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In light of the emerging importance of the gut microbiome in human health, there is a need to improve the colonization efficiency of therapeutic bacteria called probiotics. Despite their recognized potential, artificially administered bacteria exhibit poor colonization in the intestine, limiting their therapeutic efficacy. Addressing this challenge requires innovative strategies; however, reported examples are limited. In nature, including in the intestinal tract, bacteria live via biofilm formation. Recently, it has been reported that RNase I, a member of the RNase T2 family conserved among almost all species, including bacteria, inhibits biofilm formation in Escherichia coli. In this study, we focus on these results and investigate the relationship between high biofilm formation and intestinal attachment using a non-settling E. coli laboratory strain as a probiotic model. The intestinal colonization abilities were evaluated through a microfluidic device mimicking the intestinal tract and through oral administration to mice. The in vitro and in vivo experiments showed that the E. coli strain lacking RNase I exhibited remarkable stability in intestinal colonization. We investigated the observation of colonization using fluorescence in situ hybridization, and inoculated E. coli cells were aggregated with the gut microbiome in the cecum and colon. This study proposes a technique to improve the intestinal colonization of bacteria by simply manipulating a single gene disruption, and it is expected to contribute to future research on the colonization of useful bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing finantial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The renal glomerulus produces primary urine from blood plasma by ultrafiltration. The ultrastructure of the glomerulus is closely related to filtration function and disease development. The ultrastructure of glomeruli has mainly been evaluated using transmission electron microscopy; however, the volume that can be observed using transmission electron microscopy is extremely limited relative to the total volume of the glomerulus. Consequently, observing structures that exist in only one location in each glomerulus, such as the vascular pole, and evaluating low-density or localized lesions are challenging tasks. Array tomography (AT) is a technique used to analyze the ultrastructure of tissues and cells via scanning electron microscopy of serial sections. In this study, we present an AT workflow that is optimized for observing complete serial sections of the whole glomerulus, and we share several analytical examples that use the optimized AT workflow, demonstrating the usefulness of this approach. Overall, this AT workflow can be a powerful tool for structural and pathological evaluation of the glomerulus. This workflow is also expected to provide new insights into the ultrastructure of the glomerulus and its constituent cells., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Objectives: We aimed to examine the mid-term results corresponding to the entry site in patients who underwent pre-emptive thoracic endovascular aortic aneurysm repair (TEVAR) for uncomplicated type B aortic dissection (TBAD)., Methods: We included 27 patients who underwent pre-emptive TEVAR for uncomplicated TBAD between September 2014 and December 2019. We divided the patients into 2 groups depending on the proximal landing zone (zone 2 group, zone ≥3 group) and retrospectively analyzed the risk of all-cause and aorta-related mortality, aortic events (rupture, open conversion, and secondary intervention), and aortic enlargement (≥5 mm)., Results: The median age of the patients was 53 (47-65) years. The median duration from the onset of uncomplicated TBAD to TEVAR was 43 (30-99) days, and the median follow-up duration was 48 (36-57) months. The maximum preoperative diameter of the dissected aorta was 40 mm in the zone 2 group and 35 mm in the zone ≥3 group (p=0.134). There was no case of hospital death or spinal cord ischemia; however, there was 1 (3.7%) case of perioperative stroke in the zone 2 group. Multivariate analysis of the risk factors for aortic enlargement following pre-emptive TEVAR for uncomplicated TBAD revealed that only zone 2 landing was an independent risk factor. The estimated Kaplan-Meier curve showed a higher rate of aortic enlargement in the zone 2 group at 4 years after pre-emptive TEVAR (46.4% vs 0%, log-rank test; p=0.011)., Conclusions: In this study on TBAD, we found that zone 2 landing was associated with aortic enlargement after pre-emptive TEVAR. In cases where the distance from the left subclavian artery to a major entry point was short, there were more cases of aortic dilatation., Clinical Impact: The effectiveness of entry closure for type B aortic dissection was demonstrated in the INSTEAD XL trial. The cause of aortic enlargement after pre-emptive endovascular treatment for type B aortic dissection remains controversial. In the present study, zone 2 landing was a risk factor for aortic enlargement after pre-emptive thoracic endovascular aortic aneurysm repair (TEVAR) for uncomplicated type B dissection. Patients with zone 2 landing should be closely followed up after pre-emptive TEVAR., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Aim: Malnutrition is a prevalent health issue among hemodialysis patients. Oral frailty, a condition characterized by impairments in multiple oral health aspects and functions, has been associated with nutritional status in the general population. We aimed to determine whether oral frailty was associated with nutritional status in hemodialysis patients aged ≥50 years. Our secondary aim was to determine the prevalence of oral frailty in this population., Methods: This cross-sectional study included hemodialysis patients who were admitted to a single medical center. According to the Oral Frailty 5-item Checklist, oral frailty is characterized by the presence of two or more of the following criteria: (i) fewer teeth, (ii) difficulty in chewing, (iii) difficulty in swallowing, (iv) dry mouth, and (v) low articulatory oral motor skills. Patients' nutritional status was assessed using the Geriatric Nutritional Risk Index (GNRI). Multivariable ordinal logistic regression analysis was performed to assess the association between oral frailty and nutritional status (classified into three categories according to the GNRI: >98, 92-98, and <92)., Results: In total, 152 hemodialysis patients (55 women and 97 men) with a mean age of 70.4 years were included. The prevalence of oral frailty was 61.2%. After adjusting for health characteristics and sociodemographic background, oral frailty was associated with poor nutritional status according to the GNRI (odds ratio = 2.04, 95% confidence interval = 1.02-4.09)., Conclusions: In this study, approximately 60% of hemodialysis patients aged ≥50 years exhibited oral frailty, and hemodialysis patients with oral frailty had poor nutritional status according to the GNRI. Geriatr Gerontol Int 2024; 24: 891-897., (© 2024 Japan Geriatrics Society.)

