Experimental pathology by intravital microscopy and genetically encoded fluorescent biosensors

The invention of two-photon excitation microscopes widens the potential application of intravital microscopy (IVM) to the broad field of experimental pathology. Moreover, the recent development of fluorescent protein-based, genetically encoded biosensors provides an ideal tool to visualize the cell function in live animals. We start from a brief review of IVM with two-photon excitation microscopes and genetically encoded biosensors based on the principle of Förster resonance energy transfer (FRET). Then, we describe how IVM using biosensors has revealed the pathogenesis of several disease models

Two-Photon AMPK and ATP Imaging Reveals Metabolic Recovery in Mouse Rod Photoreceptor Cells

The title of publisher's version: Two-photon AMPK and ATP imaging reveals the bias between rods and cones in glycolysis utility.In vertebrates, retinal rod and cone photoreceptor cells rely significantly on glycolysis. Lactate released from photoreceptor cells fuels neighboring retinal pigment epithelium cells and Müller glial cells through oxidative phosphorylation. To understand this highly heterogeneous metabolic environment around photoreceptor cells, single-cell analysis is needed. Here, we visualized cellular AMP-activated protein kinase (AMPK) activity and ATP levels in the retina by two-photon microscopy. Transgenic mice expressing a hyBRET-AMPK biosensor were used for measuring the AMPK activity. GO-ATeam2 transgenic mice were used for measuring the ATP level. Temporal metabolic responses were successfully detected in the live retinal explants upon drug perfusion. A glycolysis inhibitor, 2-deoxy-d-glucose (2-DG), activated AMPK and reduced ATP. These effects were clearly stronger in rods than in cones. Notably, rod AMPK and ATP started to recover at 30 min from the onset of 2-DG perfusion. Consistent with these findings, ex vivo electroretinogram recordings showed a transient slowdown in rod dim flash responses during a 60-min 2-DG perfusion, whereas cone responses were not affected. Based on these results, we propose that cones surrounded by highly glycolytic rods become less dependent on glycolysis, and rods also become less dependent on glycolysis within 60 min upon the glycolysis inhibition

Functional visualization of NK Cell-mediated killing of metastatic single tumor cells

ナチュラルキラー（NK）細胞による転移がん細胞殺傷の可視化 --NK細胞とがん細胞の肺毛細血管上での戦いを実況中継する--. 京都大学プレスリリース. 2022-02-07.Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically-encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hrs of arrival, but not thereafter. Intravital dynamic imaging revealed that 50% of NK-tumor cell encounters lead to tumor cell death in the first 4 hrs after tumor cell arrival, but after 24 hrs of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24 hrs period, the probability of ERK activation in NK cells upon encountering the tumor cells was decreased from 68% to 8%, which correlated with the loss of the activating ligand CD155/PVR/Necl5 from the tumor cell surface. Thus, by quantitatively visualizing the NK-tumor cell interaction at the early stage of metastasis, we have revealed the crucial parameters of NK cell immune surveillance in the lung

Improved Security Evaluation Techniques for Imperfect Randomness from Arbitrary Distributions

Dodis and Yu (TCC 2013) studied how the security of cryptographic primitives that are secure in the ideal model in which the distribution of a randomness is the uniform distribution, is degraded when the ideal distribution of a randomness is switched to a real-world (possibly biased) distribution that has some lowerbound on its min-entropy or collision-entropy. However, in many constructions, their security is guaranteed only when a randomness is sampled from some non-uniform distribution (such as Gaussian in lattice-based cryptography), in which case we cannot directly apply the results by Dodis and Yu. In this paper, we generalize the results by Dodis and Yu using the Rényi divergence, and show how the security of a cryptographic primitive whose security is guaranteed when the ideal distribution of a randomness is a general (possibly non-uniform) distribution $Q$, is degraded when the distribution is switched to another (real-world) distribution $R$. More specifically, we derive two general inequalities regarding the Rényi divergence of $R$ from $Q$ and an adversary\u27s advantage against the security of a cryptographic primitive. As applications of our results, we show (1) an improved reduction for switching the distributions of distinguishing problems with public samplability, which is simpler and much tighter than the reduction by Bai et al. (ASIACRYPT 2015), and (2) how the differential privacy of a mechanism is degraded when its randomness comes from not an ideal distribution $Q$ but a real-world distribution $R$. Finally, we show methods for approximate-sampling from an arbitrary distribution $Q$ with some guaranteed upperbound on the Rényi divergence (of the distribution $R$ of our sampling methods from $Q$)

