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Essential amino acids (EAAs) are important for the maintenance of brain functions. Therefore, the yeast Saccharomyces cerevisiae that accumulate EAAs would help elderly people ingest appropriate levels of EAAs, which in turn could slow neurodegeneration, extend the healthy lifespan and improve quality of life. Here, we isolated two mutant strains, ETH-80 and ETH-129, that accumulate the EAA methionine. Both strains were derived from a diploid laboratory yeast by conventional mutagenesis and carry a novel mutation in the MET13 gene, which encodes the Ser443Phe variant of methylenetetrahydrofolate reductase. Enzymatic analysis revealed that the Ser443Phe substitution abolished the sensitivity to S-adenosyl methionine (SAM)-mediated inhibition even in the presence of 2 mM SAM, while increasing the activity for NADPH-dependent reduction. Furthermore, yeast cells expressing the Ser443Phe variant showed a fourfold increase in intracellular methionine content compared to the wild-type Met13. These findings will be useful for the future development of methionine-accumulating yeast strains., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

A high-fat diet (HFD) causes inflammation with an increase in microglial activity in the hypothalamic arcuate nucleus (ARC) and ventral tegmental area (VTA), resulting in insulin resistance in both regions. This leads to a deterioration in glucose and energy metabolism. The effect of voluntary exercise on HFD-induced inflammation in the central nervous system (CNS) remains unclear. To clarify the effects of voluntary exercise on the CNS, 8-week-old male C57BL6 mice were fed a chow diet (CHD) or HFD for 4 weeks; each group was further divided into running exercise (EX+) on a wheel and no exercise (EX-) groups. The expression of the inflammatory cytokine, tumor necrosis factor alpha (TNFα), in the ARC and VTA was significantly increased in the HFD/EX- group, with an increase of microglial activity noted, compared to the CHD/EX- group. The expression of TNFα was significantly suppressed, with a decrease of microglial activity, in the HFD/EX+ compared to HFD/EX- group. Insulin resistance in the ARC and VTA was improved with the suppression of TNFα expression. The HFD/EX- group showed significant weight gain and impaired glucose metabolism compared to the CHD/EX- group. The HFD/EX+ group showed an improvement in glucose and energy metabolism compared to the HFD/EX- group. In addition, voluntary wheel running suppressed HFD-induced inflammation in the ARC, with a decrease in microglial activity observed independently of weight changes. Our data suggest that voluntary exercise prevents obesity and improves glucose metabolism by suppressing inflammation in the ARC and VTA under HFD conditions., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

The precise onset of flowering is crucial to ensure successful plant reproduction. The gene FLOWERING LOCUS T ( FT ) encodes florigen, a mobile signal produced in leaves that initiates flowering at the shoot apical meristem. In response to seasonal changes, FT is induced in phloem companion cells located in distal leaf regions. Thus far, a detailed molecular characterization of the FT -expressing cells has been lacking. Here, we used bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate gene expression in FT -expressing cells and other phloem companion cells. Our bulk nuclei RNA-seq demonstrated that FT -expressing cells in cotyledons and in true leaves differed transcriptionally. Within the true leaves, our snRNA-seq analysis revealed that companion cells with high FT expression form a unique cluster in which many genes involved in ATP biosynthesis are highly upregulated. The cluster also expresses other genes encoding small proteins, including the flowering and stem growth inducer FPF1-LIKE PROTEIN 1 (FLP1) and the anti-florigen BROTHER OF FT AND TFL1 (BFT). In addition, we found that the promoters of FT and the genes co-expressed with FT in the cluster were enriched for the consensus binding motifs of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of the paralogous NIGT1.2 and NIGT1.4 repressed FT expression and significantly delayed flowering under nitrogen-rich conditions, consistent with NIGT1s acting as nitrogen-dependent FT repressors. Taken together, our results demonstrate that major FT -expressing cells show a distinct expression profile that suggests that these cells may produce multiple systemic signals to regulate plant growth and development., Competing Interests: Competing Interest Statement: The authors declare no competing interest.

Purpose: The purpose of this study was to examine the location where the femoral artery contacts the vastus medialis at the adductor tendon hiatus, which is important when using the subvastus approach in medial closed wedge distal femoral osteotomy. We evaluated the correlation between differences in height, vastus medialis morphology, and lower limb alignment., Methods: Sixty knees (16 male, 44 female) that underwent plain computer tomography (CT) were included. Using the radiographic hip-knee-ankle (HKA) angle as a reference, the knees were divided into three groups of 20 knees: valgus, varus, and neutral. The mechanical lateral distal femoral angle (mLDFA) and distance from the medial femoral epicondyle to the centre of the femoral head (D1) were measured on full-length weight-bearing anteroposterior radiographs. The first cross-section on CT where the vastus medialis muscle and femoral artery connect was defined as the cross-sectional image for measurement. The direct distance from the medial epicondyle to the measured cross-sectional image (D2) was measured in the coronal view. The ratio of the vastus medialis muscle width to the femoral posterior wall width was defined as the vastus medialis muscle coverage ratio (CR). Correlations between each measurement and group were evaluated., Results: There was a positive correlation between D1 and D2 in the overall, neutral, and varus groups; however, there was no correlation in the valgus group. A positive correlation was observed in terms of the relationship between CR and D2 in the overall and valgus groups. In addition, there was no statistically significant difference in the correlation between the mLDFA and D2, with patient height as a control variable overall and in all groups., Conclusion: In the valgus group, distance to the adductor hiatus was correlated with vastus medialis coverage. Overhang of the vastus medialis may be an important influencing factor of femoral and popliteus artery position., Level of Evidence: Level III, retrospective cohort study., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Journal of Experimental Orthopaedics published by John Wiley & Sons Ltd on behalf of European Society of Sports Traumatology, Knee Surgery and Arthroscopy.)

To the best of our knowledge, enzymes that catalyse intramolecular Diels-Alder ([4+2] cycloaddition) reactions are frequently reported in natural product biosynthesis; however, no native enzymes utilising Lewis acid catalysis have been reported. Verticilactam is a representative member of polycyclic macrolactams, presumably produced by spontaneous cycloaddition. We report that the intramolecular [4+2] cycloadditions can be significantly accelerated by ferredoxins (Fds), a class of small iron-sulphur (Fe-S) proteins. Through iron atom substitution by Lewis acidic gallium (Ga) iron and computational calculations, we confirm that the ubiquitous Fe-S cluster efficiently functions as Lewis acid to accelerate the tandem [4+2] cycloaddition and Michael addition reactions by lowering free energy barriers. Our work highlights Nature's ingenious strategy to generate complex molecule structures using the ubiquitous Fe-S protein. Furthermore, our study sheds light on the future design of Fd as a versatile Lewis acid catalyst for [4+2] cycloaddition reactions., (© 2024. The Author(s).)