Background: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad., Results: To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice., Conclusions: Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes., (© 2024. The Author(s).)

Pneumolysin (Ply) is an indispensable cholesterol-dependent cytolysin for pneumococcal infection. Although Ply-induced disruption of pneumococci-containing endosomal vesicles is a prerequisite for the evasion of endolysosomal bacterial clearance, its potent activity can be a double-edged sword, having a detrimental effect on bacterial survivability by inducing severe endosomal disruption, bactericidal autophagy, and scaffold epithelial cell death. Thus, Ply activity must be maintained at optimal levels. We develop a highly sensitive assay to monitor endosomal disruption using NanoBiT-Nanobody, which shows that the pneumococcal sialidase NanA can fine-tune Ply activity by trimming sialic acid from cell-membrane-bound glycans. In addition, oseltamivir, an influenza A virus sialidase inhibitor, promotes Ply-induced endosomal disruption and cytotoxicity by inhibiting NanA activity in vitro and greater tissue damage and bacterial clearance in vivo. Our findings provide a foundation for innovative therapeutic strategies for severe pneumococcal infections by exploiting the duality of Ply activity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aim: This study examined the associations between geriatric factors and decreased opportunities for conversation among older adults amid a period of self-restraint during the COVID-19 pandemic., Methods: A cross-sectional questionnaire-based survey was carried out in October 2020. The participants were 204 residents aged ≥65 years staying at a private care home in Kyoto city, Japan. Logistic regression analysis was carried out with the reduction of conversation opportunities as the dependent variable, and geriatric factors as independent variables after adjusting for age and sex. We compared the decreased frequency of opportunities between residents in the assisted living wing and in the nursing care wing of the private care home., Results: The percentages of respondents who reported a decrease in the opportunities for conversation among themselves were 43.9% for residents in the assisted living wing and 19.7% for those in the nursing care wing. After adjusting for age and sex, the opportunities for conversation was significantly associated with the basic activities of daily living (OR 1.07, 95% CI 1.01-1.12), instrumental self-maintenance (OR 1.25, 95% CI 1.08-1.46), intellectual activity (OR 1.35, 95% CI 1.09-1.66), depression (OR 1.13, 95% CI 1.04-1.23), depressive mood (OR 3.83, 95% CI 1.98-7.42), decreased motivation (OR 3.11, 95% CI 1.58-6.12), appetite loss (OR 4.32, 95% CI 1.54-12.07), swallowing function (OR 1.05, 95% CI 1.00-1.10), chewing difficulty (OR 2.50, 95% CI 1.31-4.75) and eating alone (OR 2.5, 95% CI 1.35-4.62)., Conclusion: Decreased opportunities for conversation was more perceived among older adults with higher daily functioning, suggesting that it is associated with depressed mood, oral function and solitary eating. Geriatr Gerontol Int 2024; 24: 385-391., (© 2024 Japan Geriatrics Society.)

