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Leguminous plants establish a symbiosis with rhizobia to enable nitrogen fixation in root nodules under the control of the presumed root-to-shoot-to-root negative feedback called autoregulation of nodulation. In Lotus japonicus , autoregulation is mediated by CLE-RS genes that are specifically expressed in the root, and the receptor kinase HAR1 that functions in the shoot. However, the mature functional structures of CLE-RS gene products and the molecular nature of CLE-RS/HAR1 signalling governed by these spatially distant components remain elusive. Here we show that CLE-RS2 is a post-translationally arabinosylated glycopeptide derived from the CLE domain. Chemically synthesized CLE-RS glycopeptides cause significant suppression of nodulation and directly bind to HAR1 in an arabinose-chain and sequence-dependent manner. In addition, CLE-RS2 glycopeptide specifically produced in the root is found in xylem sap collected from the shoot. We propose that CLE-RS glycopeptides are the long sought mobile signals responsible for the initial step of autoregulation of nodulation. Symbiotic bacteria form nodules with plant roots and this is controlled by CLE-RS genes found in the plant. In this study, the CLE-RS2 gene product is shown to be a glycopeptide that can travel from the roots to the shoot of plants and binds to the receptor kinase HAR1.

We demonstrate label-free dynamic optical coherence tomography (D-OCT)-based visualization and quantitative assessment of patterns of tumor spheroid response to three anti-cancer drugs. The study involved treating human breast adenocarcinoma (MCF-7 cell-line) with paclitaxel (PTX), tamoxifen citrate (TAM), and doxorubicin (DOX) at concentrations of 0 (control), 0.1, 1, and 10 µM for 1, 3, and 6 days. In addition, fluorescence microscopy imaging was performed for reference. The D-OCT imaging was performed using a custom-built OCT device. Two algorithms, namely logarithmic intensity variance (LIV) and late OCT correlation decay speed (OCDS l ) were used to visualize the tissue dynamics. The spheroids treated with 0.1 and 1 µM TAM appeared similar to the control spheroid, whereas those treated with 10 µM TAM had significant structural corruption and decreasing LIV and OCDS l over treatment time. The spheroids treated with PTX had decreasing volumes and decrease of LIV and OCDS l signals over time at most PTX concentrations. The spheroids treated with DOX had decreasing and increasing volumes over time at DOX concentrations of 1 and 10 µM, respectively. Meanwhile, the LIV and OCDS l signals decreased over treatment time at all DOX concentrations. The D-OCT, particularly OCDS l , patterns were consistent with the fluorescence microscopic patterns. The diversity in the structural and D-OCT results among the drug types and among the concentrations are explained by the mechanisms of the drugs. The presented results suggest that D-OCT is useful for evaluating the difference in the tumor spheroid response to different drugs and it can be a useful tool for anti-cancer drug testing.

This study aims at demonstrating label-free drug-response-patterns assessment of different tumor spheroids and drug types by dynamic optical coherence tomography (D-OCT). The study involved human breast cancer (MCF-7) and colon cancer (HT-29) spheroids. The MCF-7 and HT-29 spheroids were treated with paclitaxel (Taxol; PTX) and the active metabolite of irinotecan SN-38, respectively. The drugs were applied with 0 (control), 0.1, 1, and 10 μM concentrations and the treatment durations were 1, 3, and 6 days. A swept-source OCT microscope equipped with a repeated raster scanning protocol was used to scan the spheroids. Logarithmic intensity variance (LIV) and late OCT correlation decay speed (OCDS l ) algorithms were used to visualize the tumor spheroid dynamics. LIV and OCDS l images visualized different response patterns of the two types of spheroids. In addition, spheroid morphology, LIV, and OCDS l quantification showed different time-courses among the spheroid and drug types. These results may indicate different action mechanisms of the drugs. The results showed the feasibility of D-OCT for the evaluation of drug response patterns of different cell spheroids and drug types and suggest that D-OCT can perform anti-cancer drug testing.

The Japanese rhinoceros beetle Trypoxylus dichotomus is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as a research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10 × Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. We focused on the genes related to horn formation and obtained new insights into the evolution of the gene repertoire and sexual dimorphism as exemplified by the sex-specific splicing pattern of the doublesex gene. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism.