The arginine transporter Can1 is a multifunctional protein of the conventional yeast Saccharomyces cerevisiae . Apart from facilitating arginine uptake, Can1 plays a pivotal role in regulating proline metabolism and maintaining cellular redox balance. Here, we report a novel function of Can1 in the control of yeast biofilm formation. First, the S. cerevisiae CAN1 gene knockout strain displayed a significant growth delay compared to the wild-type strain. Our genetic screening revealed that the slow growth of the CAN1 knockout strain is rescued by a functional deficiency of the FLO8 gene, which encodes the master transcription factor associated with biofilm formation, indicating that Can1 is involved in biofilm formation. Intriguingly, the CAN1 knockout strain promoted the Flo11-dependent aggregation, leading to higher biofilm formation. Furthermore, the CAN1 knockout strain of the pathogenic yeast Candida glabrata exhibited slower growth and higher biofilm formation, similar to S. cerevisiae . More importantly, the C. glabrata CAN1 gene knockout strain showed severe toxicity to macrophage-like cells and nematodes. The present results could help to elucidate both the molecular mechanism underlying yeast biofilm formation and the role it plays. Future investigations may offer insights that contribute to development of antibiofilm agents., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Nishimura, Tanahashi, Nakagami, Morioka and Takagi.)

Objective: To examine communication in home medical care., Methods: Conversations that happened during home medical care involving physicians at nine clinics were recorded and analyzed using the Roter Interaction Analysis System (RIAS). Additional categories were developed to code aspects of home medical care., Results: Overall, 55 conversations were analyzed. The mean age of the patients was 82.9 ± 10.1 years old. The most common triad was physician, patient, and patient's companion. Information about home medical care professionals who were not present during the conversation was provided by the physician in 21 cases (38.2%), the patient in nine cases (16.4%), and companions in 21 (39.6%) cases., Conclusion: In home medical care, the participants mentioned home medical care professions who were not present at the time, suggesting that these conversations may have facilitated interprofessional collaboration., Practice Implications: Physicians should be aware that during home medical care, the presence of multiple attendants and other medical professionals contributing to communicate with the patient., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Acetic acid is a common inhibitor present in lignocellulose hydrolysate, which inhibits the ethanol production by yeast strains. Therefore, the cellulosic ethanol industry requires yeast strains that can tolerate acetic acid stress. Here we demonstrate that overexpressing a yeast native arginase-encoding gene, CAR1 , renders Saccharomyces cerevisiae acetic acid tolerance. Specifically, ethanol yield increased by 27.3% in the CAR1 -overexpressing strain compared to the control strain under 5.0 g/L acetic acid stress. The global intracellular amino acid level and compositions were further analyzed, and we found that CAR1 overexpression reduced the total amino acid content in response to acetic acid stress. Moreover, the CAR1 overexpressing strain showed increased ATP level and improved cell membrane integrity. Notably, we demonstrated that the effect of CAR1 overexpression was independent of the spermidine and proline metabolism, which indicates novel mechanisms for enhancing yeast stress tolerance. Our studies also suggest that CAR1 is a novel genetic element to be used in synthetic biology of yeast for efficient production of fuel ethanol., 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., (© 2024 The Authors.)

Streptomyces sp. SN-593, a reveromycin producer, was previously thought to belong to the genus Streptomyces based on its morphological and chemotaxonomic characteristics. In this paper, we re-considered its taxonomic position according to the current criteria. Phylogenetic analysis using 16S rRNA gene sequences showed that the strain belongs to the genus Actinacidiphila. In multilocus sequence and phylogenomic analyses, the strain SN-593 represented a distinct evolutionary lineage within this genus, and its closest neighbor was A. yanglinensis. Digital DNA-DNA hybridization indicated that the strain shares less than 32% DNA-DNA relatedness with the type strains of closely related species, confirming this strain is a novel genomospecies. According to its phenotypic distinctiveness from the closest neighbor, we propose Actinacidiphila reveromycinica sp. nov. for the strain SN-593T. Additionally, as Streptomyces acidipaludis belonged to the genus Actinacidiphila in these analyses, it should be transferred to the genus, for which Actinacidiphila acidipaludis comb. nov. is proposed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Humans and mammals need to ingest essential amino acids (EAAs) for protein synthesis. In addition to their importance as nutrients, EAAs are involved in brain homeostasis. However, elderly people are unable to efficiently consume EAAs from their daily diet due to reduced appetite and variations in the contents of EAAs in foods. On the other hand, strains of the yeast Saccharomyces cerevisiae that accumulate EAAs would enable elderly people to intakegest adequate amounts of EAAs and thus might slow down the neurodegenerative process, contributing to the extension of their healthy lifespan. In this study, we isolated a mutant (strain HNV-5) that accumulates threonine, an EAA, derived from a diploid laboratory yeast by conventional mutagenesis. Strain HNV-5 carries a novel mutation in the HOM3 gene encoding the Ala462Thr variant of aspartate kinase (AK). Enzymatic analysis revealed that the Ala462Thr substitution significantly decreased the sensitivity of AK activity to threonine feedback inhibition even in the presence of 50 mM threonine. Interestingly, Ala462Thr substitution did not affect the catalytic ability of Hom3, in contrast to previously reported amino acid substitutions that resulted in reduced sensitivity to threonine feedback inhibition. Furthermore, yeast cells expressing the Ala462Thr variant showed an approximately threefold increase in intracellular threonine content compared to that of the wild-type Hom3. These findings will be useful for the development of threonine-accumulating yeast strains that may improve the quality of life in elderly people.IMPORTANCEFor humans and mammals, essential amino acids (EAAs) play an important role in maintaining brain function. Therefore, increasing the intake of EAAs by using strains of the yeast Saccharomyces cerevisiae that accumulate EAAs may inhibit neurodegeneration in elderly people and thus contribute to extending healthy lifespan and improving their quality of life. Threonine, an EAA, is synthesized from aspartate. Aspartate kinase (AK) catalyzes the first step in threonine biosynthesis and is subject to allosteric regulation by threonine. Here, we isolated a threonine-accumulating mutant of S. cerevisiae by conventional mutagenesis and identified a mutant gene encoding a novel variant of AK. In contrast to previously isolated variants, the Hom3 variant exhibited AK activity that was insensitive to feedback inhibition by threonine but retained its catalytic ability. This resulted in increased production of threonine in yeast. These findings open up the possibility for the rational design of AK to increase threonine productivity in yeast., Competing Interests: The authors declare no conflict of interest.