Correlative fluorescent and electron microscopic images of the same section of epoxy (or other polymer)-embedded samples, hereafter referred to as 'in-resin CLEM', have been developed to improve the positional accuracy and Z-axis resolution limitations of conventional correlative light and electron microscopy (CLEM). High-pressure freezing and quick-freezing substitution result in in-resin CLEM of acrylic-based resin-embedded cells expressing green fluorescent protein, yellow fluorescent protein, mVenus and mCherry, which are sensitive to osmium tetroxide. The identification of osmium-resistant fluorescent proteins leads to the development of in-resin CLEM of Epon-embedded cells. Using subtraction-based fluorescence microscopy with a photoconvertible fluorescent protein, mEosEM-E, its green fluorescence can be observed in thin sections of Epon-embedded cells, and two-color in-resin CLEM using mEosEM-E and mScarlet-H can be performed. Green fluorescent proteins, CoGFP variant 0 and mWasabi, and far-red fluorescent proteins, mCherry2 and mKate2, are available for in-resin CLEM of Epon-embedded cells using the standard procedure for Epon-embedding with additional incubation. Proximity labeling is applied to in-resin CLEM to overcome the limitations of fluorescent proteins in epoxy resin. These approaches will contribute significantly to the future of CLEM analysis., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Astrocytes interact with not only synapses but also brain blood vessels through perivascular astrocyte endfeet (PV-AEF) to form the neurovascular unit (NVU). However, PV-AEF components have not been fully identified. Here, we biochemically isolated blood vessels from mouse brain homogenates and purified PV-AEF. The purified PV-AEF were observed in different sizes, similar to PV-AEF on brain blood vessels. Mass spectrometry analysis identified 9,762 proteins in the purified PV-AEF, including cell adhesion molecules, nectin-2δ, Kirrel2, and podoplanin. Immunofluorescence microscopic analysis revealed that nectin-2δ and podoplanin were concentrated mainly in arteries/arterioles and veins/venules of the mouse brain, whereas Kirrel2 was mainly in arteries/arterioles. Nectin-2α/δ, Kirrel2, and podoplanin were preferentially observed in large sizes of the purified PV-AEF. Furthermore, Kirrel2 potentially has cell adhesion activity of cultured astrocytes. Collectively, these results indicate that PV-AEF have heterogeneity in sizes and molecular components, implying different roles of PV-AEF in NVU function depending on vascular regions., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

In autophagy, a membrane cisterna called the isolation membrane expands, bends, becomes spherical, and closes to sequester cytoplasmic constituents into the resulting double-membrane vesicle autophagosome for lysosomal/vacuolar degradation. Here, we discover a mechanism that allows the isolation membrane to expand with a large opening to ensure non-selective cytoplasm sequestration within the autophagosome. A sorting nexin complex that localizes to the opening edge of the isolation membrane plays a critical role in this process. Without the complex, the isolation membrane expands with a small opening that prevents the entry of particles larger than about 25 nm, including ribosomes and proteasomes, although autophagosomes of nearly normal size eventually form. This study sheds light on membrane morphogenesis during autophagosome formation and selectivity in autophagic degradation., (© 2023. Springer Nature Limited.)

Endoplasmic reticulum-mitochondrial contact sites (ERMCS) play an important role in mitochondrial dynamics, calcium signaling, and autophagy. Disruption of the ERMCS has been linked to several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). However, the etiological role of ERMCS in these diseases remains unclear. We previously established tyrosine hydroxylase reporter ( TH- GFP) iPSC lines from a PD patient with a PRKN mutation to perform correlative light-electron microscopy (CLEM) analysis and live cell imaging in GFP-expressing dopaminergic neurons. Here, we analyzed ERMCS in GFP-expressing PRKN- mutant dopaminergic neurons from patients using CLEM and a proximity ligation assay (PLA). The PLA showed that the ERMCS were significantly reduced in PRKN- mutant patient dopaminergic neurons compared to the control under normal conditions. The reduction of the ERMCS in PRKN -mutant patient dopaminergic neurons was further enhanced by treatment with a mitochondrial uncoupler. In addition, mitochondrial calcium imaging showed that mitochondrial Ca 2+ flux was significantly reduced in PRKN -mutant patient dopaminergic neurons compared to the control. These results suggest a defect in calcium flux from ER to mitochondria is due to the decreased ERMCS in PRKN -mutant patient dopaminergic neurons. Our study of ERMCS using TH- GFP iPSC lines would contribute to further understanding of the mechanisms of dopaminergic neuron degeneration in patients with PRKN mutations., 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 Yokota, Yoshino, Hosoi, Hashimoto, Kakuta, Shiga, Ishikawa, Okano, Hattori, Akamatsu and Koike.)

Background: Bronchiolar adenoma/ciliated muconodular papillary tumor (BA/CMPT) is listed in the World Health Organization (WHO) Classification 5th edition as a rare benign tumor with papillary growth of ciliary, goblet, and basal cells., Case Presentation: The patient was a 67-year-old female in whom a nodular shadow of 20 mm in diameter in the right lower lobe S10 center was found in chest computed tomography (CT) for examination of dorsal pain. Positron emission tomography/computed tomography (PET-CT) showed the accumulation of 18 F-fluorodeoxyglucose (FDG) with a standardized uptake value (SUV) max of 13.0. Primary lung cancer was suspected, and surgery was scheduled as a therapeutic strategy. Thoracoscopic resection of the right lower lobe was performed, and possible BA/CMPT or adenocarcinoma was suggested in the differential diagnosis by the intra-perioperative rapid pathologic diagnosis. The final diagnosis was BA/CMPT. Histological findings indicated that the cause of the high FDG-PET scan might be due to many inflammatory cell infiltration in the tumor., Conclusions: We report a resected case of BA/CMPT with exceptionally high FDG accumulation in PET., (© 2023. The Author(s).)

