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Initial studies on the immunogenicity of SARS-CoV-2 (CoV-2) S glycoprotein ("spike") as a protein subunit vaccine suggested sub-optimal efficacy in mammals. Although protein engineering efforts have produced CoV-2 spike protein sequences with greatly improved immunogenicity, additional strategies for improving the immunogenicity of CoV-2 protein subunit vaccines are scaffolding and the use of adjuvants. Comparisons of the effectiveness of engineered protein-only and engineered protein-nanoparticles vaccines have been rare. To explore this knowledge gap, we inoculated mice with two doses of either sequence-optimized trimeric spike protein or one of several sequence-optimized spike nanoparticles. We measured their immune response up to two months after the first dose. We also measured the immune response and protection against live virus in hamsters inoculated with either sequence-optimized trimeric spike protein or a liposome-based sequence-optimized spike nanoparticle. We found that in the presence of adjuvant, the antibody and neutralization titers elicited by spike-nanoparticles were not significantly greater than those elicited by spike-only in mice, even at doses as low as 0.1 µg/animal. Hamsters vaccinated with spike-only or spike-nanoparticles were equally protected from live virus one month after their first inoculation. These results suggest that sequence-optimized protein subunit vaccines in the form of individual prefusion-stabilized trimers can be as effective in improving immunogenicity as scaffolded forms., (© 2024. The Author(s).)

Dietary protein intake modulates growth, reproduction, and longevity by stimulating amino acid (AA)-sensing pathways. Essential AAs are often considered as limiting nutrients during protein scarcity, and the role of dietary non-essential AAs (NEAAs) is less explored. Although tyrosine has been reported to be crucial for sensing protein restriction in Drosophila larvae, its effect on adult physiology and longevity remains unclear. Here, using a synthetic diet, we perform a systematic investigation of the effect of single NEAA deprivation on nutrient-sensing pathways, reproductive ability, starvation resistance, feeding behavior, and life span in adult female flies. Specifically, dietary tyrosine deprivation decreases internal tyrosine levels and fecundity, influences AA-sensing machineries, and extends life span. These nutritional responses are not observed under higher total AA intake or in infertile female flies, suggesting a context-dependent influence of dietary tyrosine. Our findings highlight the unique role of tyrosine as a potentially limiting nutrient, underscoring its value for dietary interventions aimed at enhancing health span.

Female mosquitoes engage in blood feeding from their hosts to facilitate egg maturation but cease feeding once a sufficient blood meal has been acquired. Abdominal distention has been proposed as a contributing factor; however, it has also been suggested that there are chemical controls. In this study, we focus on negative chemical regulators of blood feeding, particularly those present in the host blood. Serum derived from animal blood inhibits the feeding of ATP, a phagostimulant of blood feeding in Aedes aegypti. Fibrinopeptide A (FPA), a 16-amino acid peptide cleaved from fibrinogen during blood coagulation, serves as an inhibitory factor in the serum. Our findings suggest that blood-feeding arrest in female mosquitoes is triggered by the detection of FPA in the host blood, which increases as blood coagulation proceeds in the mosquito's midgut, highlighting the role of host-derived substances as negative regulators of mosquito behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective . Time-of-flight (TOF) is an important factor that directly affects the image quality of PET systems, and various attempts have been made to improve the coincidence resolving time (CRT) of PET detectors. For independent readout detectors, the timing is acquired for each silicon photomultiplier (SiPM), so they are less sensitive to diffused scintillation light, resulting in a better CRT. Further improvement can be expected if the light can be focused on a single SiPM. However, existing SiPM arrays have a thin protective cover on the SiPM and the gap between the SiPMs is filled with either air or the protective cover, so the light must diffuse through the cover. In this work, we investigated optical crosstalk in the protective cover to improve the CRT. Approach . We used 3.1 × 3.1 × 20 mm 3 fast LGSO crystals and 3 mm square 8 × 8 multi pixel photon counter (MPPC) arrays. Pitch of the MPPCs was 3.2 mm and thickness of the protective cover on them was 150 μ m. To reduce diffusion of scintillation light in the protective cover, the part of the inactive areas on the MPPC array were optically separated using reflective material. Specifically, 50, 100, 150, and 350 μ m deep grid-shaped slits were made along the inactive area of the MPPCs and they were filled with BaSO 4 powder as the reflective material. Main results . Coincidence counts were measured with a pair of TOF detectors, and the CRT was shorter with a deeper slit depth. The CRT before improvement was 235 ps, and using the cover having the 350 μ m deep slits filled with reflective material lowered the CRT to 211 ps. Significance . Up to 10% of the scintillation light was diffused to other MPPCs by the protective cover, and the CRT was degraded by 10% due to optical crosstalk of the cover. The proposed method promises to improve the CRT of the TOF detector., (© 2024 Institute of Physics and Engineering in Medicine.)

Life activities are supported by the intricate metabolic network that is fueled by nutrients. Nutritional and genetic studies in model organisms have determined that dietary restriction and certain mutations in the insulin signaling pathway lead to lifespan extension. Subsequently, the detailed mechanisms of aging as well as various nutrient signaling pathways and their relationships have been investigated in a wide range of organisms, from yeast to mammals. This review summarizes the roles of nutritional and metabolic signaling in aging and lifespan with a focus on amino acids, the building blocks of organisms. We discuss how lifespan is affected by the sensing, transduction, and metabolism of specific amino acids and consider the influences of life stage, sex, and genetic background on the nutritional control of aging. Our goal is to enhance our understanding of how nutrients affect aging and thus contribute to the biology of aging and lifespan.