Transient anoxia causes amnesia and neuronal death. This is attributed to enhanced glutamate release and modeled as anoxia-induced long-term potentiation (aLTP). aLTP is mediated by glutamate receptors and nitric oxide (·NO) and occludes stimulation-induced LTP. We identified a signaling cascade downstream of ·NO leading to glutamate release and a glutamate-·NO loop regeneratively boosting aLTP. aLTP in entothelial ·NO synthase (eNOS)-knockout mice and blocking neuronal NOS (nNOS) activity suggested that both nNOS and eNOS contribute to aLTP. Immunostaining result showed that eNOS is predominantly expressed in vascular endothelia. Transient anoxia induced a long-lasting Ca 2+ elevation in astrocytes that mirrored aLTP. Blocking astrocyte metabolism or depletion of the NMDA receptor ligand D-serine abolished eNOS-dependent aLTP, suggesting that astrocytic Ca 2+ elevation stimulates D-serine release from endfeet to endothelia, thereby releasing ·NO synthesized by eNOS. Thus, the neuro-glial-endothelial axis is involved in long-term enhancement of glutamate release after transient anoxia., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Arginine is a proteinogenic amino acid that organisms additionally exploit both for nitrogen storage and as a stress protectant. The location of arginine, whether intra- or extracellular, is important in maintaining physiological homeostasis. Here, we identified an arginine transporter ortholog of the emerging fungal pathogenic Candida glabrata. Blast searches revealed that the C. glabrata genome contains two potential orthologs of the Saccharomyces cerevisiae arginine transporter gene CAN1 (CAGL0J08162g and CAGL0J08184g). We then found that CAGL0J08162g is stably located on the plasma membrane and performs cellular uptake of arginine. Moreover, CAGL0J08162-disrupted cells of C. glabrata showed a partial resistance to canavanine, a toxic analog of arginine. Our data suggest that CAGL0J08162g is a key arginine transporter in the pathogenic C. glabrata (CgCan1).

Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal phosphate (PLP)-dependent biosynthesis of cysteine persulfide (CysSSH) and related supersulfides by cysteinyl-tRNA synthetase (CARS). Here, we investigated the physiological role of CysSSH in budding yeast (Saccharomyces cerevisiae) by generating a PLP-binding site mutation K109A in CRS1 (the yeast ortholog of CARS), which decreased the synthesis of CysSSH and related supersulfides and also led to reduced chronological aging, effects that were associated with an increased endoplasmic reticulum stress response and impaired mitochondrial bioenergetics. Reduced chronological aging in the K109A mutant could be rescued by using exogenous supersulfide donors. Our findings indicate important roles for CARS in the production and metabolism of supersulfides-to mediate mitochondrial function and to regulate longevity., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Recent studies have shown a role of inflammation in muscle atrophy and sarcopenia. However, no anti-inflammatory pharmacotherapy has been established for the treatment of sarcopenia. Here, we investigate the potential role of PPARα and its ligands on inflammatory response and PGC-1α gene expression in LPS-treated C2C12 myotubes. Knockdown of PPARα, whose expression was upregulated upon differentiation, augmented IL-6 or TNFα gene expression. Conversely, PPARα overexpression or its activation by ligands suppressed 2-h LPS-induced cytokine expression, with pemafibrate attenuating NF-κB or STAT3 phosphorylation. Of note, reduction of PGC-1α gene expression by LPS treatment for 24 hours was partially reversed by fenofibrate. Our data demonstrate a critical inhibitory role of PPARα in inflammatory response of C2C12 myotubes and suggest a future possibility of PPARα ligands as a candidate for anti-inflammatory therapy against sarcopenia., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tomohiro Tanaka reports a relationship with Kowa Company Ltd that includes: speaking and lecture fees. All the other 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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: In recent years, technologies promoting the digitization of self-monitoring of blood glucose (SMBG) records including app-cloud cooperation systems have emerged. Studies combining these technological interventions with support from remote health care professionals have reported improvements in glycemic control., Objective: To assess the use of an app-cloud cooperation system linked with SMBG devices in clinical settings, we evaluated its effects on outpatient management of diabetes without remote health care professional support., Methods: In this multicenter, open-label, and single-armed prospective study, 48 patients with diabetes (including type 1 and type 2) at 3 hospitals in Japan treated with insulin or glucagon-like peptide 1 receptor agonists and performing SMBG used the app-cloud cooperation system for 24 weeks. The SMBG data were automatically uploaded to the cloud via the app. The patients could check their data, and their attending physicians reviewed the data through the cloud prior to the patients' regular visits. The primary outcome was changes in glycated hemoglobin (HbA 1c ) levels., Results: Although HbA 1c levels did not significantly change in all patients, the frequency of daily SMBG following applying the system was significantly increased before induction at 12 (0.60 per day, 95% CI 0.19-1.00; P=.002) and 24 weeks (0.43 per day, 95% CI 0.02-0.84; P=.04). In the subset of 21 patients whose antidiabetic medication had not been adjusted during the intervention period, a decrease in HbA 1c level was observed at 12 weeks (P=.02); however, this significant change disappeared at 24 weeks (P=.49). The Diabetes Treatment Satisfaction Questionnaire total score and "Q4: convenience" and "Q5: flexibility" scores significantly improved after using the system (all P<.05), and 72% (33/46) patients and 76% (35/46) physicians reported that the app-cloud cooperation system helped them adjust insulin doses., Conclusions: The digitization of SMBG records and sharing of the data by patients and attending physicians during face-to-face visits improved self-management in patients with diabetes., Trial Registration: Japan Registry of Clinical Trials (jRCT) jRCTs042190057; https://jrct.niph.go.jp/en-latest-detail/jRCTs042190057., (©Tomoko Handa, Takeshi Onoue, Tomoko Kobayashi, Ryutaro Maeda, Keigo Mizutani, Ayana Yamagami, Tamaki Kinoshita, Yoshinori Yasuda, Shintaro Iwama, Takashi Miyata, Mariko Sugiyama, Hiroshi Takagi, Daisuke Hagiwara, Hidetaka Suga, Ryoichi Banno, Yoshinori Azuma, Takatoshi Kasai, Shuko Yoshioka, Yachiyo Kuwatsuka, Hiroshi Arima. Originally published in JMIR Diabetes (https://diabetes.jmir.org), 19.01.2024.)

Nitric oxide (NO) is a natural reactive nitrogen species (RNS) that alters proteins, DNA, and lipids and damages biological activities. Although microorganisms respond to and detoxify NO, the regulation of the cellular metabolic mechanisms that cause cells to tolerate RNS toxicity is not completely understood. We found that the proline and arginine auxotrophic proA5 and argB2 mutants of the fungus Aspergillus nidulans require more arginine and proline for normal growth under RNS stress that starves cells by accumulating fewer amino acids. Fungal transcriptomes indicated that RNS stress upregulates the expression of the biosynthetic genes required for global amino acids, including proline and arginine. A mutant of the gene disruptant, cpcA , which encodes the transcriptional regulation of the cross-pathway control of general amino acid synthesis, did not induce these genes, and cells accumulated fewer amino acids under RNS stress. These results indicated a novel function of CpcA in the cellular response to RNS stress, which is mediated through amino acid starvation and induces the transcription of genes for general amino acid synthesis. Since CpcA also controls organic acid biosynthesis, impaired intermediates of such biosynthesis might starve cells of amino acids. These findings revealed the importance of the mechanism regulating amino acid homeostasis for fungal responses to and survival under RNS stress.