In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same Epon-embedded ultrathin section. This method offers the advantage of high positional accuracy compared to standard CLEM. However, it requires the expression of recombinant proteins. In order to detect the localization of endogenous target(s) and their localized ultrastructures of Epon-embedded samples using in-resin CLEM, we investigated whether immunological and affinity-labeling using fluorescent dyes applied to in-resin CLEM of Epon-embedded cells. The orange fluorescent (λ em ∼550 nm) and far-red (λ em ∼650 nm) fluorescent dyes examined maintained a sufficient level of fluorescent intensity after staining with osmium tetroxide and subsequent dehydration treatment with ethanol. Immunological in-resin CLEM of mitochondria and the Golgi apparatus was achieved using anti-TOM20, anti-GM130 antibodies, and fluorescent dyes. Two-color in-resin CLEM revealed that wheat germ agglutinin-puncta showed the ultrastructures of multivesicular body-like structures. Finally, taking the advantage of high positional accuracy, volume in-resin CLEM of mitochondria in the semi-thin section (2 μm thick) of Epon-embedded cells was performed by focused ion beam scanning electron microscopy. These results suggested that the application of immunological reaction and affinity-labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells is suitable for analyzing the localization of endogenous targets and their ultrastructures by scanning and transmission electron microscopy., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Abnormal α-synuclein aggregation is a key pathological feature of a group of neurodegenerative diseases known as synucleinopathies, which include Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy (MSA). The pathogenic β-sheet seed conformation of α-synuclein is found in various tissues, suggesting potential as a biomarker, but few studies have been able to reliably detect these seeds in serum samples. In this study, we developed a modified assay system, called immunoprecipitation-based real-time quaking-induced conversion (IP/RT-QuIC), which enables the detection of pathogenic α-synuclein seeds in the serum of individuals with synucleinopathies. In our internal first and second cohorts, IP/RT-QuIC showed high diagnostic performance for differentiating PD versus controls (area under the curve (AUC): 0.96 (95% confidence interval (CI) 0.95-0.99)/AUC: 0.93 (95% CI 0.84-1.00)) and MSA versus controls (AUC: 0.64 (95% CI 0.49-0.79)/AUC: 0.73 (95% CI 0.49-0.98)). IP/RT-QuIC also showed high diagnostic performance in differentiating individuals with PD (AUC: 0.86 (95% CI 0.74-0.99)) and MSA (AUC: 0.80 (95% CI 0.65-0.97)) from controls in a blinded external cohort. Notably, amplified seeds maintained disease-specific properties, allowing the differentiation of samples from individuals with PD versus MSA. In summary, here we present a novel platform that may allow the detection of individuals with synucleinopathies using serum samples., (© 2023. The Author(s).)

Malperfusion of the celiac artery alone, with acute aortic dissection, rarely requires early intervention. A 57-year-old woman had thrombosed type A acute aortic dissection with celiac malperfusion, for which total arch replacement was performed after percutaneous balloon angioplasty of the celiac artery. The endovascular treatment minimized extensive hepatic infarction, which saved the patient's life., (© 2023. The Author(s).)

While occlusal status has been reported to be related to cognitive function, little is known about the influence of age on that relationship. The present study examined the associations of tooth loss and occlusal status with dementia in the older people, as well as the effects of age on those relationships. A total of 196 older participants (median age: 84 years) were enrolled. Occlusal status was assessed using functional tooth units (FTU), calculated based on the number of paired natural or artificial teeth. Logistic regression analysis was then performed using dementia as the objective variable, and FTU or number of teeth as explanatory variables. The results showed that higher FTU was associated with lower risk of dementia. Furthermore, when stratified by median age, the association was greater for those aged less than 84 years. On the other hand, there was no significant association of number of present teeth with dementia. These results suggest that the risk of dementia is lower for individuals with better occlusion and that occlusal factor may have a greater effect on dementia onset in younger older people. It is thus recommended that both occlusal function and age be incorporated as factors in programs developed for dementia prevention.

Although hair loss is not a horrible disease, it sometimes reduces the patients' quality of life (QOL) and increases their mental stress. Currently, there is no effective treatment for hair loss. It is known that honeybee propolis has various biological activities, including stimulating the proliferation of hair matrix keratinocytes. However, little is known with the hair promoting activity of stingless bee propolis. Hence, this study investigates the hair growth-promoting activity of Philippines stingless bee propolis extract and the underlying a molecular mechanism of promoting hair growth. For the evaluation of hair growth stimulating activity, 99.5% ethanolic extract of Philippines stingless bee propolis is examined using the simple shaving model in C57BL/6N mice. Melaninization of dorsal skin and histological analysis of hair follicles (HFs) revealed that propolis promotes hair growth by stimulating HFs development. The expression of mRNA (Wnt3a, Ctnnb1/β-catenin, Lef1, and Bmp2) and protein (WNT3A and β-catenin) of selected Wnt/β-catenin associated genes explains Philippines stingless bee propolis promoting HFs development by activating Wnt/β-catenin signaling pathway. These results suggest that the treatment of propolis strongly promotes hair growth by stimulating the development of HFs via activation of Wnt/β-catenin signaling pathway. This further indicates the potential of Philippines stingless bee propolis as a novel promising agricultural product for hair growth.