The gut microbiota is crucial for intestinal health, including gastrointestinal (GI) motility. How commensal bacterial species influence GI motility has not been fully elucidated. A major factor of GI motility is the gut contraction promoting the propulsive movement of orally ingested materials. Here, we developed a method to monitor and quantify gut contractions in living Drosophila melanogaster larvae. We found that the culture medium of an isolated strain Lactiplantibacillus plantarum Lsi promoted gut contraction in vivo , which was not observed in Leuconostoc sp. Leui nor Acetobacter persici Ai culture medium. To identify bacteria-derived metabolites, we performed metabolome analysis of the culture media by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of the 66 metabolites detected, we found that some metabolites changed in a species-specific manner. Among them, acetylcholine was specifically produced by L. plantarum. Feeding exogenous acetylcholine increased the frequency of gut contractions, which was blocked by D-tubocurarine, an inhibitor of nicotinic acetylcholine receptors. In this study, we propose a mechanism by which the gut microbiota influences Drosophila gut motility. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Vaccination is an important factor in public health. The recombinant bacillus Calmette Guérin (rBCG) vaccine, which expresses foreign antigens, is expected to be a superior vaccine against infectious diseases. Here, we report a new recombination platform in which the BCG Tokyo strain is transformed with nucleotide sequences encoding foreign protein fused with the MPB70 immunogenic protein precursor. By RNA-sequencing, mpb70 was found to be the most transcribed among all known genes of BCG Tokyo. Small oligopeptide, namely, polyhistidine tag, was able to be expressed in and secreted from rBCG through a process in which polyhistidine tag fused with intact MPB70 were transcribed by an mpb70 promoter. This methodology was applied to develop an rBCG expressing the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2. Immunoblotting images and mass spectrometry data showed that RBD was also secreted from rBCG. Sera from mice vaccinated with the rBCG showed a tendency of weak neutralizing capacity. The secretion was retained even after a freeze-drying process. The freeze-dried rBCG was administered to and recovered from mice. Recovered rBCG kept secreting RBD. Collectively, our recombination platform offers stable secretion of foreign antigens and can be applied to the development of practical rBCGs., (© 2024 The Societies and John Wiley & Sons Australia, Ltd.)

The insect epidermis forms the exoskeleton and determines the body size of an organism. How the epidermis acts as a metabolic regulator to adapt to changes in dietary protein availability remains elusive. Here, we show that the Drosophila epidermis regulates tyrosine (Tyr) catabolism in response to dietary protein levels, thereby promoting metabolic homeostasis. The gene expression profile of the Drosophila larval body wall reveals that enzymes involved in the Tyr degradation pathway, including 4-hydroxyphenylpyruvate dioxygenase (Hpd), are upregulated by increased protein intake. Hpd is specifically expressed in the epidermis and is dynamically regulated by the internal Tyr levels. Whereas basal Hpd expression is maintained by insulin/IGF-1 signalling, Hpd induction on high-protein diet requires activation of the AMP-activated protein kinase (AMPK)-forkhead box O subfamily (FoxO) axis. Impairment of the FoxO-mediated Hpd induction in the epidermis leads to aberrant increases in internal Tyr and its metabolites, disrupting larval development on high-protein diets. Taken together, our findings uncover a crucial role of the epidermis as a metabolic regulator in coping with an unfavourable dietary environment., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Methionine restriction (MetR) extends lifespan in various organisms, but its mechanistic understanding remains incomplete. Whether MetR during a specific period of adulthood increases lifespan is not known. In Drosophila, MetR is reported to extend lifespan only when amino acid levels are low. Here, by using an exome-matched holidic medium, we show that decreasing Met levels to 10% extends Drosophila lifespan with or without decreasing total amino acid levels. MetR during the first four weeks of adult life only robustly extends lifespan. MetR in young flies induces the expression of many longevity-related genes, including Methionine sulfoxide reductase A (MsrA), which reduces oxidatively-damaged Met. MsrA induction is foxo-dependent and persists for two weeks after cessation of the MetR diet. Loss of MsrA attenuates lifespan extension by early-adulthood MetR. Our study highlights the age-dependency of the organismal response to specific nutrients and suggests that nutrient restriction during a particular period of life is sufficient for healthspan extension., (© 2023. The Author(s).)

Objective . A crosshair light sharing (CLS) PET detector as a TOF-DOI PET detector with high spatial resolution has been developed. To extend that work, a detector calibration method was developed to achieve both higher coincidence resolving time (CRT) and DOI resolution. Approach . The CLS PET detector uses a three-layer reflective material in a two-dimensional crystal array to form a loop structure within a pair of crystals, enabling a CRT of about 300 ps and acquisition of DOI from multi-pixel photon counter (MPPC) output ratios. The crystals were 1.45 × 1.45 × 15 mm 3 fast LGSO, and the crystal array was optically coupled to an MPPC array. It is important to reduce as many inter-crystal scattering (ICS) events as possible in advance for the accurate detector calibration. DOI information is also expected to improve the CRT because it can estimate the time delay due to the detection depth of crystals. Main results . Using crystal identification and light collection rate of the highest MPPC output reduces the number of ICS events, and CRT is improved by 26%. In addition, CRT is further improved by 13% with a linear correction of time delay as a function of energy. The DOI is ideally estimated from the output ratio of only the MPPC pairs optically coupled to the interacted crystals, which is highly accurate, but the error is large due to light leakage in actual use. The previous method, which also utilizes light leakage to calculate the output ratio, is less accurate, but the error can be reduced. Using the average of the two methods, it is possible to improve the DOI resolution by 12% while maintaining the smaller error. Significance . By applying the developed calibration method, the CLS PET detector achieves the CRT of 251 ps and the DOI resolution of 3.3 mm., (© 2023 IOP Publishing Ltd.)

Super-resolution vibrational microscopy is promising to increase the degree of multiplexing of nanometer-scale biological imaging because of the narrower spectral linewidth of molecular vibration compared to fluorescence. However, current techniques of super-resolution vibrational microscopy suffer from various limitations including the need for cell fixation, high power loading, or complicated detection schemes. Here, we present reversible saturable optical Raman transitions (RESORT) microscopy, which overcomes these limitations by using photoswitchable stimulated Raman scattering (SRS). We first describe a bright photoswitchable Raman probe (DAE620) and validate its signal activation and depletion characteristics when exposed to low-power (microwatt level) continuous-wave laser light. By harnessing the SRS signal depletion of DAE620 through a donut-shaped beam, we demonstrate super-resolution vibrational imaging of mammalian cells with excellent chemical specificity and spatial resolution beyond the optical diffraction limit. Our results indicate RESORT microscopy to be an effective tool with high potential for multiplexed super-resolution imaging of live cells.

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Nakano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Detecting multiple enzyme activities simultaneously with high spatial specificity is a promising strategy to investigate complex biological phenomena, and Raman imaging would be an excellent tool for this purpose due to its high multiplexing capabilities. We previously developed activatable Raman probes based on 9CN-pyronins, but specific visualization of cells with target enzyme activities proved difficult due to leakage of the hydrolysis products from the target cells after activation. Here, focusing on rhodol bearing a nitrile group at the position of 9 (9CN-rhodol), we established a novel mechanism for Raman signal activation based on a combination of aggregate formation (to increase local dye concentration) and the resonant Raman effect along with the bathochromic shift of the absorption, and utilized it to develop Raman probes. We selected the 9CN-rhodol derivative 9CN-JCR as offering a suitable combination of increased stimulated Raman scattering (SRS) signal intensity and high aggregate-forming ability, resulting in good retention in target cells after probe activation. By using isotope-edited 9CN-JCR-based probes, we could simultaneously detect β-galactosidase, γ-glutamyl transpeptidase, and dipeptidyl peptidase-4 activities in live cultured cells and distinguish cell regions expressing target enzyme activity in Drosophila wing disc and fat body ex vivo .