A cell population characterized by the release of glucose repression and known as [ GAR + ] emerges spontaneously in the yeast Saccharomyces cerevisiae . This study revealed that the [ GAR + ] variants exhibit retarded alcoholic fermentation when glucose is the sole carbon source. To identify the key to the altered glucose response, the gene expression profile of [ GAR + ] cells was examined. Based on RNA-seq data, the [ GAR + ] status was linked to impaired function of the Cyc8p-Tup1p complex. Loss of Cyc8p led to a decrease in the initial rate of alcoholic fermentation under glucose-rich conditions via the inactivation of pyruvate decarboxylase, an enzyme unique to alcoholic fermentation. These results suggest that Cyc8p can become inactive to attenuate alcoholic fermentation. These findings may contribute to the elucidation of the mechanism of non-genetic heterogeneity in yeast alcoholic fermentation.

Aims: In our previously reported randomized controlled trial in patients with noninsulin-treated type 2 diabetes, the use of flash glucose monitoring (FGM) improved glycated hemoglobin (HbA1c), and the improvement was sustained after the cessation of glucose monitoring. In this post-hoc analysis, we examined data from our trial to identify the factors that influenced FGM efficacy., Methods: We analyzed data for 48 of 49 participants of the FGM group who completed the trial to clarify the changes in various parameters and factors related to HbA1c improvement with the use of FGM., Results: Analyses of the FGM data during the 12-week FGM provision period showed that the weekly mean blood glucose levels considerably decreased as early as at 1 week compared with the baseline values, and this decline continued for 12 weeks. An enhancement in the Diabetes Treatment Satisfaction Questionnaire regarding "willingness to continue the current treatment" score was significantly associated with the improvement in HbA1c at 12 (p = 0.009) and 24 weeks (p = 0.012)., Conclusions: Glycemic control was improved soon after FGM initiation, accompanied by improved satisfaction with continuation of the current treatment in patients with noninsulin-treated type 2 diabetes., Competing Interests: Declaration of Competing Interest HA reports grants and speakers’ honoraria from Abbott Japan outside the submitted work. No other potential conflicts of interest relevant to this article were reported., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Proline, which is a predominant amino acid in grape musts, is involved in the taste and flavor of foods and beverages. The yeast Saccharomyces cerevisiae poorly utilizes proline in wine-making processes, leading to a nitrogen deficiency during fermentation and proline accumulation in wine. Previous studies have shown that the protein kinase A (PKA) pathway is involved in inhibitory mechanisms of proline utilization. In this study, we screened the PKA pathway-related genes that regulate proline utilization. Using a yeast culture collection of disrupted strains associated with the downstream of the PKA cascade, we revealed that the stress-responsive transcription factor genes MSN2/4 regulate proline utilization. Moreover, we found that Msn2/4 up-regulate the SHY1 gene during the cell growth of the wine fermentation model, which may cause the inhibition of proline utilization. The SHY1-deleted strain of the commercial wine yeast clearly showed proline consumption and average ethanol production under the wine fermentation model. The present data indicate that the PKA-Msn2/4-Shy1 cascade controls the inhibition of proline utilization under wine-making processes. Our study could hold promise for the development of wine yeast strains that can efficiently reduce proline during wine fermentation., (Copyright © 2023 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Hydroponic cultivation of Solanum lycopersicum (tomato) is important, and high tomato production depends on the use of nitrogen and phosphate fertilizers. We had developed a microbial fertilizer (MF), which is mainly composed of nitrate. To investigate the effect of MF on plant growth, hydroponic tomato was grown with MF or commercial inorganic fertilizer (IF), and the microbiomes of the rhizosphere and the liquid phase were analyzed by confocal microscopy and high-throughput sequencing. Plant biomass and biofilm formation were increased by growth in MF compared to IF. The microbial community structures of tomato roots and hydroponic water differed between the two conditions, and three operational taxonomic units (OTUs) dominated in plants grown with MF. The three OTUs were related to Rudaea spp., Chitinophaga spp., and Stenotrophobacter terrae, which are reported to be disease-suppressive epiphytic or endophytic microbes of plant roots. Because these three OTUs also predominated in the MF itself, they were likely provided to the rhizosphere or endophytic environments of tomato roots via hydroponic water. KEY POINTS: • Microbial fertilizer for hydroponic growth enhanced biofilm formation on tomato root. • Microbial fertilizer contains tomato-root epiphytic or endophytic microbes. • Microbial fertilizer provided beneficial microbes to the rhizosphere and endophytic environments of tomato roots via hydroponic water., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Although proline is the most or second most abundant amino acid in wort and grape must, it is not fully consumed by the yeast Saccharomyces cerevisiae during alcoholic fermentation, unlike other amino acids. Our previous studies showed that arginine, the third most abundant amino acid in wort, inhibits the utilization of proline in most strains of S. cerevisiae . Furthermore, we found that some non- Saccharomyces yeasts utilized proline in a specific artificial medium with arginine and proline as the only nitrogen source, but these yeasts were not suitable for beer fermentation due to their low alcohol productivity. For yeasts to be useful for brewing, they need to utilize proline and produce alcohol during fermentation. In this study, 11 S. cerevisiae strains and 10 non- Saccharomyces yeast strains in the Phaff Yeast Culture Collection were identified that utilize proline effectively. Notably, two of these S. cerevisiae strains , UCDFST 40-144 and 68-44, utilize proline and produce sufficient alcohol in the beer fermentation model used. These strains have the potential to create distinctive beer products that are specifically alcoholic but with a reduction in proline in the finished beer.

