protein conformation might change with stimulating or suppressing molecules, or even whether temperature and chilli sensing shared the same molecular mechanism.Here's where the high-speed atomic force microscopy comes inAtomic force microscopy (AFM) senses the topology of surfaces through the effect of distance on the forces on a nanosized tip positioned directly above the surface. The microscope was first invented in 1986 but gained a new lease of life through work at Kanazawa University that enabled it to capture topologies at high speed thereby providing a window into the dynamics of structures.Sumino, Hattori and colleagues used high-speed AFM to image the TRPV1 receptor both in its unbound state and when bound to ligand molecules that either stimulate, that is agonist molecules, or suppress - antagonist molecules - the protein's activity. They used the molecule resiniferatoxin, which is 1000 times hotter than capsaicin, as the agonist and for the antagonist they used capsazepine, which blocks the pain of capsaicin.From the structures captured the researchers were able to observe fluctuations in the conformation of both the bound and unbound states of TRPV1. They found that resiniferaNanoLSI Podcast website

Kanazawa Univesity NanoLSI Podcast:Dynamic 3D structure extraction from HS-AFM imagesTranscript of this podcast Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by  Holger Flechsig and Toshio Ando at the Kanazawa University NanoLSI. The research described in this podcast was published in the journal Current Opinion in Structural Biology in April 2023 Kanazawa University NanoLSI website  https://nanolsi.kanazawa-u.ac.jp/en/ Dynamic 3D structure extraction from high-speed atomic force microscopy images  By allowing the direct observation of biomolecules in dynamic action, high-speed atomic force microscopy or AFM has opened a new avenue to dynamic structural biology. A vast number of successful applications within the past 15 years have provided unique insights into essential biological processes at the nanoscale – visualizing, for example, how molecular motors execute their specific functions. Some intrinsic limitations of AFM imaging are that only the surface topography can be acquired, and that the AFM tip is too large to resolve details below the nanometer scale. To facilitate the interpretation and understanding of high-speed AFM observations, post-experimental analysis and computational methods play an increasingly important role. In their review paper published in the Current Opinion in Structural Biology journal Holger Flechsig a computational scientist at the NanoLSI at Kanazawa University and Toshio Ando, a Distinguished Professor at NanoLSI, provide an overview of developments in this topical field of interdisciplinary research. Computational modeling and simulations already allow the reconstruction of 3D conformations with atomistic resolution from topographic resolution-limited AFM images. Furthermore, quantitative analysis methods allow for example automated recognition of biomolecular shape changes from topographic images, or feature assignment including the identification of amino acid residues on the molecular surface.So how is all this implemented?The developed computational methods are often implemented in open-access software, allowing for convenient applications by the broad Bio-AFM community to complement experimental observations. In that regard, the BioAFMviewer software project initiated at Kanazawa University in 2020 has gained significant attention and plays an important role in a plethora of collaboration projects.Combining high-speed AFM and computational modeling will elevate the understanding of how proteins function in atomistic detail. An ambitious future goal is the application of molecular modeling to reconstruct atomistic-level 3D molecular movies from high-speed AFM topographic movies.ReferenceHolger Flechsig and Toshio Ando. Protein dynamics by the combination of high-speed AFM and computational modelingNanoLSI Podcast website

Kanazawa University NanoLSI Podcast:Scanning probe simultaneously captures structural and ion concentration changesTranscript of this podcast Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research led by Yasufumi Takahashi and Takeshi Fukuma at the Kanazawa University NanoLSI. The research described in this podcast was published in the Journal of the American Chemistry Society Au in February 2023 Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Scanning probe simultaneously captures structural and ion concentration changesResearchers at Kanazawa University report in the Journal of the American Chemistry Society Gold how they have developed operando  scanning ion conductance microscopy to allow simultaneous measurements of changes in the anode surface topography of a lithium ion battery during use, as well as the varying ion concentration with depth. Combining both types of information should help researchers evaluate the correlation between the two to design better batteriesLithium ion batteries dominate the energy storage sector from the scale of small portable devices to electric vehicles and even grid-scale electricity suppliers. Research is constantly ongoing to improve their energy density, charging speed, lifetime and safety, among other attributes. Here an understanding of not just the changes that go on in lithium ion batteries but how different parameters might interact can be extremely advantageous. Now researchers led by Yasufumi Takahashi and Takeshi Fukuma at Kanazawa University in Japan report simultaneous measurements of topography and ion concentration profiles by developing their operando scanning ion conductance microscopy (operando SICM). The combined data they retrieve can enable evaluation of correlations between the two parameters for improving future battery performance.So what sorts of processes in batteries might we want to shed light on?As Takahashi and Fukuma list in their report on the work, several processes are involved in the charging and discharging of lithium batteries, including the transport, solvation or desolvation, and intercalation of lithium ions, as well as structural changes and expansion in the electrodes, and the formation and deposition of by-products. These all occur out of equilibrium under applied electric potentials. “Capturing such multi-step and time-dependent changes with a relevant spatiotemporal resolution enables optimizing the operating conditions,” they point out highlighting design features that might benefit such as the structure of electrodes and separator, and tailoring additives to ensure proper solid−electrolyte interphase formation. As a result, numerous techniques have been used to investigate how both the surface topography and the ion concentration of the anode or cathode change during charging or discharging, all with different limitations.A key advantage of scanning ion conductance microscopy is that it can measure surface morphology and properties, including changes in ion concentration with depth in the electrolyte. Until now, however, no-one had retrieved simultaneous topographical and ion concentration data of a battery during charging and discharging.So how does scanning ion conductance microscopy work anyway, and how did the researchers get it take both types of data simultaneously?Scanning ion conductance microscopy uses a nanopipette containing an ionic solution as the scanning probe. The nanopipette acts as a probe and monitors changes in ion current from which it is possible to visualise ion concentrations as well as the distance to a surface. To take both measurements simultaneously, the researcNanoLSI Podcast website

Kien Xuan NGO: Interdisciplinary research to address important problems in modern biologyAssistant Professor Kien Xuan Ngo is member of Toshio Ando's Nanometrology group at the Kanazawa NanoLSI. In this podcast is describes his research on structural biology for clarifying the functions of cytoskeletal proteins—such as actin and microtubules—and so-called ‘ABC transporters'. He is combining his expertise in biochemistry, biophysics, and mathematical simulations to address important problems in modern biology. The Kanazawa University NanoLSI Podcast offers updates of the latest news and research at the WPI-NanoLSI Kanazawa University.The Nano Life Science Institute (NanoLSI) at Kanazawa University was established in 2017 as part of the World Premier International (WPI) Research Center Initiative of the Ministry of Education, Culture, Sports, Science and Technology (MEXT).Researchers at the NanoLSI are combining their cutting-edge expertise in scanning probe microscopy to establish ‘nano-endoscopic techniques' to directly image, analyze, and manipulate biomolecules for insights into mechanisms governing life phenomena such as diseases.Further informationWPI-NanoLSI Kanazawa University websitehttps://nanolsi.kanazawa-u.ac.jp/en/NanoLSI Podcast website