Postfixation with osmium tetroxide and Epon embedding are essential for the preservation and visualization of subcellular ultrastructures via electron microscopy. These chemical treatments diminish the fluorescent intensity of most fluorescent proteins in cells, creating a problem for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded mammalian cultured cells. We found that two green and two far-red fluorescent proteins retain their fluorescence after chemical fixation with glutaraldehyde, osmium tetroxide-staining, dehydration, and polymerization of Epon resins. Consequently, we could observe the fluorescence of fluorescent proteins in ultrathin sections of Epon-embedded cells via fluorescence microscopy, investigate ultrastructures of the cells in the same sections via electron microscopy, and correlate the fluorescent image with the electron microscopic image without chemical or physical distortion of the cells. In other words, referred as "in-resin CLEM" of Epon-embedded samples. This technique also improves the Z-axis resolution of fluorescent images. In this chapter, we introduce the detailed protocol for in-resin CLEM of Epon-embedded mammalian cultured cells using these fluorescent proteins., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novel genome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Priority substances likely to pollute water can be characterized by mid-infrared spectroscopy based on their specific absorption spectral signature. In this work, the detection of volatile aromatic molecules in the aqueous phase by evanescent-wave spectroscopy has been optimized to improve the detection efficiency of future in situ optical sensors based on chalcogenide waveguides. To this end, a hydrophobic polymer was deposited on the surface of a zinc selenide prism using drop and spin-coating methods. To ensure that the water absorption bands will be properly attenuated for the selenide waveguides, two polymers were selected and compared: polyisobutylene and ethylene/propylene copolymer coating. The system was tested with benzene, toluene, and ortho-, meta-, and para-xylenes at concentrations ranging from 10 ppb to 40 ppm, and the measured detection limit was determined to be equal to 250 ppb under these analytical conditions using ATR-FTIR. The polyisobutylene membrane is promising for pollutant detection in real waters due to the reproducibility of its deposition on selenide materials, the ease of regeneration, the short response time, and the low ppb detection limit, which could be achieved with the infrared photonic microsensor based on chalcogenide materials. To improve the sensitivity of future infrared microsensors, the use of metallic nanostructures on the surface of chalcogenide waveguides appears to be a relevant way, thanks to the plasmon resonance phenomena. Thus, in addition to preliminary surface-enhanced infrared absorption tests using these materials and a functionalization via a self-assembled monolayer of 4-nitrothiophenol, heterostructures combining gold nanoparticles/chalcogenide waveguides have been successfully fabricated with the aim of proposing a SEIRA microsensor device., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Objective: To determine whether periodontal status is associated with oral function, including masticatory performance and occlusal force, among community-dwelling older adults., Background: Although a potential association between periodontal status and oral function has been reported, variations in the root surface area (RSA) of each tooth have not been considered., Methods: We used data from a population-based study involving community-dwelling older adults. The RSA with periodontal ligament (RSA-PL), which quantifies the RSA attached to the periodontal ligament and alveolar bone, was calculated based on full-mouth periodontal examination. Masticatory performance was assessed using spectrophotometric measurement of the color (a* value) of color-changing chewing gum. The bilateral maximal occlusal force (MOF) was measured using a pressure-sensitive sheet and dedicated software. The associations of the RSA-PL, a quantitative marker of periodontal tissue support, with the a* value and MOF were assessed using linear regression models., Results: The analyses included data from 250 adults [60.8% women; age, mean (standard deviation) 82.5 (5.0) years]. On average, the study participants had an RSA-PL of 26.3 cm 2 , a* value of 25.0, and an MOF value of 555.1 N. After adjustments for potential confounders, including dentition status, age, sex, dental visit regularity, smoking status, physical activity level, depressive symptoms, a history of stroke or diabetes mellitus, and body mass index, the RSA-PL was found to be associated with the a* value [coefficient (per 1 cm 2 increase): 0.16, 95% confidence interval (CI) 0.10-0.22)] and MOF (coefficient: 9.2, 95% CI 5.3-13.1)., Conclusion: This study demonstrated that greater amounts of remaining tooth-supporting structures, indicated by higher RSA-PL values, were associated with better masticatory performance and a higher occlusal force among community-dwelling older adults., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Retrograde transport of lysosomes is recognised as a critical autophagy regulator. Here, we found that acrolein, an aldehyde that is significantly elevated in Parkinson's disease patient serum, enhances autophagy by promoting lysosomal clustering around the microtubule organising centre via a newly identified JIP4-TRPML1-ALG2 pathway. Phosphorylation of JIP4 at T217 by CaMK2G in response to Ca 2+ fluxes tightly regulated this system. Increased vulnerability of JIP4 KO cells to acrolein indicated that lysosomal clustering and subsequent autophagy activation served as defence mechanisms against cytotoxicity of acrolein itself. Furthermore, the JIP4-TRPML1-ALG2 pathway was also activated by H 2 O 2 , indicating that this system acts as a broad mechanism of the oxidative stress response. Conversely, starvation-induced lysosomal retrograde transport involved both the TMEM55B-JIP4 and TRPML1-ALG2 pathways in the absence of the JIP4 phosphorylation. Therefore, the phosphorylation status of JIP4 acts as a switch that controls the signalling pathways of lysosoma l distribution depending on the type of autophagy-inducing signal., (©2022 The Authors. Published under the terms of the CC BY 4.0 license.)