Commensal microbes in animals have a profound impact on tissue homeostasis, stress resistance, and ageing. We previously showed in Drosophila melanogaster that Acetobacter persici is a member of the gut microbiota that promotes ageing and shortens fly lifespan. However, the molecular mechanism by which this specific bacterial species changes lifespan and physiology remains unclear. The difficulty in studying longevity using gnotobiotic flies is the high risk of contamination during ageing. To overcome this technical challenge, we used a bacteria-conditioned diet enriched with bacterial products and cell wall components. Here, we demonstrate that an A. persici-conditioned diet shortens lifespan and increases intestinal stem cell (ISC) proliferation. Feeding adult flies a diet conditioned with A. persici, but not with Lactiplantibacillus plantarum, can decrease lifespan but increase resistance to paraquat or oral infection of Pseudomonas entomophila, indicating that the bacterium alters the trade-off between lifespan and host defence. A transcriptomic analysis using fly intestine revealed that A. persici preferably induces antimicrobial peptides (AMPs), while L. plantarum upregulates amidase peptidoglycan recognition proteins (PGRPs). The specific induction of these Imd target genes by peptidoglycans from two bacterial species is due to the stimulation of the receptor PGRP-LC in the anterior midgut for AMPs or PGRP-LE from the posterior midgut for amidase PGRPs. Heat-killed A. persici also shortens lifespan and increases ISC proliferation via PGRP-LC, but it is not sufficient to alter the stress resistance. Our study emphasizes the significance of peptidoglycan specificity in determining the gut bacterial impact on healthspan. It also unveils the postbiotic effect of specific gut bacterial species, which turns flies into a "live fast, die young" lifestyle., Competing Interests: The authors declare no competing interests., (Copyright: © 2023 Onuma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Visualizing small individual biomolecules at subcellular resolution in live cells and tissues can provide valuable insights into metabolic activity in heterogeneous cells, but is challenging. Methods: Here, we used stimulated Raman scattering (SRS) microscopy to image deuterated methionine (d-Met) incorporated into Drosophila tissues in vivo . Results : Our results demonstrate that SRS can detect a range of previously uncharacterized cell-to-cell differences in d-Met distribution within a tissue at the subcellular level. Discussion : These results demonstrate the potential of SRS microscopy for metabolic imaging of less abundant but important amino acids such as methionine in tissue., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Spratt, Mizuguchi, Akaboshi, Kosakamoto, Okada, Obata and Ozeki.)

Context: Indonesia, Japan, Korea, Thailand., Objective: To culturally validate and translate the Scientific Exercise Guidelines for Adults with Spinal Cord Injury (SEG-SCI) for use in four Asian countries., Design: Systematic Review., Participants: N/A., Methods: A systematic review was conducted to identify all published English- and local-language studies conducted in Indonesia, Japan, Korea, and Thailand, testing the effects of exercise training interventions on fitness and cardiometabolic health in adults with acute or chronic SCI. Protocols and results from high-quality controlled studies were compared with the SEG-SCI. Forward and backward translation processes were used to translate the guidelines into Bahasa Indonesian, Japanese, Korean and Thai languages., Results: Fifteen studies met the review criteria. At least one study from each country implemented exercise prescriptions that met or exceeded the SEG-SCI. Two were controlled studies. In those two studies, relative to control conditions, participants in exercise conditions achieved significant improvements in fitness or cardiometabolic health outcomes only when the exercise intervention protocol met or exceeded the SEG-SCI. During the language translation processes, end-users confirmed that SEG-SCI language and terminology were clear., Conclusion: Clinical researchers in Indonesia, Japan, Korea and Thailand have implemented exercise protocols that meet or exceed the SCI-SEG. Results of high-quality studies align with the SEG-SCI recommendations. Based on this evidence, we recommend that the SEG-SCI be adopted in these countries. The cultural validation and translation of the SEG-SCI is an important step towards establishing consistent SCI exercise prescriptions in research, clinical and community settings around the world.

Objective. In carbon ion therapy, the visualization of the range of incident particles in a patient body is important for treatment verification. In-beam positron emission tomography (PET) imaging is one of the methods to verify the treatment in ion therapy due to the high quality of PET images. We have shown the feasibility of in-beam PET imaging of radioactive 15 O and 11 C ion beams for range verification using our OpenPET system. Recently, we developed a whole gamma imager (WGI) that can simultaneously work as PET, single gamma ray and triple gamma ray imaging. The WGI has high potential to detect the location of 10 C, which emits positrons with a simultaneous gamma ray of 718 keV, within the patient's body during ion therapy. Approach. In this work, we focus on investigating the performance of WGI for 10 C imaging and its feasibility for range verification in carbon ion therapy. First, the performance of the WGI was studied to image a 10 C point source using the Geant4 toolkit. Then, the feasibility of WGI was investigated for an irradiated polymethyl methacrylate (PMMA) phantom with a 10 C ion beam at the carbon therapy facility of the Heavy Ion Medical Accelerator in Chiba. Main results. The average spatial resolution and sensitivity for the simulated 10 C point source at the centre of the field of view were 5.5 mm FWHM and 0.010%, respectively. The depth dose of the 10 C ion beam was measured, and the triple gamma image of 10 C nuclides for an irradiated PMMA phantom was obtained by applying a simple back projection to the detected triple gammas. Significance. The shift between Bragg peak position and position of the peak of the triple gamma image in an irradiated PMMA phantom was 2.8 ± 0.8 mm, which demonstrates the capability of triple gamma imaging using WGI for range verification of 10 C ion beams., (© 2022 Institute of Physics and Engineering in Medicine.)