Holger Flechsig: Computational biophysics to visualize the dynamics of proteins  Assistant Professor Holger Flechsig is a member of the Computational Molecular Physics group at the NanoLSI WPI Kanazawa University.  Here he describes his research on answering the questions, “How do proteins work.” Specially, on molecular machines and motors, protein allostery, proteins interactions and cellular scale phenomena using multi-scale molecular dynamics simulations that enable us to produce molecular movies to visualize the dynamic motion of proteins. The Kanazawa University NanoLSI Podcast offers updates of the latest news and research at the WPI-NanoLSI Kanazawa University. The Nano Life Science Institute (NanoLSI) at Kanazawa University was established in 2017 as part of the World Premier International (WPI) Research Center Initiative of the Ministry of Education, Culture, Sports, Science and Technology (MEXT). Researchers at the NanoLSI are combining their cutting-edge expertise in scanning probe microscopy to establish ‘nano-endoscopic techniques' to directly image, analyze, and manipulate biomolecules for insights into mechanisms governing life phenomena such as diseases. Further information WPI-NanoLSI Kanazawa University websitehttps://nanolsi.kanazawa-u.ac.jp/en/NanoLSI Podcast website

Kanazawa University NanoLSI Podcast: The offshoot of cells visualized in real timeTranscript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Richard Wong and colleagues at the Kanazawa University NanoLSI. The research described in this podcast was published in the Journal of Extracellular Vesicles, in November 2022 Kanazawa University NanoLSI website https://nanolsi.kanazawa-u.ac.jp/en/The offshoot of cells visualized in real timeIn a study recently published in the Journal of Extracellular Vesicles, researchers from Kanazawa University use high-speed microscopy to capture the dynamics of nanosized sacs released from cells.Small extracellular vesicles (sEVs) are tiny sacs released by cells to deliver chemical messengers to other cells. Since sEVs are compatible with biological tissue they are being investigated as carriers for nanodrugs. However, the impact of physiological stress—such as changes in temperature—on the structure of sEVs is obscure. A research team led by Richard Wong and Keesiang Lim at Kanazawa University has now used an advanced form of microscopy to elucidate these changes in real time.The temperature, acid, and salt levels in our bodies can fluctuate with factors such as disease. Thus, research on sEVs for drug development requires a deeper understanding of how stressful environments affect the vesicles' structure. For their study, the team first isolated sEVs from cells. Next, using a technique known as high-speed atomic force microscopy (HS-AFM) the structure of sEVs was revealed to be either spherical or ellipsoidal in shape. HS-AFM also enabled the researchers to accurately measure the sizes of sEVs without rupturing or damaging the vesicular membranes.The effect of varying temperatures on sEVs was the first parameter assessed. At temperatures higher than normal (37°C) body temperature the vesicles showed deformations in shape coupled with a loss of elasticity of their membranes. On the other hand, sEVs in cold conditions (4°C) had a reduced ability to release any internal material effectively.The researchers then studied the effects of pH (acid levels) on sEVs. The physiological pH of the bloodstream is 7.4. A pH less than 7 indicates acidic conditions and anything more than that is termed alkaline. The sEVs seemed to maintain their shape in acidic conditions (pH 4) but in alkaline conditions (pH 10) they were deformed. However, at a pH of 4 the sEVs were smaller in size suggesting their internal contents had been lost.Now, salt levels (known as osmotic pressure) at a concentration of 0.15 M are healthy. However, changes in osmotic pressure can have detrimental effects on cells. As conditions were gradually changed it was found that the spherical nature of sEVs decreased at high salt concentrations (1.8 M) but seemed to remain intact at low concentrations (0 M). After a while, vesicles in high osmotic conditions showed ruptured membranes.An understanding of these dynamics is imperative to formulating sEVs as pharmaceutical aids in different disease conditions. This study established HS-AFM as a useful tool to depict changes in sEVs under various physiological conditions in real time. “In summary, our study demonstrates the feasibility of HS-AFM for structural characterization and assessment of nanoparticles,” concludes the team.ReferenceElma Sakinatus Sajidah, Keesiang Lim, Tomoyoshi Yamano, Goro Nishide, Yujia Qiu, Takeshi Yoshida, Hanbo Wang, Akiko Kobayashi, Masaharu Hazawa, Firli Dewi, Rikinari Hanayama, Toshio Ando, Richard Wong. Spatiotemporal tracking of small extracellular vesicle nanotopology in response to physicochemical strNanoLSI Podcast website

Kanazawa University NanoLSI Podcast: Biological lasso: Enhanced drug delivery to the brainTranscript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Kunio Matsumoto and colleagues at the Kanazawa University NanoLSI. The research described in this podcast was published in the journal Nature Biomedical Engineering in November 2022 Kanazawa University NanoLSI website https://nanolsi.kanazawa-u.ac.jp/en/Biological lasso: Enhanced drug delivery to the brainIn a study recently published in the journal Nature Biomedical Engineering, researchers from Kanazawa University use a method called “lasso-grafting” to design therapeutics with enhanced longevity and brain penetration. Cell growth and repair are stimulated by biomolecules known as cytokines and growth factors. Unfortunately, delivering adequate concentrations of these molecules to the brain for treating neurological conditions like Alzheimer's disease is challenging as they are either cleared out of the blood very quickly or do not penetrate neural tissue effectively. A research team led by Kunio Matsumoto and Katsuya Sakai at Kanazawa University in collaboration with Junichi Takagi, Osaka University and Hiroaki Suga, the University of Tokyo has now used a technique called “lasso-grafting” to design molecules that replicate growth factors with longer retention in the body and brain penetration.The team synthesized a molecular entity comprising two components: macrocyclic peptides inserted into antibody fragments (known as Fc). Macrocyclic peptides are truncated proteins which can be engineered to resemble growth factors. Using lasso-grafting, a method previously developed by the researchers, the selected peptides were inserted into loops found on Fc. Now, lasso-grafting ensures that the macrocyclic peptides are easily exposed while keeping the structural integrity and function of both the peptide and Fc intact. Fc was used for this purpose as it remains in the body long enough and can easily add functionality of the Fab of choice.Using this process, a designer molecule replicating the hepatocyte growth factor (HGF) was first created. HGF binds a docking protein known as Met on the surface of cells to initiate signaling for cell growth and survival. Thus, aMD4 and aMD5, two macrocyclic peptides that can also bind to Met were first identified. They were then grafted into various sites on Fc until optimum insertion sites were found. When exposed to cells, Fc(aMD4) and Fc(aMD5) indeed latched onto Met receptors and initiated cellular signaling akin to HGF (Fig. 1b). Next, the longevity of Fc(aMD4) compared to Fc and HGF alone, was examined. When administered to mice, concentrations of HGF dwindled significantly after an hour while Fc(aMD4) persisted at levels enough to activate Met, for up to 200 hours. Markers for cellular replication were also active in these mice. Fc(aMD4) thus showed longevity and bioactivity.  The final step was to determine the brain penetration of these designer molecules. For this purpose, aMD4 was inserted into an Fc of anti-transferrin receptor (TfR) antibody which accumulates in the mouse brain after peripheral administration (Fig. 1c). Indeed, TfR(aMD4) showed high accumulation and retention within the brain tissues of mice compared to Fc(aMD4) alone.This study depicts a novel strategy of inducing the effects of growth factors and cytokines with enhanced retention in brain tissues. What's more, based on the macrocyclic peptides and antibodies selected, this technique can be applied to imitate several growth factors. “Thus, lasso-grafting enables the design of protein therapeutics with thNanoLSI Podcast website