HSF (heat shock transcription factor or heat shock factor) was discovered as a transcription factor indispensable for heat shock response. Although four classical HSFs were discovered in mammals and two major HSFs, HSF1 and HSF2, were cloned in the same year of 1991, only HSF1 was intensively studied because HSF1 can give rise to heat shock response through the induction of various HSPs' expression. On the other hand, HSF2 was not well studied for some time, which was probably due to an underestimate of HSF2 itself. Since the beginning of the 21st century, HSF2 research has progressed and many biologically significant functions of HSF2 have been revealed. For example, the roles of HSF2 in nervous system protection, inflammation, maintenance of mitosis and meiosis, and cancer cell survival and death have been gradually unveiled. However, we feel that the fact HSF2 has a relationship with various factors is not yet widely recognized; therefore, the biological significance of HSF2 has been underestimated. We strongly hope to widely communicate the significance of HSF2 to researchers and readers in broad research fields through this review. In addition, we also hope that many readers will have great interest in the molecular mechanism in which HSF2 acts as an active transcription factor and gene bookmarking mechanism of HSF2 during cell cycle progression, as is summarized in this review.

Approximately 20% of the community-dwelling Japanese elderly (≥65 years) experience falling annually, with injury frequency rising with age. Increased nursing home admission/hospitalization risk influences healthy aging and QOL. Nutrition for musculoskeletal health is necessary, though the relationship of falling with nutritional status in the elderly is largely unknown. We investigated falling incidents and nutritional status, including a Japanese-style diet in a community-dwelling cohort. Using a cross-sectional design, 186 subjects (median age 83.0 years, males/females 67/119) were analyzed. Oral and systemic health conditions were assessed. A brief-type self-administered diet history questionnaire (BDHQ) was given for nutritional status. Analysis of covariance (adjusted for gender, age, BMI, articular disease/osteoporosis history, present tooth number, educational level) and the Japanese-Mediterranean diet (jMD) score adapted for Japan were used. The jMD score and falling incidents were significantly associated, with point increases related to a significantly decreased falling risk of 28% (OR: 0.72; 95%CI: 0.57−0.91). Of the 13 jMD food components, fish, eggs, and potatoes had a significant relationship with reduced falling, while significant associations of intake of animal protein, potassium, magnesium, zinc, and cholesterol (p < 0.05) were also observed. The results suggest that the jMD dietary pattern is an important factor for the prevention of falling incidents in elderly individuals.

Internet addiction (IA) negatively affects adolescents' lifestyle behaviors. Inappropriate lifestyle behaviors could have negative effects on dental health. This cross-sectional study aimed to test whether IA was indirectly associated with dental caries through unhealthy lifestyle behaviors among high school students in southwest Japan. IA was characterized by a Young's Internet Addiction Test score of ≥ 50, unhealthy lifestyle behaviors by a cumulative count of 8 different lifestyle behaviors (termed the unhealthy lifestyle behavior index [ULBI]), and dental caries by the number of decayed, missing, and filled permanent teeth (DMFT). Poisson regression and linear regression models were fitted to the relationship, with IA as the exposure, the ULBI as the mediator, and the DMFT as the outcome. The natural indirect effect (NIE) and the proportion mediated by the ULBI were estimated by performing a mediation analysis. Overall, 1562 high school students were included. IA was observed in 406 participants and was associated with a larger DMFT. The ULBI significantly mediated the association between IA and the DMFT (NIE: incidence rate ratio = 1.05, 95% confidence interval = 1.03-1.07, proportion mediated = 64.3%). Dental caries was more common in our cohort of high school students with IA, which is partially explained by these students having unhealthy lifestyle behaviors., (© 2022. The Author(s).)

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite Plasmodium berghei also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.

