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To investigate the functional role of S100A4 in advanced colorectal carcinoma (Ad-CRC) and locally advanced rectal carcinoma (LAd-RC) receiving neoadjuvant chemoradiotherapy (NCRT). We analyzed histopathological and immunohistochemical sections from 150 patients with Ad-CRC and 177 LAd-RC patients treated with NCRT. S100A4 knockout (KO) HCT116 cells were also used. S100A4 expression was absent in normal mucosa but increased progressively from colorectal adenoma to carcinoma, suggesting that S100A4 regulation is an early event in colorectal carcinogenesis. In Ad-CRC, high S100A4 expression correlated with high tumor budding and nuclear β-catenin, deep invasion, lymph-vascular involvement, and unfavorable prognosis. In NCRT-treated LAd-RC, high S100A4 expression was associated with poor treatment response and short progression-free survival. S100A4 KO decreased the proliferation of HCT116 cells through activation of the p53/p21 waf1 axis, and sensitized cells to adriamycin-induced apoptosis. Levels of the apoptotic marker, cleaved poly (ADP-ribose) polymerase 1, were significantly higher in samples with low S100A4 and wild type p53. Finally, we observed a direct interaction between S100A4 and p53. In conclusion, S100A4 expression engenders aggressive behavior in Ad-CRC through association with β-catenin-driven tumor buddings. S100A4 exerts anti-apoptotic and proliferative effects via inhibition of p53 in LAd-RC patients receiving NCRT, which leads to chemoradioresistance and poor prognosis., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Consent for publication: Not applicable. Ethics approval: This study was conducted in accordance with the principles expressed in the Declaration of Helsinki 1964 and later versions. This study was approved by the Kitasato University Medical Ethics Committee (B19-155). This article does not contain any studies with animals performed by any of the authors. Informed consent: was obtained from all subjects involved in the study., (© 2024. The Author(s).)

The patient was a 51-year-old man. He was diagnosed with cStage Ⅲb descending colon cancer after a positive stool occult blood test, and the patient was referred to our department for laparoscopic left hemicolectomy with D3 lymph node dissection. Intraoperatively, we incidentally found a mass lesion at the end of the ileum and a large hard enlarged lymph node in the nearby mesentery of the small intestine, so we additionally performed a partial resection of the small intestine and dissection of the regional lymph node. Pathological findings showed that the small intestinal lesion was adenocarcinoma of the small intestine. Follow-up abdominal contrast CT showed an enlarged lymph node within the small intestinal mesentery at the origin of the ileocolic artery. The patient was suspected to have lymph node recurrence of small intestinal cancer, and the patient had radical resection with ileal resection with D3 lymph node dissection. Pathological findings showed lymph node recurrence of small intestinal cancer, and postoperative adjuvant chemotherapy(4 couses of CAPOX therapy)was performed. As of 3 years and 10 months postoperatively, the patient is alive without recurrence.

Background: The acini, the secretory endpieces of the salivary glands, are composed of serous and/or mucous acinar cells and surrounded by myoepithelial cells. Myoepithelial cells are multipolar, stellate cells with long processes encircling the acini and intercalated ducts. These cells contract to facilitate salivary secretion and transport., Highlight: In rat major salivary gland acini, the morphology of myoepithelial cells varies across glands: parotid glands lack myoepithelial cells, submandibular glands contain "slender"-shaped cells, and sublingual glands contain "macho"-shaped cells. These morphological variations are thought to depend on the salivary viscosity. Myoepithelial cells in the intercalated ducts exhibit minimal variation across the major salivary glands, with processes oriented parallel to the duct axis. These cells are covered by a thin collagen layer and small fibroblasts, collectively termed "the peri-intercalated duct sheath." Ebner's glands, located beneath the circumvallate and foliate papillae and containing numerous taste buds, develop myoepithelial cells in both the acini and intercalated ducts to facilitate vigorous saliva secretion, enhancing gustatory sensitivity., Conclusions: The morphology of myoepithelial cells is influenced by their functional roles under different anatomical, physiological, and pathological conditions. Increased thickness and branching occur to adapt to salivary viscosity and/or enhance secretion. In the intercalated ducts, myoepithelial cells support salivary transport with the aid of the surrounding collagen layer and fibroblasts in "the peri-intercalated duct sheath"., Competing Interests: Declaration of Competing Interest All authors are required to disclose any COI within the period of 12 months prior to the submission of any manuscript in the subject matter of which any company, entity, or organization has an interest., (Copyright © 2024. Published by Elsevier B.V.)