Kanazawa University NanoLSI Podcast: Chemists uncover cracks in the amour of cellulose nanocrystalsTranscript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Takeshi Fukuma and colleagues at the Kanazawa University NanoLSI. The research described in this podcast was published in the journal Science Advances in October 2022Kanazawa University NanoLSI website https://nanolsi.kanazawa-u.ac.jp/en/Chemists uncover cracks in the amour of cellulose nanocrystalsChemists in Japan, Canada and Europe have uncovered flaws in the surface structure of cellulose nanocrystals—an important step toward deconstructing cellulose to produce renewable nano-materials relevant to biochemical products, energy solutions, and biofuels.The findings—published in Science Advances—are the most detailed look yet at the surface chemistry and structure of individual cellulose nanocrystal (CNC) particles.The team, led by researchers at Kanazawa University, applied three-dimensional atomic force microscopy (3D-AFM) and molecular dynamics simulations to individual CNC fibres in water. The high-resolution scanning revealed new details of the cellulose chain arrangements on the CNCs surfaces.“This is an essential step towards understanding the mechanisms of CNC degradation, which is crucial for biomass conversion, with relevance to renewable nanomaterials and chemical production,” said Professor Takeshi Fukuma, Director of the Nano Life Science Institute at Kanazawa University.For the most part, the structure of a single CNC fibre showed honeycomb or zigzag chain arrangements on crystalline portions, interspersed with disordered, non-crystalline regions at irregular intervals. The researchers uncovered structural defects associated with the non-crystalline regions of the surface.“This is a great example of an international collaboration developed at the Nano Life Science Institute at Kanazawa University,” said University of British Columbia Professor Mark MacLachlan, Canada Research Chair in Supramolecular Materials and co-author on the paper. “It is important to visualize the surface and defects in these natural structures in order to advance their applications.”Chemists with Professor MacLachlan's lab at UBC helped devise the experiment, and synthesized and purified the cellulose nanocrystals for the project. Computational studies and modeling were undertaken by a team from Finland, led by Professor Adam Foster.The study also modelled the three-dimensional arrangement of water molecules near the CNC surface—which might offer material scientists additional clues to how the CNC surface might respond to molecular adsorption, diffusion and chemical reactions.ReferenceAyhan Yurtsever*, Pei-Xi Wang, Fabio Priante, Ygor Morais Jaques, Keisuke Miyazawa, Mark J. MacLachlan, Adam S. Foster, Takeshi Fukuma*. Molecular insights on the crystalline cellulose-water interfaces via three-dimensional atomic force microscopy. Science Advances 8, eabq0160 (2022).https://doi.org/10.1126/sciadv.abq0160Original release by UBC  https://science.ubc.ca/news/chemists-uncover-cracks-amour-cellulose-nanocrystals NanoLSI Podcast website

Kanazawa University NanoLSI Podcast: Elucidating the structure of nanomaterials found in crustaceansTranscript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Ayhan Yurtsever and Takeshi Fukuma at the Kanazawa University NanoLSI. The research described in this podcast was published in the journal Small Methods in June 2022Kanazawa University NanoLSI website https://nanolsi.kanazawa-u.ac.jp/en/Elucidating the structure of nanomaterials found in crustaceansIn a study recently published in the journal Small Methods, researchers from Kanazawa University used 3D atomic force microscopy to reveal the structural chemistry of chitin, a nanosized biomolecule derived from certain crustaceans.What do crabs, shrimps, and fungi have in common? Besides being culinary delicacies, they all contain a biomolecule called chitin on their external coating. Attributed to its strength and non-toxic nature, chitin is gaining popularity in bioengineering applications such as drug delivery systems. However, scientists are still uncertain about the exact structure of chitin and its interactions with water—a medium that it frequently comes in contact with during chemical processes. Now, a research team led by Ayhan Yurtsever and Takeshi Fukuma at Kanazawa University NanoLSI has used a form of high-resolution microscopy to understand the chemical nature of chitin's surface and its reaction patterns with water.The researchers first isolated chitin from shrimp shells for their experiments. Its surface was then scrutinized using 3D atomic force microscopy (AFM) in addition to a traditional electron microscope. Both these techniques revealed a homogenous layer of highly organized needle-shaped crystals on the surface. The researchers took a closer look at  the crystals—the primary points of interaction with water molecules. They saw that in addition to their highly crystalline nature, the crystals were surprisingly devoid of any structural disarray. This led the group to conclude that hydrochloric acid (which aided in the extraction of chitin from shrimp cells) was indeed successful in removing all other particles from the chitin surface, keeping only the crystals intact. The structural integrity of chitin was not compromised.Finally, the team used simulations to observe the chemistry between the chitin and water molecules and found that the latter formed well-organized layers encapsulating the chitin surface. A closer analysis of their interface revealed robust chemical bonds between the two molecules. However, the water layer had a heterogeneous appearance with sporadic absences of water molecules. This pattern led the team to believe that the chitin surface consisted of two types of molecular groups: those that interacted with water and those that did not. Knowledge of this pattern is useful in formulating future chemical reactions with chitin.“These findings provide important insights into chitin NC structures at the molecular level, which is critical for developing the properties of chitin-based nanomaterials,” concludes the team. The chemical treatment of chitin, which is often a prerequisite for formulating functional nanomaterials, can be developed better with the structural knowledge of chitin nanocrystals in mind.BackgroundChitin: Chitin is a naturally occurring polymer found in the shells of crustaceans and the outer wall of fungi. It is responsible for providing strength to these external coatings of organisms.In recent years, chitin has been used as a nanomaterial across engineering and medical fields. It has been used as reinforcement material, for water purificatiNanoLSI Podcast website