Aim: To clarify older adults' preferences for and actual situations of artificial hydration and nutrition (AHN) in end-of-life care in a care home., Methods: Participants were residents of a care home who had completed advance directives regarding preferred methods of AHN from 2009 to 2018. Advance directives alone were available from April 2009 to June 2016 (Wave 1), and advance care planning for AHN including advance directives was introduced in July 2016 (Wave 2). AHN preferences included (i) intensive methods (percutaneous endoscopic gastrostomy, nasogastric tube feeding and total parenteral nutrition), (ii) drip infusion, and (iii) oral intake only. Participants were followed until the end of 2020, and we checked whether decisions about AHN were based on older adults' preferences., Results: In total, 272 participants had completed advance directives. Most participants preferred "oral intake only" (59.5%), followed by drip infusion (32.0%) and intensive methods (8.5%) in advance directives. Ninety of the 272 participants completed advance directives twice; 83.3% did not change their AHN preferences from Wave 1 to Wave 2. By the end of 2020, 93 of the 272 participants died in the care home. AHN was provided according to older adults' preferences in 48.9% (oral intake only), in 51.4% (drip infusion) and in 55.6% (intensive methods) of cases respectively., Conclusions: Most participants preferred oral intake only, and their preferences were reflected in decisions about actual situations of AHN in end-of-life care. To prepare for advanced dementia and senility, early advance care planning for AHN should be promoted. Geriatr Gerontol Int 2022; 22: 581-587., (© 2022 The Authors. Geriatrics & Gerontology International published by John Wiley & Sons Australia, Ltd on behalf of Japan Geriatrics Society.)

Neuronal ceroid lipofuscinosis is one of many neurodegenerative storage diseases characterized by excessive accumulation of lipofuscins. CLN10 disease, an early infantile neuronal ceroid lipofuscinosis, is associated with a gene that encodes cathepsin D (CtsD), one of the major lysosomal proteases. Whole body CtsD-knockout mice show neurodegenerative phenotypes with the accumulation of lipofuscins in the brain and also show defects in other tissues including intestinal necrosis. To clarify the precise role of CtsD in the central nervous system (CNS), we generated a CNS-specific CtsD-knockout mouse (CtsD-CKO). CtsD-CKO mice were born normally but developed seizures and their growth stunted at around postnatal day 23 ± 1. CtsD-CKO did not exhibit apparent intestinal symptoms as those observed in whole body knockout. Histologically, autofluorescent materials were detected in several areas of the CtsD-CKO mouse's brain, including: thalamus, cerebral cortex, hippocampus, and cerebellum. Expression of ubiquitin and autophagy-associated proteins was also increased, suggesting that the autophagy-lysosome system was impaired. Microglia and astrocytes were activated in the CtsD-CKO thalamus, and inducible nitric oxide synthase (iNOS), an inflammation marker, was increased in the microglia. Interestingly, deposits of proteinopathy-related proteins, phosphorylated α-synuclein, and Tau protein were also increased in the thalamus of CtsD-CKO infant mice. Considering these results, we propose thatt the CtsD-CKO mouse is a useful mouse model to investigate the contribution of cathepsin D to the early phases of neurodegenerative diseases in relation to lipofuscins, proteinopathy-related proteins and activation of microglia and astrocytes., (© 2022. The Author(s).)

Biotin ligases have been developed as proximity biotinylation enzymes for analyses of the interactome. However, there has been no report on the application of proximity labeling for in-resin correlative light-electron microscopy of Epon-embedded cells. In this study, we established a proximity-labeled in-resin CLEM of Epon-embedded cells using miniTurbo, a biotin ligase. Biotinylation by miniTurbo was observed in cells within 10 min following the addition of biotin to the medium. Using fluorophore-conjugated streptavidin, intracellular biotinylated proteins were labeled after fixation of cells with a mixture of paraformaldehyde and glutaraldehyde. Fluorescence of these proteins was resistant to osmium tetroxide staining and was detected in 100-nm ultrathin sections of Epon-embedded cells. Ultrastructures of organelles were preserved well in the same sections. Fluorescence in sections was about 14-fold brighter than that in the sections of Epon-embedded cells expressing mCherry2 and was detectable for 14 days. When mitochondria-localized miniTurbo was expressed in the cells, mitochondria-like fluorescent signals were detected in the sections, and ultrastructures of mitochondria were observed as fluorescence-positive structures in the same sections by scanning electron microscopy. Proximity labeling using miniTurbo led to more stable and brighter fluorescent signals in the ultrathin sections of Epon-embedded cells, resulting in better performance of in-resin CLEM., (© 2022. The Author(s).)