Background: In 2020, an incident involving spoiled salad dressing from a commercial source occurred. Upon opening the bottle, the contents exploded from gas that seemed to have fermented inside the bottle. For safety concerns, we sought to investigate the bacteria from the salad dressing in order to notify the company that made the product and relevant authorities., Methods: Anaerobic and carbon dioxide culture methods were used. To determine species of colonies, MALDI-TOF-MS and 16S rRNA whole sequencing were performed., Results: There were no colonies grown in anaerobic condition; however, we obtained three colonies from the carbon dioxide atmosphere. We determined the first colony as Alkalihalobacillus clausii ( Bacillus clausii ), the second as Bacillus spp. such as B. australimaris , B. safensis or B. safensis subsp. osmophilus and the third as B. paralicheniformis . Phylogenic tree analysis using the16S rRNA sequence revealed these colonies to be in a proximity of known gas-producing species. The NCBI database search revealed that a key gas production pathway gene, pyruvate formate-lyase (pfl), of which the gene product catalyzes pyruvate to formate conversion, exists in B. paralicheniformis. Formate dehydrogenase (FdhH) produces CO 2 from formate that the coding gene fdhF positive bacteria can participate in gas production when formate is present in the culture. And we found fdhF from A. clausii , B. australimaris/B. safensis and B. paralicheniformis . Furthermore, under butanediol producing pathway, genes coding two enzymes involved in CO 2 production, namely als and ald, existed in B. australimaris / B. safensis and B paralicheniformis , whereas A. clausii possessed als., Conclusion: Candidate species A. clausii, B. australimaris / B. safensis and B. paralicheniformis from spoiled salad dressing were thought to produce CO 2 gas each from their own enzymes, or in combination, which caused the explosion upon opening. The endospore forming nature of Bacillus should alert us to be cautious when considering food producing process regulations where we need to thoroughly heat any product during manufacture in order to inactivate any bacteria as there is the possibility of this type of dangerous occurrence., Competing Interests: The authors declare no conflict of interest., (©2022 Tottori University Medical Press.)

The intake of dietary protein regulates growth, metabolism, fecundity and lifespan across various species, which makes amino acid (AA)-sensing vital for adaptation to the nutritional environment. The general control nonderepressible 2 (GCN2)-activating transcription factor 4 (ATF4) pathway and the mechanistic target of rapamycin complex 1 (mTORC1) pathway are involved in AA-sensing. However, it is not fully understood which AAs regulate these two pathways in living animals and how they coordinate responses to protein restriction. Here we show in Drosophila that the non-essential AA tyrosine (Tyr) is a nutritional cue in the fat body necessary and sufficient for promoting adaptive responses to a low-protein diet, which entails reduction of protein synthesis and mTORC1 activity and increased food intake. This adaptation is regulated by dietary Tyr through GCN2-independent induction of ATF4 target genes in the fat body. This study identifies the Tyr-ATF4 axis as a regulator of the physiological response to a low-protein diet and sheds light on the essential function of a non-essential nutrient., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The small biomolecule methionine (Met) is a fundamental amino acid required for a vast range of biological processes such as protein synthesis, cancer metabolism, and epigenetics. However, it is still difficult to visualize the subcellular distribution of small biomolecules including Met in a minimally invasive manner. Here, we demonstrate stimulated Raman scattering (SRS) imaging of cellular uptake of deuterated methionine (d 8 -Met) in live HeLa cells by way of comparison to the previously used alkyne-labeled Met analogue─homopropargylglycine (Hpg). We show that the solutions of d 8 -Met and Hpg have similar SRS signal intensities. Furthermore, by careful image analysis with background subtraction, we succeed in the SRS imaging of cellular uptake of d 8 -Met with a much greater signal intensity than Hpg, possibly reflecting the increased and minimally invasive uptake kinetics of d 8 -Met compared with Hpg. We anticipate that d 8 -Met and other deuterated biomolecules will be useful for investigating metabolic processes with subcellular resolution.

Shiga toxin-producing Escherichia coli (STEC) causes proximal tubular defects in the kidney. However, factors altered by Shiga toxin (Stx) within the proximal tubules are yet to be shown. We determined Stx receptor Gb3 in murine and human kidneys and confirmed the receptor expression in the proximal tubules. Stx2-injected mouse kidney tissues and Stx2-treated human primary renal proximal tubular epithelial cell (RPTEC) were collected and microarray analysis was performed. We compared murine kidney and RPTEC arrays and selected common 58 genes that are differentially expressed vs. control (0 h, no toxin-treated). We found that the most highly expressed gene was GDF15, which may be involved in Stx2-induced weight loss. Genes associated with previously reported Stx2 activities such as src kinase Yes phosphorylation pathway activation, unfolded protein response (UPR) and ribotoxic stress response (RSR) showed differential expressions. Moreover, circadian clock genes were differentially expressed, suggesting Stx2-induced renal circadian rhythm disturbance. Circadian rhythm-regulated proximal tubular Na + -glucose transporter SGLT1 (SLC5A1) was down-regulated, indicating proximal tubular functional deterioration, and mice developed glucosuria confirming proximal tubular dysfunction. Stx2 alters gene expression in murine and human proximal tubules through known activities and newly investigated circadian rhythm disturbance, which may result in proximal tubular dysfunctions.

Leptospirosis is a zoonosis caused by the pathogenic bacterium Leptospira. The Microscopic Agglutination Test (MAT) is widely used as the gold standard for diagnosis of leptospirosis. In this method, diluted patient serum is mixed with serotype-determined Leptospires, and the presence or absence of aggregation is determined under a dark-field microscope to calculate the antibody titer. Problems of the current MAT method are 1) a requirement of examining many specimens per sample, and 2) a need of distinguishing contaminants from true aggregates to accurately identify positivity. Therefore, increasing efficiency and accuracy are the key to refine MAT. It is possible to achieve efficiency and standardize accuracy at the same time by automating the decision-making process. In this study, we built an automatic identification algorithm of MAT using a machine learning method to determine agglutination within microscopic images. The machine learned the features from 316 positive and 230 negative MAT images created with sera of Leptospira-infected (positive) and non-infected (negative) hamsters, respectively. In addition to the acquired original images, wavelet-transformed images were also considered as features. We utilized a support vector machine (SVM) as a proposed decision method. We validated the trained SVMs with 210 positive and 154 negative images. When the features were obtained from original or wavelet-transformed images, all negative images were misjudged as positive, and the classification performance was very low with sensitivity of 1 and specificity of 0. In contrast, when the histograms of wavelet coefficients were used as features, the performance was greatly improved with sensitivity of 0.99 and specificity of 0.99. We confirmed that the current algorithm judges the positive or negative of agglutinations in MAT images and gives the further possibility of automatizing MAT procedure., Competing Interests: The authors have declared that no competing interests exist.