Kanazawa University NanoLSI Podcast: Simulating 3D-AFM images for systems not in equilibriumTranscript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Takeshi Fukuma and his co-researchers at the Kanazawa University NanoLSI. The research described in this podcast was published in the Journal of Physical Chemistry Letters in June 2022Learn more about their research at the WPI Kanazawa Nano Life Science Institutehttps://nanolsi.kanazawa-u.ac.jp/en/research/researchers/ Simulating 3D-AFM images for systems not in equilibriumResearchers at Kanazawa University report in The Journal of Physical Chemistry Letters how to simulate 3D atomic force microscopy images of out-of-equilibrium systems involving biomolecules.  The approach makes use of a celebrated equation from thermodynamics applicable to non-equilibrium situations.Three-dimensional atomic force microscopy (3D-AFM) is a technique used for probing the distribution of solvent molecules at solid–liquid interfaces.  Initially applied for studying situations where the solvent is water, the method is now also being used for other molecules.  A recent development is to use 3D-AFM for resolving the organization of biopolymers such as chromosomes or proteins within cells.  Due to the complexity of such systems, however, simulations of the 3D-AFM imaging process are needed to assist with its interpretation.  Simulation methods developed so far have assumed that the probed system is in equilibrium during the AFM scan cycle.  This limits their validity to situations where the solvent molecules move much faster than the scanning probe.  Now, Takeshi Fukuma from Kanazawa University NanoLSI and colleagues have developed a 3D-AFM simulation approach that works for non-equilibrium systems, making it applicable to measurements where molecular motion happens on timescales comparable to or larger than that of the AFM probing cycle.The basic principle of AFM is to make a very small tip, attached to a cantilever, scan a sample's surface.  The tip's response to height differences in the scanned surface provides structural information of the sample.  In 3D-AFM, the tip is made to penetrate the sample, and the force experienced by the tip is the result of interactions with nearby (parts of) molecules.  For a given horizontal (xy) position of the tip, the dependence of the force F on the tip's vertical (z) position as it penetrates into the sample is captured in a force–distance (F versus z) curve.  Combining all force–distance curves obtained during the xy scans gives the 3D-AFM image.Fukuma and colleagues considered the situation where an AFM tip probes a globular biopolymer, and modeled both tip and molecule as beads connected by springs (2000 beads for the molecule, 50 beads for the tip).  They calculated the force–distance curves by using the so-called Jarzynski equality, an equation that relates the free energy difference between two states of a system to the work (proportional to the force) required to go from one state to the other.  Importantly, the equality holds for non-equilibrium situations.The researchers were able to show that the simulations reproduced the internal structure of the biopolymer, with some fiber features being clearly observable.  They also looked at how the scanning velocity affects the simulation results, and found that there is an optimum velocity range for the vertical (z) scan.  Finally, Fukuma and colleagues simulated 3D-AFM images of cytoskeleton fibers for which experimentally obtained 3D-AFM images exist, and found that the simulations agree well with the expeNanoLSI Podcast website

 Hanae Sato is an Associate Professor at the Nano Life Science Institute (WPI-NanoLSI), Kanazawa University. Here, she describes her research on molecular biology and clarifying cellular quality control mechanisms of nonsense-mediated messenger RNA decay and potential therapeutic applications.Details here: https://nanolsi.kanazawa-u.ac.jp/en/research/researchers/hanae-sato/The Kanazawa University NanoLSI Podcast offers updates of the latest news and research at the WPI-NanoLSI Kanazawa University.The Nano Life Science Institute (NanoLSI) at Kanazawa University was established in 2017 as part of the World Premier International (WPI) Research Center Initiative of the Ministry of Education, Culture, Sports, Science and Technology (MEXT).Researchers at the NanoLSI are combining their cutting-edge expertise in scanning probe microscopy to establish ‘nano-endoscopic techniques' to directly image, analyze, and manipulate biomolecules for insights into mechanisms governing life phenomena such as diseases.Further informationWPI-NanoLSI Kanazawa University websitehttps://nanolsi.kanazawa-u.ac.jp/en/NanoLSI Podcast website

Kanazawa University NanoLSI Podcast: Heat and manipulate, one cell at a timeTranscript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Satoshi Arai and his co-researchers at the Kanazawa University NanoLSI. The research described in this podcast was published in the journal ACS Nano, in June 2022Learn more about their research here: WPI Kanazawa Nano Life Science Institutehttps://nanolsi.kanazawa-u.ac.jp/en/research/researchers/ Original article Ferdinandus, Madoka Suzuki, Cong Quang Vu, Yoshie Harada, Satya Ranjan Sarker, Shin'ichi Ishiwata, Tetsuya Kitaguchi, and Satoshi Arai.  Modulation of Local Cellular Activities using a Photothermal Dye-Based Subcellular-Sized Heat Spot, ACS Nano 16, 9004–9018 (2022).DOI: 10.1021/acsnano.2c00285URL: https://pubs.acs.org/doi/10.1021/acsnano.2c00285 Heat and manipulate, one cell at a timeResearchers at NanoLSI at Kanazawa University report in ACS Nano the development of a nanoparticle that acts as both a heater and a thermometer.  Inserting the nanoparticle in living cells results in a heat spot that, by switching it on and off, enables the controlled modulation of local cellular activities. Being able to heat nano-sized regions in biological tissues is key for several biomedical applications.  Indeed, many biological processes are temperature-sensitive, and the ability to locally modify temperature provides a way to manipulate cellular activity.  A notable application is the destruction of cancer cells by heating them.  In addition to the need for an in-tissue local heating mechanism, it also important to be able to instantaneously measure the generated temperature.  Satoshi Arai from NanoLSI at Kanazawa University and colleagues have now engineered a nanoparticle that is both a nanoheater and a nanothermometer at the same time.  They successfully showed that the insertion of a single, controllable heat spot in tissue can be very effective in modifying cellular function.The nanoparticle, called “nanoHT” by the scientists — an abbreviation of “nanoheater-thermometer” — is essentially a polymer matrix embedding a dye molecule used for sensing temperature, and another dye molecule  for releasing heat.  The latter happens through the conversion of light into thermal energy. That is, shining a near-infrared laser (with a wavelength of 808 nanometer) onto heat-releasing dye molecule results in fast heating, with a stronger increase in temperature for higher laser power.Temperature sensing is based on the thermal fluorescence effect of the dye molecule used for sensing temperature.  When irradiated with light of one wavelength, the molecule emits light at another wavelength — fluorescence.  The higher the temperature, the less intense the fluorescence becomes.  This inverse relationship can be used to measure temperature.  Arai and colleagues tested the performance of nanoHT as a thermometer, and established that it can determine temperatures with a resolution of 0.8 °C and less.The researchers then performed experiments with human cells called HeLa cells.  They looked at the effect of heating through nanoHT, and found that at a temperature increment of about 11.4 °C, the heated HeLa cells died within a few seconds.  This finding suggests that nanoHT could be used to induce cell death in cancer cells.Arai and colleagues also studied how nanoHT can be used to affect the behavior of muscles.  They introduced the nanoparticle into myotube, a type of fiber present in muscle tissue.  UpNanoLSI Podcast website