The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscopy/electron microscopy (LM/EM) imaging with a glutaraldehyde-resistant tissue clearing method, Sca l eSF. Our multi-scale neuronal imaging incorporates (1) brain-wide macroscopic observation, (2) mesoscopic circuit mapping, (3) microscopic subcellular imaging, and (4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied this technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Objective　Continuous oral health care and regular dental visits are important for maintaining oral health. As a result of the high communicability of the coronavirus disease (COVID-19), individuals are reluctant to visit medical institutions. It is as yet unclear how the oral health of the population has been affected by the interruption of regular medical services during the COVID-19 pandemic. This study aimed to examine the association between the status of regular dental visits and changes in oral health among high school students during the pandemic using data from routine medical checkups conducted at a school, as well as data obtained from a questionnaire-based survey conducted simultaneously with medical checkups.Methods　This study included 878 participants (comprising freshmen and sophomores) during the 2019 academic year from a high school in Fukuoka prefecture, Japan. Data on the frequency of dental checkups and anxiety related to dental visits during the pandemic was obtained through a questionnaire-based survey. Changes in dental health and gingival status between 2019 and 2020 were determined using data from routine medical checkups at the school. Poisson regression analysis with robust standard errors was used to evaluate the association between changes in dental health and gingival status and the status of regular dental visits.Results　Of 878 participants, 417 (47.5%) did not have regular dental visits, 320 (36.4%) continued to have regular dental visits, and 141 (16.1%) had interrupted regular dental visits during the pandemic. In the interrupted regular dental visit group, 30.5% of participants were anxious about visiting a dental institution, which was a significantly higher proportion than other groups. Of the 521 participants who did not have gingival inflammation at the time of dental checkup in 2019, gingival inflammation at the time of dental checkup in 2020 was observed in 31.0% of participants having no regular dental visits, 20.2% participants having regular dental visits, and 38.2% participants having interrupted regular dental visits. The multivariable adjusted incidence rate ratios were 1.95 [95% confidence interval (CI), 1.34-2.84] in the interrupted regular dental visit group and 1.50 (95%CI,1.07-2.10) in the no regular dental visit group. There was no significant association between interruption of regular dental visits and changes in dental health status.Conclusion　The study results showed that there was a significant association between interruptions in regular dental visits and anxiety related to dental visits during the COVID-19 pandemic, and that a higher proportion of participants who had interruptions in regular dental visits had gingival inflammation at the time of the medical checkup at the school.

Aim: Recent studies have reported that the albumin-to-globulin ratio (AGR) may be a useful inflammatory-nutritional biomarker to predict postoperative complications and poor prognosis in various types of patients with cancer. However, its prognostic value in patients with esophageal cancer is still unclear. We aimed to examine the utility of the AGR for predicting the short- and long-term outcomes in patients with esophageal cancer who underwent curative resection., Patients and Methods: This was a retrospective cohort analysis reviewing the medical records of consecutive patients who underwent esophagectomy for clinical stage I to III esophageal cancer at Yokohama City University. A total of 105 patients were identified between 2005 and 2018. The overall survival (OS), recurrence-free survival (RFS), and postoperative complication rates were compared between patients with high AGR (>1.48) and those with low AGR (≤1.48) group., Results: A total of 57 and 48 patients were classified into the high and low AGR groups, respectively. There was no significant difference between the two groups in the rate of overall postoperative complications of more than Clavien-Dindo grade 3 (50.9% vs. 54.2%, p=0.85). The long-term findings showed that 5-year OS and RFS rates were significantly better for the group with a high AGR (67.2% vs. 33.8%, p<0.001 and 51.6% vs. 28.5%, p=0.003, respectively)., Conclusion: This study suggests that a low preoperative AGR is a risk factor for poor RFS and OS in patients who are planning to undergo curative surgery for esophageal cancer. AGR may be a useful biomarker for establishing treatment strategies to improve patients' survival., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Intestinal inflammation can be accompanied by osteoporosis, but their relationship, mediated by immune responses, remains unclear. Here, we investigated a non-IgE-mediated food-allergic enteropathy model of ovalbumin (OVA) 23-3 mice expressing OVA-specific T-cell-receptor transgenes. Mesenteric lymph nodes (MLNs) and their pathogenic CD4 + T cells were important to enteropathy occurrence and exacerbation when the mice were fed an egg-white (EW) diet. EW-fed OVA23-3 mice also developed bone loss and increased CD44 hi CD62L lo CD4 + T cells in the MLNs and bone marrow (BM); these changes were attenuated by MLN, but not spleen, resection. We fed an EW diet to F1 cross offspring from OVA23-3 mice and a mouse line expressing the photoconvertible protein KikGR to track MLN CD4 + T cells. Photoconverted MLN CD44 hi CD62L lo CD4 + T cells migrated predominantly to the BM; pit formation assay proved their ability to promote bone damage via osteoclasts. Significantly greater expression of IL-4 mRNA in MLN CD44 hi CD62L lo CD4 + T cells and bone was observed in EW-fed OVA23-3 mice. Anti-IL-4 monoclonal antibody injection canceled bone loss in the primary inflammation phase in EW-fed mice, but less so in the chronic phase. This novel report shows the specific inflammatory relationship, via Th2-dominant-OVA-specific T cells and IL-4 production, between MLNs and bone, a distant organ, in food-allergic enteropathy., (© 2021. The Author(s), under exclusive licence to Society for Mucosal Immunology.)