Circulating tumor cells (CTCs) are associated with cancer metastasis and prognosis but their scarcity in whole blood prevents their use as a diagnostic tool. The purpose of the present study was to establish a novel approach to capture and cultivate CTCs using a microfilter device. The present study was a prospective study of patients with pancreatic cancer at the University of Tsukuba Hospital (Tsukuba, Japan). From each patient, 5 ml of whole blood was collected into an EDTA collection tube. Whole blood was filtered to isolate CTCs and cells captured on the microfilter were cultured in place. A total of 15 patients were enrolled. CTCs and/or CTC clusters were detected in 2 of 6 cases on day 0. In all cases, CTCs and/or formed clusters and/or colonies were observed during long-term culture periods of up to 103 days. In samples where CTCs were not immediately evident, CTC clusters and colonies emerged after long-term culture. To confirm activity of the cultured CTCs on the filters, staining with Calcein AM was performed and epithelial cellular adhesion molecule-positive cells were observed. The system enables the capture and culture of CTCs. Cultured CTCs may be used for patient-specific drug susceptibility testing and genomic profiling of cancer.

Aromatase is an estrogen synthetic enzyme that plays important roles in brain functions. To quantify aromatase expression in the brain by positron emission tomography (PET), we had previously developed [ 11 C]cetrozole, which showed high specificity and affinity. To develop more efficient PET tracer(s) for aromatase imaging, we synthesized three analogs of cetrozole. We synthesized meta-cetrozole, nitro-cetrozole, and iso-cetrozole, and prepared the corresponding 11 C-labeled tracers. The inhibitory activities of these three analogs toward aromatase were evaluated using marmoset placenta, and PET imaging of brain aromatase was performed using the 11 C-labeled tracers in monkeys. The most promising analog in the monkey study, iso-cetrozole, was evaluated in the human PET study. The highest to lowest inhibitory activity of the analogs toward aromatase in the microsomal fraction from marmoset placenta was in the following order: iso-cetrozole, nitro-cetrozole, cetrozole, and meta-cetrozole. This order showed good agreement with the order of the binding potential (BP) of each 11 C-labeled analog to aromatase in the rhesus monkey brain. A human PET study using [ 11 C]iso-analog showed a similar distribution pattern of binding as that of [ 11 C]cetrozole. The time–activity curves showed that elimination of [ 11 C]iso-cetrozole from brain tissue was faster than that of 11 C-cetrozole, indicating more rapid metabolism of [ 11 C]iso-cetrozole. [ 11 C]Cetrozole has preferable metabolic stability for brain aromatase imaging in humans, although [ 11 C]iso-cetrozole might also be useful to measure aromatase level in living human brain because of its high binding potential.

Objective: The high frequency of prescribing oral antimicrobial agents, such as third-generation cephalosporins, macrolides, and fluoroquinolones, in local clinics is a major issue that can lead to the emergence of antimicrobial-resistant bacteria in the community. Hospitals have recently adopted the days of antibiotic spectrum coverage (DASC), which considers the antibacterial spectrum as a novel indicator of appropriate antimicrobial use. Although it has been used in inpatient settings, its applicability in community pharmacy settings remains unclear. Materials and Methods: The aim of this study was to determine whether the DASC is a valuable indicator of appropriate antimicrobial use in community pharmacies. We tabulated the use of antimicrobials dispensed at one of our pharmacies in Tokyo from 1 January 2018, to 31 December 2023. The DASC/100 prescriptions were calculated using the Antibiotic Spectrum Coverage score, which quantifies the extent of antimicrobial activity against key organisms. Higher scores indicate broader-spectrum agents, whereas lower scores indicate narrower-spectrum agents. Additionally, the days of therapy (DOT) value was calculated, along with the DOT/100 prescriptions, based on the dispensed prescriptions. Subgroup analyses were conducted for pediatric children aged < 6 years and the elderly (≥65 years). Results: The DASC/DOT was used to assess the appropriate use of antimicrobials. The DOT/100 and DASC/100 prescriptions in 2021–2023 were 50.1% and 51.5% lower, respectively, than those before 2020 (p < 0.05). During the same period, the DASC/DOT decreased by 0.7%, indicating that, despite the decrease in the number of antimicrobial prescriptions, the antimicrobial spectrum did not become narrower. In children < 6 years of age, DOT and DASC values declined significantly, possibly indicating a decrease in the number of antibiotic prescriptions for viral upper respiratory tract infections. In the elderly (≥65 years), the DASC/DOT remained relatively high, indicating their continued reliance on broad-spectrum agents, such as fluoroquinolones and macrolides. Conclusions: This study demonstrates the feasibility and benefits of the DASC/DOT as a spectrum-based indicator for appropriate antimicrobial use in community pharmacies. Therefore, the DASC/DOT serves as a practical and spectrum-sensitive indicator of outpatient antimicrobial use to guide antimicrobial stewardship in community settings. Furthermore, age-specific analyses highlighted the importance of targeted interventions to promote the judicious use of broad-spectrum antimicrobials, particularly among the elderly (≥65 years old).