The effects of the chemical components of fine particulate matter (PM 2.5 ) have been drawing attention. However, information regarding the impact of low PM 2.5 concentrations is limited. Hence, we aimed to investigate the short-term effects of the chemical components of PM 2.5 on pulmonary function and their seasonal differences in healthy adolescents living on an isolated island without major artificial sources of air pollution. A panel study was repeatedly conducted twice a year for one month every spring and fall from October 2014 to November 2016 on an isolated island in the Seto Inland Sea, which has no major artificial sources of air pollution. Daily measurements of peak expiratory flow (PEF) and forced expiratory volume in 1 s (FEV 1 ) were performed in 47 healthy college students, and the concentrations of 35 chemical components of PM 2.5 were analyzed every 24 h. Using a mixed-effects model, the relationship between pulmonary function values and concentrations of PM 2.5 components was analyzed. Significant associations were observed between several PM 2.5 components and decreased pulmonary function. Among the ionic components, sulfate was strongly related to decreases in PEF and FEV 1 (-4.20 L/min [95 % confidence interval (CI): -6.40 to -2.00] and - 0.04 L [95 % CI: -0.05 to -0.02] per interquartile range increase, respectively). Among the elemental components, potassium induced the greatest reduction in PEF and FEV 1 . Therefore, PEF and FEV 1 were significantly reduced as the concentrations of several PM 2.5 components increased during fall, with minimal changes observed during spring. Several chemical components of PM 2.5 were significantly associated with decreased pulmonary function among healthy adolescents. The concentrations of PM 2.5 chemical components differed by season, suggesting the occurrence of distinct effects on the respiratory system depending on the type of component., Competing Interests: Declaration of competing interest 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., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Proline is the most abundant amino acid in wine and beer, because the yeast Saccharomyces cerevisiae hardly assimilates proline during fermentation processes. Our previous studies showed that arginine induces endocytosis of the proline transporter Put4, resulting in inhibition of proline utilization. We here report a possible role of arginine sensing in the inhibition of proline utilization. We first found that two basic amino acids, ornithine, and lysine, inhibit proline utilization by inducing Put4 endocytosis in a manner similar to arginine, but citrulline does not. Our genetic screening revealed that the arginine transporter Can1 is involved in the inhibition of proline utilization by arginine. Intriguingly, the arginine uptake activity of Can1 was not required for the arginine-dependent inhibition of proline utilization, suggesting that Can1 has a function beyond its commonly known function of transporting arginine. More importantly, our biochemical analyses revealed that Can1 activates signaling cascades of protein kinase A in response to extracellular arginine. Hence, we proposed that Can1 regulates proline utilization by functioning as a transceptor possessing the activity of both a transporter and receptor of arginine., (© 2023 John Wiley & Sons Ltd.)

Breeding yeast strains for industrial alcoholic fermentation requires laborious screening due to the lack of in vivo modification strategies. Here we show that quiescence-specific cell wall thickening via synthesis of a major component, 1,3-β-glucan, critically antagonizes cellular fermentation ability by sequestering the available cytoplasmic carbon sources. This study provides insights into glycolytic control and reports an effective and reliable rational fermentation design., (© 2023. The Author(s).)

Metallothionein (MT), which is a small metal-binding protein with cysteine-rich motifs, functions in the detoxification of heavy metals in a variety of organisms. Even though previous studies suggest that MT is involved in the tolerance mechanisms against nitrosative stress induced by toxic levels of nitric oxide (NO) in mammalian cells, the physiological functions of MT in relation to NO have not been fully understood. In this study, we analyzed the functions of MT in nitrosative stress tolerance in the yeast Saccharomyces cerevisiae . Our phenotypic analyses showed that deletion or overexpression of the MT-encoding gene, CUP1 , led to higher sensitivity or tolerance to nitrosative stress in S. cerevisiae cells, respectively. We further examined whether the yeast MT Cup1 in the cell-free lysate scavenges NO. These results showed that the cell-free lysate containing a higher level of Cup1 degraded NO more efficiently. On the other hand, the transcription level of CUP1 was not affected by nitrosative stress treatment. Our findings suggest that the yeast MT Cup1 contributes to nitrosative stress tolerance, possibly as a constitutive rather than an inducible defense mechanism., Competing Interests: Conflict of Interest: No potential conflict of interest was reported by the authors., (Copyright: © 2023 Yoshikawa et al.)

In natural long days, the florigen gene FLOWERING LOCUS T (FT) shows a bimodal expression pattern with morning and dusk peaks in Arabidopsis. This pattern differs from the one observed in the laboratory, and little is known about underlying mechanisms. A red : far-red (R : FR) ratio difference between sunlight and fluorescent light causes this FT pattern mismatch. We showed that bimodal FT expression patterns were induced in a day longer than 14 h with sunlight R : FR (= c. 1) conditions. By circadian gating experiments, we found that cumulative exposure of R : FR-adjusted light (R : FR ratio was adjusted to 1 with FR supplement) spanning from the afternoon to the next morning required full induction of FT in the morning. Conversely, only 2 h of R : FR adjustment in the late afternoon was sufficient for FT induction at dusk. We identified that phytochrome A (phyA) is required for the morning FT expression in response to the R : FR adjustment on the previous day. As a part of this mechanism, we showed that PHYTOCHROME-INTERACTING FACTOR 7 contributes to FT regulation. Our results suggest that phyA-mediated high-irradiance response and the external coincidence mechanism contribute to morning FT induction under natural long-day conditions., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

The timing of flowering affects the success of sexual reproduction. This developmental event also determines crop yield, biomass, and longevity. Therefore, this mechanism has been targeted for improvement along with crop domestication. The underlying mechanisms of flowering are highly conserved in angiosperms. Central to these mechanisms is how environmental and endogenous conditions control transcriptional regulation of the FLOWERING LOCUS T (FT) gene, which initiates floral development under long-day conditions in Arabidopsis. Since the identification of FT as florigen, efforts have been made to understand the regulatory mechanisms of FT expression. Although many transcriptional regulators have been shown to directly influence FT, the question of how they coordinately control the spatiotemporal expression patterns of FT still requires further investigation. Among FT regulators, CONSTANS (CO) is the primary one whose protein stability is tightly controlled by phosphorylation and ubiquitination/proteasome-mediated mechanisms. In addition, various CO interaction partners, some of them previously identified as FT transcriptional regulators, positively or negatively modulate CO protein activity. The FT promoter possesses several transcriptional regulatory "blocks," highly conserved regions among Brassicaceae plants. Different transcription factors bind to specific blocks and affect FT expression, often causing topological changes in FT chromatin structure, such as the formation of DNA loops. We discuss the current understanding of the regulation of FT expression mainly in Arabidopsis and propose future directions related to this topic., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