Kanazawa University NanoLSI Podcast: Chemical fixation causes aggregation artefactTranscript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Takehiko Ichikawa and his co-researchers at the Kanazawa University NanoLSI. The research described in this podcast was published in the journal Communications Biology, in May 2022.Learn more about their research here: WPI Kanazawa Nano Life Science Institutehttps://nanolsi.kanazawa-u.ac.jp/en/research/researchers/Original article Takehiko Ichikawa, Dong Wang, Keisuke Miyazawa, Kazuki Miyata, Masanobu Oshima, and Takeshi Fukuma.  Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins, Communications Biology 5, 487 (2022).DOI: 10.1038/s42003-022-03437-2URL: https://www.nature.com/articles/s42003-022-03437-2Chemical fixation causes aggregation artefactResearchers at Kanazawa University report in Communications Biology that using common chemicals for fixing living cell samples for microscopy studies causes membrane proteins to aggregate.For histological investigations of biological tissues, i.e. anatomical studies under the microscope, samples are usually fixated to prevent them from decaying. Fixation is typically done by immersing or perfusing the sample in a chemical — aldehydes and alcohols are common fixatives. It has been speculated that membrane proteins moving to some extent on a cell membrane can form aggregates during fixation. Yet, detailed cell surface studies with nanometer-scale resolution are necessary to obtain definitive insights into this potential issue. Now, Takehiko Ichikawa and colleagues at the NanoLSI at Kanazawa University have performed atomic force microscopy (AFM) studies of living mammalian cell surfaces. By comparing non-fixated and fixated samples, they found that fixation indeed leads to structural changes.The researchers developed a method of using microporous silicon nitride membranes (MPM)—that are widely used in transmission electron microscopy—for AFM imaging. Importantly, microporous silicon nitride membranes can make the cell surface flat and prevent fluctuations by supporting the area outside the observation area.  In AFM images of the surfaces of the cultured colon cancer cells on microporous silicon nitride membranes, biomolecular structures on the cell membranes showed up as protrusions, with a typical size of a few nanometers. When the cells were treated with commonly used fixatives such as paraformaldehyde, glutaraldehyde, and methanol, a few nanometer structures disappeared, and only large protrusions with diameters ranging from 20 to 100 nanometers were observed (Figure 2). The researchers performed several fluorescence experiments and concluded that large protrusions observed after fixation were formed by the aggregation of membrane proteins.The study demonstrates that the observed aggregates are artefacts resulting from the fixation process. This should call for caution among the community of researchers working with chemical fixatives. Quoting Ichikawa and colleagues: “Researchers who observe nanoscale clusters also should be careful in interpreting their experimental results when using fixed cells. We recommend that researchers use living cells as much as possible to avoid the effect of fixation when investigating nanoscale clusters […].” NanoLSI Podcast websitehttps://nanNanoLSI Podcast website

Transcript of this podcast Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Madhu Biyani from the Kanazawa University NanoLSI and her colleagues. The research described in this podcast was published in the ACS Applied Materials & Interfaces, in April 2022.Learn more about their research here: WPI Kanazawa Nano Life Science Institutehttps://nanolsi.kanazawa-u.ac.jp/en/research/researchers/Original article Madhu Biyani etal Novel DNA Aptamer for CYP24A1 Inhibition with Enhanced Antiproliferative Activity in Cancer Cells, ACS Appl. Mater. Interfaces 14, 18064-18078 (2022).DOI: 10.1021/acsami.1c22965Original article:  https://pubs.acs.org/doi/full/10.1021/acsami.1c22965Promising anticancer molecule identifiedResearchers at Kanazawa University in collaboration with a team of scientists from Toyama Prefectural University and BioSeeds Corporation report in ACS Applied Materials & Interfaces the identification of a molecule with enhanced antiproliferative activity in cancer cells.  The underlying biomolecular mechanism is the inhibition of an enzyme that is overproduced in several types of cancer.Vitamin D3 has important biological functions, including maintaining bone mineral density, which minimizes the risk of bone fracture.  But vitamin D3 is also believed to have anticancer activity, as low vitamin D3 levels and the associated overproduction of an enzyme called CYP24 are linked to a poor prognosis for cancer patients.  Molecules that limit or inhibit the action of CYP24 and molecules that mimic the function of vitamin D3 are nowadays highly researched as potential antiproliferative agents for cancer treatment.  But many of the inhibitors and D3 analogs synthesized so far have shown insufficient clinical response, as well as undesired side effects.  Now, Madhu Biyani from Kanazawa University and colleagues have identified a DNA-derived molecule that binds to and inhibits the function of CYP24 and shows promising antiproliferative activity.  The research team also provides detailed insights into the relevant molecular processes at play.The scientists screened a large number of DNA aptamers — pieces of single-stranded DNA with particular three-dimensional structures that can bind to specific target molecules and have a functional effect upon binding.  They looked for DNA aptamers that bind to CYP24 but not to the similar enzyme CYP271B, which is responsible for the synthesis of vitamin D3.An initial longlist of 18 aptamer candidates was reduced to 11 representatives with specific molecular structures.  The researchers checked the CYP24 inhibition activity of the 11 representative aptamers in vitro.  Four candidates resulting in the inhibition of CYP24 but not in the inhibition of CYP27B1 remained, of which one (Apt-7) was retained for further study.Biyani and colleagues performed simulations of Apt-7 binding to CYP24.  A molecular docking scenario was obtained, which they checked experimentally by comparing the behavior of a mixture of vitamin D3 and CYP24 with and without Apt-7.  The simulations and the experiments showed that Apt-7 results in the inhibition of CYP24 activity, and that what happens is that the aptamer likely interferes with the enzyme's active site.  The researchers also performed high-speed atomic force microscopy on the binding of CYP24 and Apt-7 in real time, confirming the molecular docking scenario obtained from simulations.Finally, the research team studied the effect of Apt-7 at the cellular level by introducing the molecule to cancer cells.  They observed significant CYP24 inhibition for a cancer cell line known to overexpress the CYP24 enzyme, thus showing antiproliferative activity.