The snow crab, Chionoecetes opilio , is a giant deep-sea brachyuran. While several decapod crustaceans generally continue to molt and grow throughout their lifetime, the snow crab has a fixed number of molts. Adolescent males continue to molt proportionately to their previous size until the terminal molt at which time an allometric increase in chela size occurs and an alteration of behavioral activities occurs, ensuring breeding success. In this study, we investigated the circulating concentrations of methyl farnesoate (an innate juvenile hormone in decapods) (MF) before or after the terminal molt in males. We then conducted eyestalk RNAseq to obtain molecular insight into the regulation of physiological changes after the terminal molt. Our analyses revealed an increase in MF titers after the terminal molt. This MF surge may be caused by suppression of the genes that encode MF-degrading enzymes and mandibular organ-inhibiting hormone that negatively regulates MF biosynthesis. Moreover, our data suggests that behavioral changes after the terminal molt may be driven by the activation of biogenic amine-related pathways. These results are important not only for elucidating the physiological functions of MFs in decapod crustaceans, which are still largely unknown, but also for understanding the reproductive biology of the snow crab.

Background We developed a Rare Cell Sorter (RCS) for collecting single cell including circulating tumor cells (CTCs). This single-institution pilot study evaluated the ability of this device to detect tumor-like cells in patients with lung cancer and confirmed their genuineness based on the epidermal growth factor receptor (EGFR) mutation concordance with tissue samples. Methods This study included patients treated for lung cancer from September 2021 to August 2022 in University of Tsukuba Hospital. Peripheral blood samples were obtained before surgery or during periodic medical checks for patients treated with drugs. We used the RCS to capture cells based on size. The cells were stained, and the Hoechst-positive, CD45-negative, and epithelial celladhesion molecule (EpCAM)- positive cells were defined as CTCs, were collected. The presumptive CTCs were counted and tested using digital droplet polymerase chain reaction for EGFR mutations and compared with the tissue EGFR status to check concordance. Results Eighteen patients were included in this study and CTCs were detected in 6 patients (33%). The CTCs from three patients showed EGFR mutation, and the EGFR mutation status of CTCs concorded with that of tissue samples in 83% of the cases (5/6). Only one CTC showed a different status from the tissue, and the concordance rate of EGFR status between CTCs and the tissue was 96% (24/25). Conclusion The ability of the RCS to detect CTCs in patients with lung cancer was demonstrated based on the concordance of EGFR status in this pilot study. This novel hybrid method of CTC recovery using the RCS has the potential to recover a wide range of CTCs regardless of EpCAM. Further validation through a large-scale study is needed.

Background/Aim: This study describes a rare cell sorter (RCS) method to detect circulating tumor cells (CTCs) and CTC clusters in whole blood without pretreatment. Patients and Methods: We collected samples from breast cancer patients at the University of Tsukuba Hospital. A total of 15 whole-blood specimens from patients with breast cancer were collected and analyzed via a microfluidics chip, fluorescence-conjugated antibody staining, and fluorescence microscopy. Of 15 total cases, eight were analyzed by RCS ver3 and seven were analyzed by RCS ver3.5 to reveal potential clinical differences in scanning methods. We then examined the HER2 status on 4 of the 15 patients using our RCS system. Results: RCS efficiently detected all subtypes of CTCs and CTC clusters from the peripheral blood of cancer patients. The concordance rate of HER2 status between tissue and CTCs in 4 tested clinical samples was 100%. Conclusion: RCS is a non-invasive method that allows for simultaneous detection of CTCs, cluster presence, and surface marker (e.g., HER2) status. Frequent sampling is, thus, possible and the large amount of data obtained will be clinically useful to predict response to therapy as well as plan adjunct support therapies in cancer patients.