Objective. Time-of-flight (TOF) and depth-of-interaction (DOI) are well recognized as important information to improve PET image quality. Since such information types are not correlated, many TOF-DOI detectors have been developed but there are only a few reports of high-resolution detectors (e.g. 1.5 mm resolution) for brain PET systems. Based on the DOI detector, which enables single-ended readout by optically coupling a pair of crystals and having a loop structure, we have developed the crosshair light sharing (CLS) PET detector that optically couples the four-loop structure, consisting of quadrisected crystals comparable in size to a photo-sensor, to four photo-sensors in close proximity arranged in a windmill shape. Even as a high-resolution detector, the CLS PET detector could obtain both TOF and DOI information. The coincidence resolving time (CRT) of the CLS PET detector needs to be further improved, however, for application to the brain PET system. Recently, a fast LGSO crystal was developed which has advantages in detection efficiency and CRT compared to the GFAG crystal. In this work, we developed the CLS PET detector using the fast LGSO crystal for the TOF-DOI brain PET system. Approach. The crystals were each 1.45 × 1.45 × 15 mm 3 and all surfaces were chemically etched. The CLS PET detector consisted of a 14 × 14 crystal array optically coupled to an 8 × 8 MPPC array. Main results. The fast LGSO array provided 10.1% energy resolution at 511 keV, 4.7 mm DOI resolution at 662 keV, and 293 ps CRT with the energy window of 440-620 keV. Significance. The developed CLS PET detector has 290% higher coincidence sensitivity, 30% better energy resolution, and 32% better time resolution compared to our previous CLS PET detector., (© 2021 Institute of Physics and Engineering in Medicine.)

An early-life inflammatory response is associated with risks of age-related pathologies. How transient immune signalling activity during animal development influences life-long fitness is not well understood. Using Drosophila as a model, we find that activation of innate immune pathway Immune deficiency (Imd) signalling in the developing larvae increases adult starvation resistance, decreases food intake and shortens organismal lifespan. Interestingly, lifespan is shortened by Imd activation in the larval gut and fat body, whereas starvation resistance and food intake are altered by that in neurons. The adult flies that developed with Imd activation show sustained Imd activity in the gut, despite complete tissue renewal during metamorphosis. The larval Imd activation increases an immunostimulative bacterial species, Gluconobacter sp., in the gut microbiome, and this dysbiosis is persistent to adulthood. Removal of gut microbiota by antibiotics in the adult fly mitigates intestinal immune activation and rescues the shortened lifespan. This study demonstrates that early-life immune activation triggers long-term physiological changes, highlighted as an irreversible alteration in gut microbiota, prolonged inflammatory intestine and concomitant shortening of the organismal lifespan., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The enteroendocrine cell (EEC)-derived incretins play a pivotal role in regulating the secretion of glucagon and insulins in mammals. Although glucagon-like and insulin-like hormones have been found across animal phyla, incretin-like EEC-derived hormones have not yet been characterised in invertebrates. Here, we show that the midgut-derived hormone, neuropeptide F (NPF), acts as the sugar-responsive, incretin-like hormone in the fruit fly, Drosophila melanogaster. Secreted NPF is received by NPF receptor in the corpora cardiaca and in insulin-producing cells. NPF-NPFR signalling resulted in the suppression of the glucagon-like hormone production and the enhancement of the insulin-like peptide secretion, eventually promoting lipid anabolism. Similar to the loss of incretin function in mammals, loss of midgut NPF led to significant metabolic dysfunction, accompanied by lipodystrophy, hyperphagia, and hypoglycaemia. These results suggest that enteroendocrine hormones regulate sugar-dependent metabolism through glucagon-like and insulin-like hormones not only in mammals but also in insects., (© 2021. The Author(s).)

We have designed and developed non-fluorescent, cell-permeable photoactivatable fluorophores, photoactivatable SPiDERs (paSPiDERs), which exhibit fluorescence activation upon light irradiation, accompanied by the generation of a quinone methide intermediate that binds covalently to intracellular proteins. The fluorescence signal is durable for 24 hours, resistant to fixation and compatible with immunostaining, and selective cell labeling can be achieved at single-cell resolution.

Leucine-rich repeat kinase 2 (LRRK2) is the causal gene of the autosomal dominant hereditary form of Parkinson's disease (PD), PARK8. We have previously reported that induced pluripotent stem cells (iPSCs) from a PARK8 patient with I2020T LRRK2 mutation replicated to some extent the pathologic phenotype evident in the brain of PD patients. In the present study, we generated gene-corrected iPSCs line, KEIUi001-A, using TALEN-mediated genome editing. KEIUi001-A retained a normal karyotype and pluripotency, i.e. the capacity to differentiate into cell types of the three germ layers. This iPSCs will be valuable for clarifying various aspects of LRRK2-related pathology., (Copyright © 2020. Published by Elsevier B.V.)

Gut microbiota impacts the host metabolome and affects its health span. How bacterial species in the gut influence age-dependent metabolic alteration has not been elucidated. Here we show in Drosophila melanogaster that allantoin, an end product of purine metabolism, is increased during aging in a microbiota-dependent manner. Allantoin levels are low in young flies but are commonly elevated upon lifespan-shortening dietary manipulations such as high-purine, high-sugar, or high-yeast feeding. Removing Acetobacter persici in the Drosophila microbiome attenuated age-dependent allantoin increase. Mono-association with A. persici , but not with Lactobacillus plantarum , increased allantoin in aged flies. A. persici increased allantoin via activation of innate immune signaling IMD pathway in the renal tubules. On the other hand, analysis of bacteria-conditioned diets revealed that L. plantarum can decrease allantoin by reducing purines in the diet. These data together demonstrate species-specific regulations of host purine levels by the gut microbiome., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Necrotic cells elicit an inflammatory response through their endogenous factors with damage-associated molecular patterns. Blocking apoptosis in Drosophila wings leads to the necrosis-driven systemic immune response by unknown mechanisms. Here, we demonstrate that immune activation in response to necrotic cells is mediated by commensal gut microbiota. Removing the microbiome attenuates hyperactivation of the innate immune signaling IMD pathway in necrosis-induced flies. Necrotic cells in wings trigger Gluconobacter expansion in the gut. An isolated Gluconobacter sp. strain is sufficient for pathological IMD activation in necrosis-induced flies, while it is not inflammatory for control animals. In addition, bacterial colonization shifts the host metabolome and shortens the lifespan of necrosis-induced flies. This study shows that local necrosis triggers a pathological systemic inflammatory response through interaction between the host and the dysbiotic gut microbiome., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Leucine-rich repeat kinase 2 (LRRK2) is the causal molecule of familial Parkinson's disease. Although the characteristics of LRRK2 have gradually been revealed, its true physiological functions remain unknown. LRRK2 is highly expressed in immune cells such as B2 cells and macrophages, suggesting that it plays important roles in the immune system. In the present study, we investigate the roles of LRRK2 in the immune functions of dendritic cells (DCs). Bone marrow-derived DCs from both C57BL/6 wild-type (WT) and LRRK2 knockout (KO) mice were induced by culture with granulocyte/macrophage-colony stimulating factor (GM/CSF) in vitro. We observed the differentiation of DCs, the phosphorylation of the transcriptional factors NF-κB, Erk1/2, and p-38 after lipopolysaccharide (LPS) stimulation and antigen-presenting ability by flow cytometry. We also analyzed the production of inflammatory cytokines by ELISA. During the observation period, there was no difference in DC differentiation between WT and LRRK2-KO mice. After LPS stimulation, phosphorylation of NF-κB was significantly increased in DCs from the KO mice. Large amounts of inflammatory cytokines were produced by DCs from KO mice after both stimulation with LPS and infection with Leishmania . CD4 + T-cells isolated from antigen-immunized mice proliferated to a significantly greater degree upon coculture with antigen-stimulated DCs from KO mice than upon coculture with DCs from WT mice. These results suggest that LRRK2 may play important roles in signal transduction and antigen presentation by DCs.