When damaged, restoring the function of the hypothalamus is currently impossible. It is unclear whether neural stem cells exist in the hypothalamus. Studies have reported that adult rodent tanycytes around the third ventricle function as hypothalamic neural stem cell-like cells. However, it is currently impossible to collect periventricular cells from humans. We attempted to generate hypothalamic neural stem cell-like cells from human embryonic stem cells (ESCs). We focused on retina and anterior neural fold homeobox (RAX) because its expression is gradually restricted to tanycytes during the late embryonic stage. We differentiated RAX::VENUS knockin human ESCs (hESCs) into hypothalamic organoids and sorted RAX + cells from mature organoids. The isolated RAX + cells formed neurospheres and exhibited self-renewal and multipotency. Neurogenesis was observed when neurospheres were transplanted into the mouse hypothalamus. We isolated RAX + hypothalamic neural stem cell-like cells from wild-type human ES organoids. This is the first study to differentiate human hypothalamic neural stem cell-like cells from pluripotent stem cells., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The symptoms of diabetes insipidus may be masked by the concurrence of adrenal insufficiency and emerge after the administration of hydrocortisone, occasionally at high doses. To elucidate the mechanism underlying polyuria induced by the administration of high-dose corticosteroids in the deficiency of arginine vasopressin (AVP), we first examined the secretion of AVP in three patients in whom polyuria was observed only after the administration of high-dose corticosteroids. Next, we examined the effects of dexamethasone or aldosterone on water balance in wild-type and familial neurohypophyseal diabetes insipidus (FNDI) model mice. A hypertonic saline test showed that AVP secretion was partially impaired in all patients. In one patient, there were no apparent changes in AVP secretion before and after the administration of high-dose corticosteroids. In FNDI mice, unlike dexamethasone, the administration of aldosterone increased urine volumes and decreased urine osmolality. Immunohistochemical analyses showed that, after the administration of aldosterone in FNDI mice, aquaporin-2 expression was decreased in the apical membrane and increased in the basolateral membrane in the collecting duct. These changes were not observed in wild-type mice. The present data suggest that treatment with mineralocorticoids induces polyuria by reducing aquaporin-2 expression in the apical membrane of the kidney in partial AVP deficiency.

The current CRISPR/Cas9 systems in the yeast Saccharomyces cerevisiae cannot be considered a non-genetic modification technology because it requires the introduction of Cas9 and sgRNA into yeast cells using plasmid expression systems. Our present study showed that the yeast genome can be edited without plasmid expression systems by using a commercially available protein transfection reagent and chemically modified sgRNAs., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Objective: Resting energy expenditure (REE) decreases if there is reduced energy intake and body weight (BW). The decrease in REE could make it difficult for patients with obesity to maintain decreased BW. This study aimed to investigate the correlation among changes in REE, energy intake, and BW during the weight loss process in patients with obesity., Materials and Methods: We conducted a retrospective cohort study of patients hospitalized for the treatment of obesity in Japan. Patients received fully controlled diet during hospitalization and performed exercises if able. REE was measured once a week using a hand-held indirect calorimetry. Energy intake was determined by actual dietary intake., Results: Of 44 inpatients with obesity, 17 were included in the analysis. Their BW decreased significantly after 1 week (-4.7 ± 2.0 kg, P < 0.001) and 2 weeks (-5.7 ± 2.2 kg, P < 0.001). The change in REE after 1 and 2 weeks was positively correlated with the energy intake/energy expenditure ratio (r = 0.66, P = 0.004 at 1 week, r = 0.71, P = 0.002 at 2 weeks). Using a regression equation (y = 0.5257x - 43.579), if the energy intake/energy expenditure ratio within the second week was 82.9%, the REE after 2 weeks was similar to the baseline level. There was no significant correlation between the change in REE and BW., Conclusion: Our data suggest that changes in REE depend on energy intake/energy expenditure ratio and that the decrease in REE can be minimized by matching energy intake to energy expenditure, even during the weight loss process.

Purpose: Few studies exist regarding sagittal alignment describing femur morphology in navigation-aided surgery. This study investigated the three-dimensional (3D) sagittal femoral alignment of the whole femur., Methods: Seventy-three consecutive patients (59 females, 14 males, mean age: 76.1 years), yielding 140 femurs, were included in this study. A computed-tomography-based patient-specific 3D femur model was used to define a mechanical axis-based reference plane. Proximal and distal femoral axis angles (PFA, DFA) to the reference plane were measured in 3D using custom software. PFA and DFA represent the proximal and distal inclination of the femoral anatomical axis in sagittal plane, respectively., Results: PFA (10.6 ± 1.5°) was greater than DFA (2.6 ± 1.6°; P < 0.0001). DFA in females (2.3 ± 1.4°) was smaller than in males (3.9 ± 1.7°; P < 0.0001)., Conclusion: This is the first report of measurement of femoral sagittal alignment related to both 3D anatomy and decision making of femoral flexion angle using navigation surgery for total knee arthroplasty. This report shows a robust DFA measurement that could be used as a template for femoral implants flexion angle when performing both conventional and navigated total knee arthroplasty., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Proline contributes to the taste and flavor of foods. The yeast Saccharomyces cerevisiae poorly assimilates proline during fermentation processes, resulting in the accumulation of proline in fermentative products. We performed here a screening of in total 1138 yeasts to obtain strains that better utilize proline. Our results suggest that proline utilization occurs in the genera of Zygoascus, Galactomyces, and Magnusiomyces., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

We isolated a new strain of the yeast Saccharomyces cerevisiae, 35a14, from banana stems in Okinawa. This strain did not belong to any industrial yeast groups in a phylogenetic tree and produced high levels of alcohol. Furthermore, awamori, an Okinawa's traditional distilled alcoholic beverage, brewed with an l-leucine overproducing mutant derived from 35a14 showed a high concentration of isoamyl acetate., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Background: Radiotherapy is an important treatment option for central nervous system malignancies. However, cranial radiation induces hippocampal dysfunction and white matter injury; this leads to cognitive dysfunction, and results in a reduced quality of life in patients. Excitatory glutamate signaling through N-methyl-d-aspartate receptors (NMDARs) plays a central role both in hippocampal neurogenesis and in the myelination of oligodendrocytes in the cerebrum., Methods: We provide a method for quantifying neurogenesis in human subjects in live brain during cancer therapy. Neuroimaging using originally created behavioral tasks was employed to examine human hippocampal memory pathway in patients with brain disorders., Results: Treatment with memantine, a non-competitive NMDAR antagonist, reversed impairment in hippocampal pattern separation networks as detected by functional magnetic resonance imaging. Hyperbaric preconditioning of the patients just before radiotherapy with memantine mostly reversed white matter injury as detected by whole brain analysis with Tract-Based Spatial Statics. Neuromodulation combined with the administration of hyperbaric oxygen therapy and memantine during radiotherapy facilitated the restoration of hippocampal function and white matter integrity, and improved higher cognitive function in patients receiving cranial radiation., Conclusions: The method described herein, for diagnosis of hippocampal dysfunction, and therapeutic intervention can be utilized to restore some of the cognitive decline experienced by patients who have received cranial radiation. The underlying mechanism of restoration is the production of new neurons, which enhances functionality in pattern separation networks in the hippocampi, resulting in an increase in cognitive score, and restoration of microstructural integrity of white matter tracts revealed by Tract-Based Spatial Statics Analysis., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The fruit-like aroma of two valine-derived volatiles, isobutanol and isobutyl acetate, has great impact on the flavour and taste of alcoholic beverages, including sake, a traditional Japanese alcoholic beverage. With the growing worldwide interest in sake, breeding of yeast strains with intracellular valine accumulation is a promising approach to meet a demand for sakes with a variety of flavour and taste by increasing the valine-derived aromas. We here isolated a valine-accumulating sake yeast mutant (K7-V7) and identified a novel amino acid substitution, Ala31Thr, on Ilv6, a regulatory subunit for acetohydroxy acid synthase. Expression of the Ala31Thr variant Ilv6 conferred valine accumulation on the laboratory yeast cells, leading to increased isobutanol production. Additionally, enzymatic analysis revealed that Ala31Thr substitution in Ilv6 decreased sensitivity to feedback inhibition by valine. This study demonstrated for the first time that an N-terminal arm conserved in the regulatory subunit of fungal acetohydroxy acid synthase is involved in the allosteric regulation by valine. Moreover, sake brewed with strain K7-V7 contained 1.5-fold higher levels of isobutanol and isobutyl acetate than sake brewed with the parental strain. Our findings will contribute to the brewing of distinctive sakes and the development of yeast strains with increased production of valine-derived compounds., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Random and rational mutagenesis of an α-amino acid ester acyl transferase from Sphingobacterium siyangensis AJ2458 (SAET) was conducted to examine the production of aspartame, an α-l-aspartyl-l-phenylalanine methyl ester. We previously reported aspartame production via combination of enzymatic and chemical methods. However, the productivity of the aspartame intermediate by SAET was approximately one-fifth that of l-alanyl-l-glutamine (Ala-Gln), whose production method has already been established. Here, to improve the enzymatic activity of SAET, we performed random mutagenesis in the gene encoding SAET and obtained 10 mutations that elevated the enzymatic activity (1.2- to 1.7-fold increase) relative to that of wild-type SAET. To further improve the activity, we performed mutagenesis to optimize the combination of the obtained mutations and finally selected one SAET variant with 10 amino acid substitutions (M35-4 SAET). An Escherichia coli strain overexpressing M35-4 SAET displayed a 5.7-fold higher activity than that of the wild-type SAET, which was almost equal to that of Ala-Gln by an E. coli strain overexpressing wild-type SAET. The Vmax value of M35-4 SAET was 2.0-fold greater, and its thermostability was higher than those of wild-type SAET. These results suggest that the obtained SAET variants contribute to improvement in aspartame production., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society.)