Signature Schemes with a Fuzzy Private Key

In this paper, we introduce a new concept of digital signature that we call \emph{fuzzy signature}, which is a signature scheme that uses a noisy string such as biometric data as a private key, but \emph{does not require user-specific auxiliary data} (which is also called a helper string in the context of fuzzy extractors), for generating a signature. Our technical contributions are three-fold: (1) We first give the formal definition of fuzzy signature, together with a formal definition of a \lq\lq setting\u27\u27 that specifies some necessary information for fuzzy data. (2) We give a generic construction of a fuzzy signature scheme based on a signature scheme that has certain homomorphic properties regarding keys and satisfies a kind of related key attack security with respect to addition, and a new tool that we call \emph{linear sketch}. (3) We specify two concrete settings for fuzzy data, and for each of the settings give a concrete instantiation of these building blocks for our generic construction, leading to two concrete fuzzy signature schemes. We also discuss how fuzzy signature schemes can be used to realize a biometric-based PKI that uses biometric data itself as a cryptographic key, which we call the \emph{public biometric infrastructure (PBI)}

Mucosa-associated invariant T cells are systemically depleted in simian immunodeficiency virus-infected rhesus macaques

Mucosa-associated invariant T (MAIT) cells contribute to host immune protection against a wide range of potential pathogens via the recognition of bacterial metabolites presented by the major histocompatibility complex class I-related molecule MR1. Although bacterial products translocate systemically in human immunodeficiency virus (HIV)-infected individuals and simian immunodeficiency virus (SIV)-infected Asian macaques, several studies have shown that MAIT cell frequencies actually decrease in peripheral blood during the course of HIV/SIV disease. However, the mechanisms underlying this proportional decline remain unclear. In this study, we characterized the phenotype, activation status, functionality, distribution, and clonotypic structure of MAIT cell populations in the peripheral blood, liver, mesenteric lymph nodes (MLNs), jejunum, and bronchoalveolar lavage (BAL) fluid of healthy and SIV-infected rhesus macaques (RMs). Low frequencies of MAIT cells were observed in the peripheral blood, MLNs, and BAL fluid of SIV-infected RMs. These numerical changes were coupled with increased proliferation and a highly public T cell receptor alpha (TCRα) repertoire in the MAIT cell compartment without redistribution to other anatomical sites. Collectively, our data show systemically decreased frequencies of MAIT cells likely attributable to enhanced turnover in SIV-infected RMs. This process may impair protective immunit

Usefulness of the Multimodal Fusion Image for Visualization of Deep Sylvian Veins

The preoperative assessment of cerebral veins is important to avoid unexpected cerebral venous infarction in the neurosurgical setting. However, information is particularly limited regarding deep Sylvian veins, which occasionally disturb surgical procedures for cerebral anterior circulation aneurysms. The predictability of detecting deep Sylvian veins and their tributaries using a modern multimodal fusion image was aimed to be evaluated. Moreover, 51 patients who underwent microsurgery for unruptured cerebral aneurysms with Sylvian fissure dissection were retrospectively reviewed. The visualization of the four components of the deep Sylvian veins in conventional computed tomography (CT) venography and multimodal fusion images was evaluated. To compare the detection accuracy among these radiological images, the sensitivity and specificity for the detection of each of the four venous structures were calculated in comparison with those of intraoperative inspections. The kappa coefficients were also measured and the inter-rater agreement for each venous structure in each radiological image was examined. In all veins, the multimodal fusion image exhibited a high detection rate without statistical difference from intraoperative inspections (P = 1.0). However, CT venography exhibited a low detection rate with a significant difference from intraoperative inspections in the common vertical trunk (P = 0.006) and attached vein (P = 0.008). The kappa coefficients of the fusion image ranged from 0.73 to 0.91 and were superior to those of CT venography for all venous structures. This is the first report to indicate the usefulness of a multimodal fusion image in evaluating deep Sylvian veins, especially for the detection of nontypical, relatively small veins with large individual variability.博士（医学）・甲第864号・令和5年3月15

Spatio-Temporal Expression Profile of Stem Cell-Associated Gene LGR5 in the Intestine during Thyroid Hormone-Dependent Metamorphosis in Xenopus laevis

The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established in the so-called postembryonic developmental period in mammals when endogenous thyroid hormone (T3) levels are high.The T3-dependent metamorphosis in anurans like Xenopus laevis resembles the mammalian postembryonic development and offers a unique opportunity to study how the adult stem cells are developed. The tadpole intestine is predominantly a monolayer of larval epithelial cells. During metamorphosis, the larval epithelial cells undergo apoptosis and, concurrently, adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and then differentiate to establish a trough-crest axis of the epithelial fold, resembling the crypt-villus axis in the adult mammalian intestine. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a well-established stem cell marker in the adult mouse intestinal crypt. Here we have cloned and analyzed the spatiotemporal expression profile of LGR5 gene during frog metamorphosis. We show that the two duplicated LGR5 genes in Xenopus laevis and the LGR5 gene in Xenopus tropicalis are highly homologous to the LGR5 in other vertebrates. The expression of LGR5 is induced in the limb, tail, and intestine by T3 during metamorphosis. More importantly, LGR5 mRNA is localized to the developing adult epithelial stem cells of the intestine.These results suggest that LGR5-expressing cells are the stem/progenitor cells of the adult intestine and that LGR5 plays a role in the development and/or maintenance of the adult intestinal stem cells during postembryonic development in vertebrates