Transcript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Rikinari Hanayama from the Kanazawa University NanoLSI and colleagues. The research described in this podcast was published in the Frontiers in Molecular Biosciences in March 2022.Learn more about their research here: WPI Kanazawa Nano Life Science Institutehttps://nanolsi.kanazawa-u.ac.jp/en/research/researchers/ReferenceHiroki Yamaguchi, Hironori Kawahara, Noriyuki Kodera, Ayanori Kumaki, Yasutake Tada, Zixin Tang, Kenji Sakai, Kenjiro Ono, Masahito Yamada, and Rikinari Hanayama.Extracellular Vesicles Contribute to the Metabolism of Transthyretin Amyloid in Hereditary Transthyretin Amyloidosis, Front. Mol. Biosci. 9, 839917 (2022).DOI: 10.3389/fmolb.2022.839917URL: https://doi.org/10.3389/fmolb.2022.839917 Biomolecular insights into protein-insolubility-related diseaseResearchers at Kanazawa University elucidate how small bio-containers enclosed by membranes are involved in a disease called ATTRv amyloidosis.  Amyloidosis is the collective name for a group of diseases characterized by the deposition of amyloids — insoluble proteins that form due to the misfolding and aggregation of soluble proteins — outside of cells.  Such depositions lead to cellular dysfunctions, and take place in patients with Alzheimer's disease, Parkinson's disease and dementia.  In the disease called hereditary (variant) transthyretin amyloidosis (abbreviated ATTRv amyloidosis), variants of the transthyretin (TTR) gene lead to TTR amyloid deposits in several organs, with symptoms including muscle weakness and cardiac failure.  It is known that the removal of amyloid proteins is promoted by so-called extracellular vesicles (EVs) — small ‘biocontainers' enclosed by a membrane — but what is unclear is whether EVs are involved in the formation and subsequent deposition of TTR amyloids in the context of ATTRv amyloidosis.  Rikinari Hanayama and colleagues from Kanazawa University have now studied the relationship between ATTRv amyloidosis and EVs, and confirm that the latter play an important role in the aggregation and deposition of TTR amyloids.The researchers first analyzed the serum of ATTRv amyloidosis patients for traces of TTR amyloid.  (Serum is blood without the clotting factors.)  They found that TTR is present in EVs derived from serum, and that the so-called V30M mutation variant of TTR aggregates at the membranes of serum-derived EVs.Hanayama and colleagues then looked at what happened when V30M-TTR amyloids were added to cell cultures, with and without serum-derived EVs.  They found that V30M-TTR amyloid aggregates are deposited on cells in a much more pronounced way when serum-derived EVs are present, indicating that serum-derived EVs promote the aggregation of V30M-TTR and their deposition on cells.From a comparison between ATTRv amyloidosis patients and healthy individuals, the scientists found that ATTRv amyloidosis is associated with a lower amount of TTR aggregates in serum-derived EVs.  The hypothesis that emerges from the experiments is that in ATTRv amyloidosis patients, the presence of V30M-TTR and EVs leads to a self-enhancing uptake of EVs; this then leads to an enhanced deposition of TTR aggregates in tissue, resulting in a decrease of TTR aggregates in serum.The findings of Hanayama and colleagues suggest that TTR in serum-derived EVs is a potential target for both ATTRv amyloidosis diagnosis and therapy.  The researchers also point to the relevance of their results on our understanding of Alzheimer's disease because TTR inhibits the nucleation of amyloid-β aggregation and its aggregation is

 Revealing atomistic structures behind AFM imaginghttps://nanolsi.kanazawa-u.ac.jp/en/achievements/revealing-atomistic-structures-behind-afm-imaging/Atomic force microscopy (AFM) enables the visualization of the dynamics of single biomolecules during their functional activity. However, all observations are restricted to regions that are accessible by a fairly big probing tip during scanning. Hence, the AFM only records images of biomolecular surfaces with limited spatial resolution, thereby missing important information that is required for a detailed understanding of the observed phenomena.To facilitate the interpretation of experimental imaging, Romain Amyot and Holger Flechsig from the Kanazawa NanoLSI have developed the mathematical framework and computational methods to reconstruct 3D atomistic structures from AFM surface scans. ==Transcript of this podcastHello and welcome to the NanoLSI podcast. In this episode we feature the latest research published by Romain Amyot and Holger Flechsig of the Computational Science group at the Kanazawa University NanoLSI.The research described in this podcast was published in the journal PLOS Computational Biology in March 2022. Revealing atomistic structures behind AFM imaginghttps://nanolsi.kanazawa-u.ac.jp/en/achievements/revealing-atomistic-structures-behind-afm-imaging/Atomic force microscopy (AFM) enables the visualization of the dynamics of single biomolecules during their functional activity. However, all observations are restricted to regions that are accessible by a fairly big probing tip during scanning. Hence, the AFM only records images of biomolecular surfaces with limited spatial resolution, thereby missing important information that is required for a detailed understanding of the observed phenomena.To facilitate the interpretation of experimental imaging, Romain Amyot and Holger Flechsig from the Kanazawa NanoLSI have developed the mathematical framework and computational methods to reconstruct 3D atomistic structures from AFM surface scans. In this paper they describe applications for high-speed AFM imaging ranging from single molecular machines, protein filaments, to even large-scale assemblies of protein lattices, and demonstrate how the full atomistic information advances the molecular understanding beyond topographic images.Their approach employs simulation AFM, which was previously established by Amyot and Flechsig and distributed within the free BioAFMviewer software package. Simulation AFM computationally emulates experimental scanning of biomolecules to translate structural data into simulation AFM topographic images that can be compared to real AFM images. The researchers implemented a procedure of automated fitting to identify the high-resolution molecular structure behind a limited-resolution experimental AFM image. It is therefore possible to retrieve full 3D atomistic information from just a scan of the protein surface obtained under AFM observations. To illustrate the potential of this achievement, Flechsig says: “Imagine that instead of just seeing the tip of an iceberg, you are now able to see everything hidden under the sea, to the extent that you can even detect impurities or density differences within its structure, helping you to explain the icebergs' coloration.”To share these developments with the global Bio-AFM community, all computational methods are embedded within the user-friendly BioAFMviewer interactive software interface. The new methods have already been applied in numerous interdisciplinary collaborations to understand expe