Background: Choroid plexus (CP) is an important tissue not only to produce cerebrospinal fluid (CSF) but also to regulate substances that are secreted into or absorbed from CSF through blood-cerebrospinal fluid barrier (BCSFB) formed by CP epithelial cells (CPECs). CPECs display signs of deterioration in aged and diseased people. However, whether CPECs in hypercholesterolemic animals develop such damage is not known., Methods: We used cholesterol-fed wild-type or Watanabe hereditary hyperlipidemic (WHHL) rabbits of identical age to determine CPEC changes in terms of morphology and protein expression/localization., Results: Compared with non-cholesterol-fed control rabbits, prolonged exposure to cholesterol reduced CPEC height and increased lipofuscin levels in CPECs, indicating cellular damage. Expression of aquaporin 1 on the apical membranes of CPECs was diminished in cholesterol-exposed rabbits, implying a reduced CSF-producing function in the CP. The rabbit macrophage-specific antibody (RAM11) immunoreaction became positive in CPECs adjacent to foam cells, indicating an alteration in this cell type., Conclusion: Cholesterol insults from the circulation (which is reflected by foam-cell accumulation in the CP) induce CPEC dysfunction, and the latter seems to be enhanced by foam cells in hypercholesterolemic rabbits.

Nutrition and light-dark cycle influence rat testicular development. With 9% casein diet (low protein diet) under normal 12 h-12 h lighting cycles (9P), juvenile rat testes undergo normal growth. On the other hand, a low protein diet with constant darkness (D9P) results in a growth arrest of rat testes. Supplementation of cystine to the low protein diet under constant darkness (D9PC) had a tendency to increase testes weight, suggesting an improvement in growth suppression. Whether the growth suppression of testes in D9P is associated with suppression of spermatogenesis has not yet been shown. We aimed to determine the effect of a low protein diet and constant darkness with or without dietary cystine in testes using a histological technique. In the histological assessment, D9P testes showed a decreased number of seminiferous tubules with elongated spermatids, indicating a functional testicular defect in this group. However, cystine supplementation resulted in enhanced spermatogenesis versus control animals (D9PC vs. D9P) implying the importance of cystine to testicular development in this condition. Furthermore, serum testosterone concentration was increased in D9PC suggesting contribution of testosterone to ameliorate spermatogenesis. From these results, we conclude that cystine supplementation to a low protein diet under constant darkness promoted an increase in testosterone which in turn benefitted spermatogenesis.

Aims: Inflammasome activation plays a pivotal role in many inflammatory diseases. Given that connexin (Cx) channels regulate numerous cellular events leading to inflammasome activation, we determined whether and how connexin affected inflammasome activation and inflammatory cell injury. Results: Exposure of mouse peritoneal macrophages (PMs) to lipopolysaccharide (LPS) plus ATP caused NLRP3 inflammasome activation, together with an increased connexin43 (Cx43). Inhibition of Cx43 blunted inflammasome activation. Consistently, PMs from the Cx43 heterozygous mouse (Cx43 +/- ) exhibited weak inflammasome activation, in comparison with those from the Cx43 +/+ mouse. Further analysis revealed that inflammasome activation was preceded by an increased reactive oxygen species (ROS) production, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase 2 (NOX2), protein carbonylation, and mitogen-activated protein kinase (MAPK) activation. Suppression of ROS with antioxidant, downregulation of NOX2 with small interfering RNA (siRNA), or inhibition of NADPH oxidase or MAPKs with inhibitors blocked Cx43 elevation and inflammasome activation. Intriguingly, suppression of Cx43 also blunted NOX2 expression, protein carbonylation, p38 phosphorylation, and inflammasome activation. In a model of acute renal injury induced by LPS, the Cx43 +/- mouse exhibited a significantly lower level of blood interleukin-1β (IL-1β), blood urea nitrogen, and urinary protein, together with milder renal pathological changes and renal expression of NLRP3 and NOX4, as compared with the Cx43 +/+ mouse. Moreover, inhibition of gap junctions suppressed IL-1β- and tumor necrosis factor-α-induced expression of NOX4 in glomerular podocytes and tubular epithelial cells. Innovation and Conclusion: Our study indicates that Cx43 contributes to inflammasome activation and the progression of renal inflammatory cell injury through modulation of intracellular redox status. Cx43 could be a novel target for the treatment of certain inflammatory diseases.

Thromboembolism is a major complication of nephrotic syndrome, with the renal vein being the most frequent site. However, the incidence of portal vein thrombosis (PVT) in patients with nephrotic syndrome is rare. We report a case of a relapsed steroid-dependent minimal change disease with incidental PVT. A 38-year-old man presented with anasarca. Elevated liver enzymes were discovered during routine blood testing within days after commencing treatment. Although drug-induced liver injuries are frequently observed with mild aminotransferase abnormality during therapy with steroid or immune-suppressive agents, imaging revealed a massive thrombus of the portal vein, which was treated by anticoagulant therapy with edoxaban. Treatment with anticoagulant therapy could normalize liver function. Two months after the initiation of treatment with edoxaban, the follow-up CT scan and ultrasound showed the disappearance of PVT. Our case suggests that much attention should be paid to PVT as a cause of liver enzyme elevation when treating patients with nephrotic syndrome.