This review discusses the growing importance of target validation within phase I (P1) trials as a new trend in drug development, especially in establishing proof of concept (POC) for first-in-class drugs. The paper describes two approaches: the P1-PIV approach, which directly evaluates the primary endpoint for a pivotal clinical study to confirm therapeutic effects during P1, and the newly introduced P1-FCTE, which assesses functional changes necessary for therapeutic effect as a novel target validation milestone in P1. By providing practical examples of first-in-class drugs, we compare the benefits, costs, hurdles and applicable therapeutic areas of these approaches. Finally, we discuss the potential of these novel approaches to facilitate POC success, shorten development timelines and ultimately increase drug discovery success rates., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The evidence of the correlation between cellular senescence and aging has increased in research with animal models. These models have been intentionally generated to target and regulate cellular senescent cells with the promoter activity of p16 Ink4a or p19 Arf , genes that are highly expressed in aging cells. However, the senolytic efficiency in various organs and cells from these models represents unexpected variation and diversity in some cases. We have generated a novel knock-in model, p16tdT-hDTR mice, which possess tdTomato and human diphtheria toxin receptor (hDTR) downstream of Cdkn2a, an endogenous p16 Ink4a gene. We successfully demonstrated that p16-derived tdTomato and hDTR expressions are observed in these mouse embryo fibroblasts and following treatment with diphtheria toxin (DT) eliminates those cells. Furthermore, we demonstrated the efficacy of eliminating p16-positive cells in vivo, and also observed a tendency to decrease their cutaneous SA-β-gal activity after subcutaneous DT injection into p16tdT-hDTR mice. In particular, comprehensive gene expression analysis in skin revealed that upregulated genes related to lipid metabolisms with aging exhibited remarkable expressions under the senolysis. These results clearly unveiled p16-positive senescent cells contribute to age-related changes in skin., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

A simple method for measuring the concentration of nano/microplastics (N/MPs) in soil, which is difficult owing to the size of the filter mesh and the resolution of the measuring instrument, was investigated. A spectrophotometer was used for the measurements and polystyrene particles were used as the N/MP samples. When measuring N/MP concentrations in soil suspensions, absorbance was measured at two wavelengths, and the best combination of wavelengths for measurement was extracted because soil particles and leached components interfere with N/MP absorbance. A wavelength combination of 220-260 nm and 280-340 nm was found to be suitable for a variety of soils. As N/MPs are adsorbed on the surface of soil particles and precipitate with soil particles in suspension, a calibration curve was created between the concentration of N/MPs in the soil suspension and the N/MP content in the soil. The calibration curve showed a linear relationship, allowing for the estimation of the concentration of N/MPs in the soil. Although other N/MP materials, such as polyethylene and polyethylene terephthalate, must also still be considered and tested, this simple method has the potential to measure N/MPs in various types of soil., 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 The Authors. Published by Elsevier Inc. All rights reserved.)

Antibody drugs play a vital role in diagnostics and therapy. However, producing antibodies from mammalian cells is challenging owing to cellular heterogeneity, which can be addressed by applying droplet-based microfluidic platforms for high-throughput screening (HTS). Here, we designed an integrated system based on disulfide-bonded redox-responsive hydrogel beads (redox-HBs), which were prepared through enzymatic hydrogelation, to compartmentalize, screen, select, retrieve, and recover selected Chinese hamster ovary (CHO) cells secreting high levels of antibodies. Moreover, redox-HBs were functionalized with protein G as an antibody-binding module to capture antibodies secreted from encapsulated cells. As proof-of-concept, cells co-producing immunoglobulin G (IgG) as the antibody and green fluorescent protein (GFP) as the reporter molecule, denoted as CHO(IgG/GFP), were encapsulated into functionalized redox-HBs. Additionally, antibody-secreting cells were labeled with protein L-conjugated horseradish peroxidase using a tyramide amplification system, enabling fluorescence staining of the antibody captured inside the beads. Redox-HBs were then applied to fluorescence-activated droplet sorting, and selected redox-HBs were degraded by reducing the disulfide bonds to recover the target cells. The results indicated the potential of the developed HTS platform for selecting a single cell viable for biopharmaceutical production., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: In recent years, alternative monitoring approaches, such as risk-based and remote monitoring techniques, have been recommended instead of traditional on-site monitoring to achieve more efficient monitoring. Remote risk-based monitoring (R2BM) is a monitoring technique that combines risk-based and remote monitoring and focuses on the detection of critical data and process errors. Direct data capture (DDC), which directly collects electronic source data, can facilitate R2BM by minimizing the extent of source documents that must be reviewed and reducing the additional workload on R2BM. In this study, we evaluated the effectiveness of R2BM and the synergistic effect of combining R2BM with DDC., Methods: R2BM was prospectively conducted with eight participants in a randomized clinical trial using a remote monitoring system that uploaded photographs of source documents to a cloud location. Critical data and processes were verified by R2BM, and later, all were confirmed by on-site monitoring to evaluate the ability of R2BM to detect critical data and process errors and the workload of uploading photographs for clinical trial staff. In addition, the reduction of the number of uploaded photographs was evaluated by assuming that the DDC was introduced for data collection., Results: Of the 4645 data points, 20.9% (n = 973, 95% confidence interval = 19.8-22.2) were identified as critical. All critical data errors corresponding to 5.4% (n = 53/973, 95% confidence interval = 4.1-7.1) of the critical data and critical process errors were detectable by R2BM. The mean number of uploaded photographs and the mean time to upload them per visit per participant were 34.4 ± 11.9 and 26.5 ± 11.8 min (mean ± standard deviation), respectively. When assuming that DDC was introduced for data collection, 45.0% (95% confidence interval = 42.2-47.9) of uploaded photographs for R2BM were reduced., Conclusions: R2BM can detect 100% of the critical data and process errors without on-site monitoring. Combining R2BM with DDC reduces the workload of R2BM and further improves its efficiency., (© 2024. The Author(s).)