24 August 2022 Changing the handedness of molecules Researchers at Kanazawa University report in the Proceedings of the National Academy of Sciences a responsive molecular system that, inverses its chirality before becoming racemic through chemical reactions.Learn more about their research here: WPI Kanazawa Nano Life Science Institutehttps://nanolsi.kanazawa-u.ac.jp/en/research/researchers/Transcript of this podcastHello and welcome to the NanoLSI podcast. In this episode we will feature the latest research published by Shigehisa Akine a member of the Supramolecular Chemistry group at the Kanazawa University NanoLSI.The research described in this podcast was published in the Proceedings of the National Academy of Sciences in March 2022.  Changing the handedness of molecules https://nanolsi.kanazawa-u.ac.jp/en/achievements/achievements-19316/Researchers at Kanazawa University report in the Proceedings of the National Academy of Sciences a responsive molecular system that, inverses its chirality before becoming racemic through chemical reactions.Molecules that can change their structure in response to a chemical or physical stimulus are called ‘responsive molecules'.  This type of molecule plays an important role in signal transduction at the nanoscale.  The typical time profile of a structural change of a responsive molecule follows an exponential relaxation.  However, molecular systems with non-typical time responses, such as e.g. chemical oscillators offer advanced functionalities and are also intensively investigated.  Shigehisa Akine and colleagues from Kanazawa University have now designed a particular responsive molecule in which the chirality (‘handedness') changes in a non-exponential fashion.  The achievement is a breakthrough in the field of responsive systems as the chirality change happens in a unimolecular system — and not as has often been the case before in supramolecular assemblies.The researchers' responsive molecule has six exchangeable sites and two forms, a ‘left-handed' and a ‘right-handed' version.  In solution, the two forms will occur in a given ratio.  Akine and colleagues started from the molecule with a particular chiral amine. They found that in a methanol solution the right-handed version was dominant.  The scientists then looked at what would happen when exchanging the so-called chiral A groups with piperidine (another form of amine).Because of the achirality of the piperidine groups, the resulting solution should become ‘racemic', which means that any effects of chirality are compensated.  This is indeed what happened, but the researchers discovered that before reaching the racemic state after two days, the solution first switched from originally P-dominant to M-dominant after 7 minutes, with maximum M-dominance after 60 – 120 minutes.  Remarkably, a similar transient chirality inversion was not observed for the reverse reaction for which the solution changed monotonically from racemic to P-dominant.Akine and colleagues note that their responsive molecule is the first unimolecular platform displaying a transient chirality inversion, and that the unique chirality change happens on the timescale of minutes to hours, which could be potentially useful for time-dependent functional materials related to human activity.  Quoting the scientists: “this result will provide an important insight into the science of autonomously driven materials.”ReferenceYoko Sakata, Shunsuke Chiba, and Shigehisa Akine, Transi

Kanazawa NanoLSI Research Podcast 26 May 2022 Small but mighty: Identifying nanosized molecules using atomic force microscopy In a recent study Mikihiro Shibata and Leonardo Puppulin at the WPI Nano Life Science Institute Kanazawa University (NanoLSI) used advanced atomic force microscopy to accurately recognize tiny cellular biomolecules. Learn more about their research here: WPI Kanazawa Nano Life Science Institutehttps://nanolsi.kanazawa-u.ac.jp/en/research/researchers/Original article:  https://pubs.acs.org/doi/10.1021/acsami.1c17708Transcript of this podcastHello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we will feature the latest research published by Mikihiro Shibata and Leonardo Puppulin. They are both members of the nanometrology group at the Kanazawa University NanoLSI.The research described in this podcast was published in the American Chemical Society, Applied Materials  Interfaces, in November 2021.  Small but mighty: Identifying nanosized molecules using atomic force microscopyIn a study recently published in the journal Applied Materials and Interfaces researchers from Kanazawa University use advanced atomic force microscopy to accurately recognize tiny cellular biomolecules. Biologists rely on a wide range of microscopy techniques to visualize biomolecules within biological cells. High-speed atomic force microscopy (HS-AFM) is one such example in which a sharp tip attached to a sensor is used for visualizing cells. Specifically, as the AFM tip scans the surface of a molecule, the pattern of signals it generates enables researchers to visualize the molecule's topography. However, recognizing individual biomolecules using HS-AFM is still in its infancy. Now, researchers at Kanazawa University report on an innovative method to facilitate this by tweaking the structures of AFM tips.The research team, led by Mikihiro Shibata and Leonardo Puppulin at the WPI Nano Life Science Institute Kanazawa University (NanoLSI), characterized a protein found on human cells known as the hepatocyte growth factor receptor (hMET). The researchers first attached aMD4, a synthetic molecule that latches onto hMET, to the HS-AFM tip using different sized linkers. Patterns of connections between this modified tip and single molecules of hMET were subsequently investigated. When hMET on a mica surface (a material typically used in HS-AFM studies) was exposed to the tip, interactions between aMD4 and the external surface of hMET were indeed observed. However, when multiple molecules of aMD4 and hMET were used, it was found that shorter and more flexible linkers enabled aMD4 to interact with two adjacent hMET molecules bringing them closer together. This observation posits practical applications in the laboratory—biologists can potentially mimic the binding of two cell surface proteins together which often leads to the induction of cellular processes. Next, the specificity of this tip for molecule recognition was examined. hMET and its mouse form are very similar in structure. However, the mouse form does not bind to aMD4. Thus, when the aMD4-loaded tips were exposed to both forms of the protein, activity was observed only with the human form. This technique could therefore be useful in the selection of specific biomolecules from a heterogenous mix as is typically seen on the cell surface. Lastly, the modified HS-AFM technique was applied when hMET was bound to a lipid surface mimicking the structural composition of cell membranes (its natural home). Similar interactions were observed in this milieu suggesting