Nitric oxide as a signaling molecule exerts cytotoxicity known as nitrosative stress at its excess concentrations. In the yeast Saccharomyces cerevisiae, the cellular responses to nitrosative stress and their molecular mechanisms are not fully understood. Here, focusing on the posttranslational modifications that are associated with nitrosative stress response, we show that nitrosative stress increased the protein S-glutathionylation level in yeast cells. Our proteomic and immunochemical analyses demonstrated that the fructose-1,6-bisphosphate aldolase Fba1 underwent S-glutathionylation at Cys112 in response to nitrosative stress. The enzyme assay using a recombinant Fba1 demonstrated that S-glutathionylation at Cys112 inhibited the Fba1 activity. Moreover, we revealed that the cytosolic glutaredoxin Grx1 reduced S-glutathionylation of Fba1 and then recovered its activity. The intracellular contents of fructose-1,6-bisphosphate and 6-phosphogluconate, which are a substrate of Fba1 and an intermediate of the pentose phosphate pathway (PPP), respectively, were increased in response to nitrosative stress, suggesting that the metabolic flow was switched from glycolysis to PPP. The cellular level of NADPH, which is produced in PPP and functions as a reducing force for nitric oxide detoxifying enzymes, was also elevated under nitrosative stress conditions, but this increase was canceled by the amino acid substitution of Cys112 to Ser in Fba1. Furthermore, the viability of yeast cells expressing Cys112Ser-Fba1 was significantly lower than that of the wild-type cells under nitrosative stress conditions. These results indicate that the inhibition of Fba1 by its S-glutathionylation changes metabolism from glycolysis to PPP to increase NADPH production, leading to nitrosative stress tolerance in yeast cells., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Ganoderma , a medicinal mushroom with various physiological activities, has been extensively investigated regarding its effectiveness. The aim of the present study was to examine the effects of a subcritical water extract of Ganoderma (SWEG) on the immune system. The use of subcritical water with a higher temperature and pressure than hot water allows efficient elution of components from natural products. As an evaluation of the effectiveness of SWEG, a cell proliferation and a cell differentiation test were carried out using A-6 cells, a model of hematopoietic stem cells. Furthermore, an oral administration test in mice was conducted to examine the effects of SWEG on the number and function of immune cells. As a result, SWEG was revealed to promote both self-renewal and differentiation into immune cells such as T cells and natural killer (NK) cells in experiments with A-6 cells. These results were not obtained in experiments using hot water extract of Ganoderma lucidum and Ganoderma sinense . The oral administration test in mice demonstrated that SWEG increased hematopoietic precursor cells, immature B cells, and NK cells in the bone marrow, and T cells in the thymus. In addition, SWEG enhanced the immune functions in the spleen by promoting granzyme B expression and NK cell activity. SWEG was demonstrated to be a food material that acts on HSCs and regulates immunity in vivo ., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Hattori et al.)

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) differentiate into AVP neurons, whereas human ESCs/iPSCs die. Human ESCs/iPSCs are generally more similar to mouse epiblast stem cells (mEpiSCs) compared to mouse ESCs. In this study, we converted human FNDI-specific iPSCs by the naive conversion kit. Although the conversion was partial, we found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation., (© 2022. The Author(s).)

Binge eating is a characteristic symptom observed in obese individuals that is related to dysfunction of dopaminergic neurons (DNs). Intermittent administration of a high-fat diet (HFD) is reported to induce binge-like eating, but the underlying mechanisms remain unclear. We generated dopaminergic neuron specific IKKβ deficient mice (KO) to examine the effects of inflammation in DNs on binge-like eating under inflammatory conditions associated with HFD. After administration of HFD for 4 weeks, mice were fasted for 24 h, and then the consumption of HFD was measured for 2 h. We also evaluated that the mRNA expressions of inflammatory cytokines, glial markers, and dopamine signaling-related genes in the ventral tegmental area (VTA) and striatum. Moreover, insulin was administered intraventricularly to assess downstream signaling. The consumption of HFD was significantly reduced, and the phosphorylation of AKT in the VTA was significantly increased in female KO compared to wild-type (WT) mice. Analyses of mRNA expressions revealed that DNs activity and inflammation in the VTA were significantly decreased in female KO mice. Thus, our data suggest that HFD-induced inflammation with glial cell activation in the VTA affects DNs function and causes abnormal eating behaviors accompanied by insulin resistance in the VTA of female mice.