Light Chain Proximal Tubulopathy (LCPT) is a rare form of paraprotein-related kidney disease in which monoclonal free light chains damage the proximal renal tubular epithelial cells. We herein report the case of a 78-year-old woman who presented with anemia and kidney dysfunction. Serum and urine protein electrophoresis analyses revealed a monoclonal IgD and λ free light chains. Proximal tubular injury and the accumulation of λ light chains were found by kidney biopsy. Electron microscopy revealed no organized structure suggestive of crystals. LCPT was caused by IgD lambda myeloma and bortezomib and dexamethasone therapy led to very good partial response (VGPR) without a worsening of the kidney function.

Tissue closure involves the coordinated unidirectional movement of a group of cells without loss of cell-cell contact. However, the molecular mechanisms controlling the tissue closure are not fully understood. Here, we demonstrate that Lamin C, the sole A-type lamin in Drosophila, contributes to the process of thorax closure in pupa. High expression of Lamin C was observed at the leading front of the migrating wing imaginal discs. Live imaging analysis revealed that knockdown of Lamin C in the thorax region affected the coordinated movement of the leading front, resulting in incomplete tissue fusion required for formation of the adult thorax. The closure defect due to knockdown of Lamin C correlated with insufficient accumulation of F-actin at the front. Our study indicates a link between A-type lamin and the cell migration behavior during tissue closure., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The intestine has direct contact with nutritional information. The mechanisms by which particular dietary molecules affect intestinal homeostasis are not fully understood. In this study, we identified S-adenosylmethionine (SAM), a universal methyl donor synthesized from dietary methionine, as a critical molecule that regulates stem cell division in Drosophila midgut. Depletion of either dietary methionine or SAM synthesis reduces division rate of intestinal stem cells. Genetic screening for putative SAM-dependent methyltransferases has identified protein synthesis as a regulator of the stem cells, partially through a unique diphthamide modification on eukaryotic elongation factor 2. In contrast, SAM in nutrient-absorptive enterocytes controls the interleukin-6-like protein Unpaired 3, which is required for rapid division of the stem cells after refeeding. Our study sheds light upon a link between diet and intestinal homeostasis and highlights the key metabolite SAM as a mediator of cell-type-specific starvation response., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Environmental stresses experienced during development exert many long-term effects upon health and disease. For example, chemical oxidants or genetic perturbations that induce low levels of reactive oxygen species can extend lifespan in several species. In some cases, the beneficial effects of low-dose oxidants are attributed to adaptive protective mechanisms such as mitohormesis, which involve long-term increases in the expression of stress response genes. Here we show in Drosophila that transient exposure to low concentrations of oxidants during development leads to an extension of adult lifespan. Surprisingly, this depends upon oxidants acting in an antibiotic-like manner to selectively deplete the microbiome of Acetobacter proteobacteria. We demonstrate that the presence of Acetobacter species, such as A. aceti, in the indigenous microbiota increases age-related gut dysfunction and shortens lifespan. This study demonstrates that low-dose oxidant exposure during early life can extend lifespan via microbiome remodelling rather than mitohormesis.

Radiotherapy (RT) is one of the most important treatments for prostate cancer. Although RT can kill cancer cells through direct and indirect effects of radiation, it occasionally induces an abscopal effect whereby localized radiation treatment is associated with elimination of metastatic cancer at a distance from the irradiated area. Thus, RT may induce an effective antitumor immune response, although the mechanism involved has remained unclear. The present was designed to evaluate this effect of RT in 36 patients with prostate cancer who provided informed consent prior to enrollment in this clinical trial. Peripheral blood samples were collected periodically after low-dose-rate (LDR) prostate brachytherapy, and lymphocyte subsets were analyzed by flow cytometry. The proportion of activated T cells (CD3+HLA-DR+, CD4+HLA-DR+ and CD8+HLA-DR+) in peripheral blood revealed a gradual and bimodal increase after LDR brachytherapy, whereas memory CD8+ T cells bimodally decreased after treatment. The ratios of activated T cells and regulatory T cells gradually increased after the treatment. Thus, LDR brachytherapy was demonstrated to induce effective immune responses in patients. This increase of activated T cells may contribute to maintenance of remission and reduction of relapse rates.

Early-life nourishment exerts long-term influences upon adult physiology and disease risk. These lasting effects of diet are well established but the underlying mechanisms are only partially understood. Here we show that restricting dietary yeast during Drosophila development can, depending upon the subsequent adult environment, more than double median lifespan. Developmental diet acts via a long-term influence upon the adult production of toxic molecules, which we term autotoxins, that are shed into the environment and shorten the lifespan of both sexes. Autotoxins are synthesised by oenocytes and some of them correspond to alkene hydrocarbons that also act as pheromones. This study identifies a mechanism by which the developmental dietary history of an animal regulates its own longevity and that of its conspecific neighbours. It also has important implications for the design of lifespan experiments as autotoxins can influence the regulation of longevity by other factors including diet, sex, insulin signalling and population density.

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 20-year-old dystonia patient with a GCH1 mutation (DYT5). Episomal vectors were used to introduce reprogramming factors (OCT3/4, SOX2, KLF4, L-MYC, LIN28, and p53 carboxy-terminal dominant-negative fragment) to the PBMCs. The generated iPSCs expressed pluripotency markers, and were capable of differentiating into derivates of all three germ layers in vitro. The iPSC line also showed a normal karyotype and preserved the GCH1 mutation. This cellular model can provide opportunities to perform pathophysiological studies for aberrant dopamine metabolism-related disorders., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

For the first time, a 15-year-old boy was found to have a slight degree of proteinuria and microscopic hematuria during annual school urinalysis screening. His kidney function had already severely deteriorated. A kidney biopsy revealed tubulointerstitial nephritis (TIN) with diffuse inflammatory cell infiltration. His medical records showed his serum creatinine level to be 0.98 mg/dL two years ago, which was abnormally high considering his age. Although the etiology of slowly progressive TIN was unclear, glucocorticoid and immunosuppressant therapy improved his kidney function. This case report suggests that all doctors should recognize the reference range for the serum creatinine level in teenagers.