Kanazawa University research: Publication of an insightful reference book on high-speed atomic force microscopy (HS-AFM) for in situ biological applications  Pioneering biophysicist Professor Toshio Ando of the NanoLSI publishes his new book on high-speed atomic force microscopy (HS-AFM) for directly monitoring the dynamics of biomolecules. The book offers easy to understand descriptions of the basic technology and in situ biological applications of liquid HS-AFM. The book is ideal for students from multidisciplinary backgrounds interested in accelerating their research on high speed, in situ monitoring of biomolecules. NanoLSI Podcast where Professor Ando describes the background to the publication of the book and his thoughts about the future of HS-AFM.Link to Nano LSI Podcasthttps://nanolsi.kanazawa-u.ac.jp/en/announcements/nanolsipodcast/Professor Toshio Ando is internationally recognized as the pioneer of high speed atomic force microscopy for biological applications. “I first became aware of atomic force microscopy in the mid-eighties after I returned to Japan following several years in the USA,” says Ando. “I had just moved to Kanazawa University and was looking for new paths to explore. I was interested in directly observing the dynamics of proteins. This is when I decided to pursue research on the development of high speed liquid AFM (HS-AFM). Now, more than 30 years later, I want to share my experiences and insights into the technology and applications of HS-AFM. This book is my way of sharing my knowledge about this subject. It is the first book on this topic and hopefully it will inspire the development of the next generation of scanning probe microscopes for biology.” Professor Ando describes how he started his research on HS-AFMhttps://nanolsi.kanazawa-u.ac.jp/wp-content/uploads/2022/05/background-to-research-on-hs-afm.mp3Professor Ando describes why he decided to write this bookhttps://nanolsi.kanazawa-u.ac.jp/wp-content/uploads/2022/05/writing-the-book.mp3The future of high speed AFMProfessor Ando shares his views about the future of HS-AFMhttps://nanolsi.kanazawa-u.ac.jp/wp-content/uploads/2022/05/evolution-of-hs-afm.mp3Ando envisages continuous evolution in both the performance and applications of HS-AFM. “The scanning speed is an area of research being addressed by many groups globally,” says Ando. “In my group we are developing new methods, that is system operation procedures, and have achieved 40 frames per second (fps). Conventional systems enable around 10 fps. I expect advances in devices used for imaging will enable image rates of 100 fps within 3 to 4 years. So this area of research is still evolving.” Ando also foresees that many proteins that have been “untouched” to-date will be imaged by high performance HS-AFM systems. “I expect many more users of HS-AFM in the future,” says Ando. About the book Professor Ando describes the contents of the book https://nanolsi.kanazawa-u.ac.jp/wp-content/uploads/2022/05/audience-and-contents.mp3‘High Speed Atomic Force Microscopy in Biology” is published by Springer and is available as an eBook or hard cover [1]. The book consists of 18 chapters and more than 300 pages that include practical hints about the preparation of cantilever tips and sample surfaces, for example, to enable first time users to succes

Professor Carsten Beta is an overseas-based principal investigator at the NanoLSI WPI Kanazawa University and faculty at the Universität Potsdam Institute of Physics and Astronomy, University of Potsdam, Germany. Here, he describes his research on biological physics on the scale of individual cells based on microscopic observations and manipulation and modelling using pattern formation in nonlinear systems.The Kanazawa University NanoLSI Podcast offers updates of the latest news and research at the WPI-NanoLSI Kanazawa University.The Nano Life Science Institute (NanoLSI) at Kanazawa University was established in 2017 as part of the World Premier International (WPI) Research Center Initiative of the Ministry of Education, Culture, Sports, Science and Technology (MEXT).Researchers at the NanoLSI are combining their cutting-edge expertise in scanning probe microscopy to establish ‘nano-endoscopic techniques' to directly image, analyze, and manipulate biomolecules for insights into mechanisms governing life phenomena such as diseases.Further informationWPI-NanoLSI Kanazawa University websitehttps://nanolsi.kanazawa-u.ac.jp/en/

Professor Shigehisa Akine is a principal investigator at the NanoLSI WPI Kanazawa University. Professor Mark MacLachlan is an overseas principal investigator at the NanoLSI WPI Kanazawa University and faculty at the University of British Columbia. In this episode of the NanoLSI Podcast they describe recent developments in their research on nanomolecular cages for biomolecular sensing and biocompatible AFM tips for chemically probing living cells, respectively.The Kanazawa University NanoLSI Podcast offers updates of the latest news and research at the WPI-NanoLSI Kanazawa University for a global audience.The Nano Life Science Institute (NanoLSI) at Kanazawa University was established in 2017 as part of the World Premier International (WPI) Research Center Initiative of the Ministry of Education, Culture, Sports, Science and Technology (MEXT).Researchers at the NanoLSI are combining their cutting-edge expertise in scanning probe microscopy to establish ‘nano-endoscopic techniques' to directly image, analyze, and manipulate biomolecules for insights into mechanisms governing life phenomena such as diseases.Further informationWPI-NanoLSI Kanazawa University websitehttps://nanolsi.kanazawa-u.ac.jp/en/

Professor Alexander S. Mikhailov is an overseas Computational Science group of the WPI NanoLSI and a professor at the Department of Physical Chemistry at the Fritz Haber Institute, Berlin. Here, he describes his research on computational molecular biophysics as a powerful approach for simulating the dynamics of complex biological structures ranging from single molecules to the cell. The Kanazawa University NanoLSI Podcast offers updates of the latest news and research at the WPI-NanoLSI Kanazawa University. The Nano Life Science Institute (NanoLSI) at Kanazawa University was established in 2017 as part of the World Premier International (WPI) Research Center Initiative of the Ministry of Education, Culture, Sports, Science and Technology (MEXT). Researchers at the NanoLSI are combining their cutting-edge expertise in scanning probe microscopy to establish ‘nano-endoscopic techniques' to directly image, analyze, and manipulate biomolecules for insights into mechanisms governing life phenomena such as diseases. Further information WPI-NanoLSI Kanazawa University website https://nanolsi.kanazawa-u.ac.jp/en/

Professor Yuri Korchev is an overseas PI of the Nanometrology group of the WPI NanoLSI and a professor at the Faculty of Medicine, Imperial College London. Here, he describes his research on cutting-edge scanning ion conductance microscopy for the life sciences in particular the development of innovative non-invasive nanopipette nanoprobes for simultaneous acquisition of 3D topography and biochemical imaging of living cells. The Kanazawa University NanoLSI Podcast offers updates of the latest news and research at the WPI-NanoLSI Kanazawa University. The Nano Life Science Institute (NanoLSI) at Kanazawa University was established in 2017 as part of the World Premier International (WPI) Research Center Initiative of the Ministry of Education, Culture, Sports, Science and Technology (MEXT). Researchers at the NanoLSI are combining their cutting-edge expertise in scanning probe microscopy to establish ‘nano-endoscopic techniques' to directly image, analyze, and manipulate biomolecules for insights into mechanisms governing life phenomena such as diseases. Further information WPI-NanoLSI Kanazawa University website https://nanolsi.kanazawa-u.ac.jp/en/