Leptin, a hormone secreted by adipocytes, exhibits therapeutic potential for the treatment of type 1 diabetes (T1D). Protein tyrosine phosphatase 1B (PTP1B) is a key enzyme that negatively regulates leptin receptor signaling. Here, the role of PTP1B in the treatment of T1D was investigated using PTP1B-deficient (knockout [KO]) mice and a PTP1B inhibitor. T1D wild-type (WT) mice induced by streptozotocin showed marked hyperglycemia compared with non-T1D WT mice. KO mice displayed significantly improved glucose metabolism equivalent to non-T1D WT mice, whereas peripheral or central administration of leptin partially improved glucose metabolism in T1D WT mice. Peripheral combination therapy of leptin and a PTP1B inhibitor in T1D WT mice improved glucose metabolism to the same level as non-T1D WT mice. Leptin was shown to act on the arcuate nucleus in the hypothalamus to suppress gluconeogenesis in liver and enhance glucose uptake in both brown adipose tissue and soleus muscle through the sympathetic nervous system. These effects were enhanced by PTP1B deficiency. Thus, treatment of T1D with leptin, PTP1B deficiency, or a PTP1B inhibitor was shown to enhance leptin activity in the hypothalamus to improve glucose metabolism. These findings suggest a potential alternative therapy for T1D., (© 2022 by the American Diabetes Association.)

Biofilms are formed by the aggregation of microorganisms into multicellular structures that adhere to surfaces. Biofilm formation by yeast is a critical issue in clinical and industrial fields because of the strong adhesion of yeast biofilm to abiotic surfaces and tissues. Here, we clarified the arginine-mediated inhibition of biofilm formation by yeast. First, we showed that arginine inhibits biofilm formation in fungi such as Saccharomyces cerevisiae, Candida glabrata, and Cladosporium cladosporioides, but not in bacteria. In regard to the underlying mechanism, biochemical analysis indicated that arginine inhibits biofilm formation by suppressing Flo11-dependent flocculation. Intriguingly, a strain with deletion of the arginine transporter-encoding CAN1 was insensitive to arginine-mediated inhibition of biofilm formation. Finally, Can1 endocytosis appeared to be required for the inhibitory mechanism of biofilm formation by arginine. The present results could help to elucidate the molecular mechanism of yeast biofilm formation and its control., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. In this study, we clarified that a glucose response is involved in an inhibitory mechanism of proline utilization in yeast. Our genetic screen showed that strains with a loss-of-function mutation on the CDC25 gene can utilize proline even under fermentation conditions. Cdc25 is a regulator of the glucose response consisting of the Ras/cAMP-dependent protein kinase A (PKA) pathway. Moreover, we found that activation of the Ras/PKA pathway is necessary for the inhibitory mechanism of proline utilization. The present data revealed that crosstalk exists between the carbon and proline metabolisms. Our study could hold promise for the development of wine yeast strains that can efficiently assimilate proline during the fermentation processes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Fungi are ubiquitously present in our living environment and are responsible for crop and infectious diseases. Developing new antifungal agents is constantly needed for their effective control. Here, we investigated fungal cellular responses to an array of antifungal compounds, including plant- and bacteria-derived antifungal compounds. The pathogenic fungus Aspergillus fumigatus generated reactive oxygen species in its hyphae after exposure to the antifungal compounds thymol, farnesol, citral, nerol, salicylic acid, phenazine-1-carbonic acid, and pyocyanin, as well as under oxidative and high-temperature stress conditions. The production of nitric oxide (NO) was determined using diaminofluorescein-FM diacetate (DAF-FM DA) and occurred in response to antifungal compounds and stress conditions. The application of reactive oxygen species or NO scavengers partly suppressed the inhibitory effects of farnesol on germination. However, NO production was not detected in the hyphae using the Greiss method. An LC/MS analysis also failed to detect DAF-FM-T, a theoretical product derived from DAF-FM DA and NO, in the hyphae after antifungal treatments. Thus, the cellular state after exposure to antifungal agents may be more complex than previously believed, and the role of NO in fungal cells needs to be investigated further., (© 2022. The Author(s).)

Evidence has accumulated that higher consumption of high-fat diets (HFDs) during the juvenile/adolescent period induces altered hippocampal function and morphology; however, the mechanism behind this phenomenon remains elusive. Using high-resolution structural imaging combined with molecular and functional interrogation, a murine model of obesity treated with HFDs for 12 weeks after weaning mice was shown to change in the glutamate-mediated intracellular calcium signaling and activity, including further selective reduction of gray matter volume in the hippocampus associated with memory recall disturbance. Dysregulation of intracellular calcium concentrations was restored by a non-competitive α-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) antagonist, followed by normalization of hippocampal volume and memory recall ability, indicating that AMPARs may serve as an attractive therapeutic target for obesity-associated cognitive decline., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Branched-chain higher alcohols (BCHAs), or fusel alcohols, including isobutanol, isoamyl alcohol, and active amyl alcohol, are useful compounds in several industries. The yeast Saccharomyces cerevisiae can synthesize these compounds via the metabolic pathways of branched-chain amino acids (BCAAs). Branched-chain amino acid aminotransaminases (BCATs) are the key enzymes for BCHA production via the Ehrlich pathway of BCAAs. BCATs catalyze a bidirectional transamination reaction between branched-chain α-keto acids (BCKAs) and BCAAs. In S. cerevisiae, there are two BCAT isoforms, Bat1 and Bat2, which are encoded by the genes BAT1 and BAT2 . Although many studies have shown the effects of deletion or overexpression of BAT1 and BAT2 on BCHA production, there have been no reports on the enhancement of BCHA production by functional variants of BCATs. Here, to improve BCHA productivity, we designed variants of Bat1 and Bat2 with altered enzyme activity by using in silico computational analysis: the Gly333Ser and Gly333Trp Bat1 and corresponding Gly316Ser and Gly316Trp Bat2 variants, respectively. When expressed in S. cerevisiae cells, most of these variants caused a growth defect in minimal medium. Interestingly, the Gly333Trp Bat1 and Gly316Ser Bat2 variants achieved 18.7-fold and 17.4-fold increases in isobutanol above that for the wild-type enzyme, respectively. The enzyme assay revealed that the catalytic activities of all four BCAT variants were lower than that of the wild-type enzyme. Our results indicate that the decreased BCAT activity enhanced BCHA production by reducing BCAA biosynthesis, which occurs via a pathway that directly competes with BCHA production. IMPORTANCE Recently, several studies have attempted to increase the production of branched-chain higher alcohols (BCHAs) in the yeast Saccharomyces cerevisiae. The key enzymes for BCHA biosynthesis in S. cerevisiae are the branched-chain amino acid aminotransaminases (BCATs) Bat1 and Bat2. Deletion or overexpression of the genes encoding BCATs has an impact on the production of BCHAs; however, amino acid substitution variants of Bat1 and Bat2 that could affect enzymatic properties-and ultimately BCHA productivity-have not been fully studied. By using in silico analysis, we designed variants of Bat1 and Bat2 and expressed them in yeast cells. We found that the engineered BCATs decreased catalytic activities and increased BCHA production. Our approach provides new insight into the functions of BCATs and will be useful in the future construction of enzymes optimized for high-level production of BCHAs.