Mixed cryoglobulinemic syndrome, which is a systemic vasculitis characterized by the immune complex deposition in small- and medium-sized arteries and most often due to chronic hepatitis C virus (HCV) infection, sometimes clinically manifests as refractory glomerulonephritis or nephritic syndrome. Patients with mixed cryoglobulinemic nephropathy who have a rapidly progressive glomerulonephritis should receive immunosuppressive therapy. After disease stabilization, patients should receive concurrent therapy for the underlying HCV infection. The standard therapy of a chronic HCV infection is IFN monotherapy or IFN combined with ribavirin; however, after the introduction of direct-acting antivirals (DAAs), the standard therapy for patients with HCV genotype 1 has dramatically changed. We report a case of HCV-associated cryoglobulinemic membranoproliferative glomerulonephritis (MPGN) successfully treated by daclatasvir and asunaprevir, which are IFN-free DAAs for HCV, in combination with angiotensin II receptor blocker without immunosuppressive therapy. The patient developed severe nephrotic syndrome with progressive kidney dysfunction. Blood examination revealed a high copy number of HCV-RNA (6.4 log IU/mL, type 1), cryoglobulinemia, paraproteinemia of IgM-κ, and hypocomplementemia. Histological analysis showed MPGN type 1. These findings were compatible with those observed in HCV-associated cryoglobulinemic MPGN. This case offers original evidence for the application of newer generation of IFN-free DAAs in the treatment of HCV-associated cryoglobulinemic nephropathy.

Living organisms experience tissue damage from both, the surrounding environment and from inside their bodies. Tissue repair/regeneration is triggered by local tissue injury to restore an injured, or lost, part of the body. Tissue damage results in a series of responses, not only locally but also systemically in distant tissues. In our recent publication, we established a "dual system" that induces spatiotemporal tissue damage simultaneously with gene manipulation in surrounding tissues. With this system, we demonstrated that appropriate regulation of methionine metabolism in the fat body is required for tissue repair in Drosophila wing discs, thus highlighting the importance of systemic damage response (SDR) in tissue repair. This "Extra View" aims to discuss our recent reports that propose methionine metabolism to be an essential part of SDR, together with related topics in several model organisms.

Background: Serum albumin concentration (SAC) is a prognostic factor that is affected by many factors such as postural change, liver function and food intake. Chronic kidney disease (CKD) patients excrete proteinuria, have low-protein diet, and receive glucocorticoid therapy. No one has evaluated the most influential factors on SAC in CKD patients., Methods: A retrospective study. Hospitalized CKD patients with less than 1 g/gCreatinine proteinuria receiving glucocorticoid therapy (n=28), with 1 or more g/gCreatinine proteinuria not receiving glucocorticoid therapy (n=36), and with 1 or more g/gCreatinine proteinuria receiving glucocorticoid therapy (n=39) were enrolled. SAC, hemoglobin, proteinuria and blood pressure at the last outpatient check-up before hospitalization, on the second day of hospitalization, at the last laboratory examination before discharge, as well as at the first outpatient follow-up after discharge were analyzed., Results: SAC decreased on the second day of hospitalization and increased at the first outpatient follow-up significantly in all groups. Unexpectedly, the change of SAC was irrelevant to the amount of proteinuria., Conclusions: SAC was affected by not only proteinuria, but also postural change, physical activity, and food in CKD patients. SAC should be analyzed by standardizing a patient's condition during phlebotomy. J. Med. Invest. 64: 146-152, February, 2017.

The terminal elimination half-life (t1/2) of empagliflozin is 13.1 hours. Accordingly, we hypothesized that the administration of empagliflozin every other day might improve glycemic control in patients with type 2 diabetes mellitus, not being inferior to the therapy every day. We investigated the clinical effects and safety of the addition of empagliflozin every day or every other day to type 2 diabetic patients with a poor control in glycemia. Thirteen Japanese patients diagnosed as type 2 diabetes mellitus recruited to this study. Subjects were divided into two groups; one was treatment with 10 mg of empagliflozin every day (Group A), the other was 10 mg of empagliflozin every other day (Group B). The comparable study of multiple clinical indexes between the 2 groups was made before and 8, 16, and 24 weeks after the treatment. After the treatment for 24 weeks, the HbA1c level was decreased both in group A (from 7.5%±1.1% to 6.5%±0.8%) and in group B (from 7.6%±0.8% to 7.2%±0.5%). This pilot trial suggested the possibility of 10-mg every other day administration with empagliflozin for Japanese patients with type 2 diabetes mellitus. J. Med. Invest. 64: 50-57, February, 2017.

There are a few stroke specialists in medically under-served areas in Japan. Consequently, in remote area patients may not receive thrombolysis with intravenous recombinant tissue plasminogen activator (iv rt-PA), the standard treatment for acute ischemic stroke. Using a mobile telestroke support system (TSS) that accesses the internet via a smart phone, we implemented iv rt-PA infusion therapy under a drip-and-ship protocol to treat the stroke patients in medically under-served areas. The physicians at the Tokushima Prefectural Kaifu Hospital (TPKH), located in rural Japan, can relay CT or MRI scans and other patient data via their smart phone to off-site stroke specialists. In the course of 34 months, we used the TSS in 321 emergencies. A total of 9 of 188 (4.8%) with acute ischemic stroke, received iv rt-PA infusion therapy using a mobile TSS; in 5 among these (55.6%), we obtained partial or complete recanalization of occluded arteries. None suffered post-treatment hemorrhage and their average NIH stroke score fell from 14.6 at the time of admission to 6.8 at 24 h post-infusion. The drip-and-ship protocol contributed to the safe and effective treatment of the stroke patients living in medically under-served rural areas., Competing Interests: The authors have no personal financial or institutional interest in any of the drugs, materials, or devices cited in this study.

Background: α-Synuclein (αSYN) has been genetically implicated in familial and sporadic Parkinson's disease (PD), and is associated with disease susceptibility, progression and pathology. Excess amounts of αSYN are toxic to neurons. In the brain, microglial αSYN clearance is closely related to neuronal survival. Leucine-rich repeat kinase 2 (LRRK2) is the one of the other genes implicated in familial and sporadic PD. While LRRK2 is known to be expressed in microglia, its true function remains to be elucidated. In this study, we investigated αSYN clearance by microglia isolated from LRRK2-knockout (KO) mice., Results: In LRRK2-KO microglia, αSYN was taken up in larger amounts and cleared from the supernatant more effectively than for microglia isolated from wild-type (WT) mice. This higher clearance ability of LRRK2-KO microglia was thought to be due to an increase of Rab5-positive endosomes, but not Rab7- or Rab11-positive endosomes. Increased engagement between Rab5 and dynamin 1 was also observed in LRRK2-KO microglia., Conclusion: LRRK2 negatively regulates the clearance of αSYN accompanied by down-regulation of the endocytosis pathway. Our findings reveal a new functional role of LRRK2 in microglia and offer a new insight into the mechanism of PD pathogenesis.

The LacZ gene, which encodes Escherichia coli β-galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ-positive cells in living organisms or tissues at single-cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single-cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)