Tandem GGGGCC repeat expansion in C9orf72 is a genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Transcribed repeats are translated into dipeptide repeat proteins via repeat-associated non-AUG (RAN) translation. However, the regulatory mechanism of RAN translation remains unclear. Here, we reveal a GTPase-activating protein, eukaryotic initiation factor 5 (eIF5), which allosterically facilitates the conversion of eIF2-bound GTP into GDP upon start codon recognition, as a novel modifier of C9orf72 RAN translation. Compared to global translation, eIF5, but not its inactive mutants, preferentially stimulates poly-GA RAN translation. RAN translation is increased during integrated stress response, but the stimulatory effect of eIF5 on poly-GA RAN translation was additive to the increase of RAN translation during integrated stress response, with no further increase in phosphorylated eIF2α. Moreover, an alteration of the CUG near cognate codon to CCG or AUG in the poly-GA reading frame abolished the stimulatory effects, indicating that eIF5 primarily acts through the CUG-dependent initiation. Lastly, in a Drosophila model of C9orf72 FTLD/ALS that expresses GGGGCC repeats in the eye, knockdown of endogenous eIF5 by two independent RNAi strains significantly reduced poly-GA expressions, confirming in vivo effect of eIF5 on poly-GA RAN translation. Together, eIF5 stimulates the CUG initiation of poly-GA RAN translation in cellular and Drosophila disease models of C9orf72 FTLD/ALS., Competing Interests: Conflict of interest The authors declare that they have no conflict of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

GGGGCC hexanucleotide repeat expansion in C9orf72 causes frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Expanded GGGGCC repeat RNA accumulates within RNA foci and is translated into toxic dipeptide repeat proteins; thus, efficient repeat RNA degradation may alleviate diseases. hnRNPA3, one of the repeat RNA-binding proteins, has been implicated in the destabilization of repeat RNA. Using APEX2-mediated proximity biotinylation, here, we demonstrate PABPC1, a cytoplasmic poly (A)-binding protein, interacts with hnRNPA3. Knockdown of PABPC1 increased the accumulation of repeat RNA and RNA foci to the same extent as the knockdown of hnRNPA3. Proximity ligation assays indicated PABPC1-hnRNPA3 and PABPC1-RNA exosomes, a complex that degrades repeat RNA, preferentially co-localized when repeat RNA was present. Our results suggest that PABPC1 functions as a mediator of polyadenylated GGGGCC repeat RNA degradation through interactions with hnRNPA3 and RNA exosome complex., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Introduction: We aimed to implement the enhanced recovery after surgery (ERAS) protocol for pediatric neuromuscular scoliosis (NMS) surgery and to examine the effectiveness of this program in this study., Methods: Subjects were children with NMS who underwent scoliosis surgery at our department by a surgeon using a single posterior approach. A series of 27 cases before the introduction of ERAS and 27 cases during program stabilization were included in the study. Patient backgrounds did not show significant differences before and after introducing ERAS. Perioperative data, complications, length of hospital stay (LOS), and readmission within 90 days were investigated and statistically analyzed., Results: When the pre- and post-ERAS induction groups were compared, no significant differences in anesthesia induction time (p=0.979), pelvic fixation (p=0.586), fusion levels (p=0.479), intraoperative hypothermia duration (p=0.154), end-of-surgery body temperature (p=0.197), operative time (p=0.18), postoperative main Cobb angle (p=0.959), main Cobb angle correction rate (p=0.91), postoperative spino-pelvic obliquity (SPO) (p=0.849), and SPO correction rate (p=0.267) were observed. However, significant differences in using V-flap technique (p=0.041), intraoperative blood loss (p=0.001), and LOS (p=0.001) were observed. Intraoperative blood loss was weakly correlated with LOS (p=0.432 and 0.001). No statistically significant difference existed between the V-flap method and LOS (p=0.265). Multiple regression analysis using LOS as the objective variable and ERAS protocols and intraoperative blood loss as explanatory variables revealed that the effect of ERAS on LOS was greater than that of intraoperative blood loss. No statistically significant differences in the readmission rates within 90 days were found., Conclusions: After the introduction of ERAS, LOS decreased without an increase in complications or readmissions within 90 days., Competing Interests: Conflicts of Interest: The authors declare that there are no relevant conflicts of interest., (Copyright © 2024 The Japanese Society for Spine Surgery and Related Research.)

Background: Emicizumab, a factor (F) VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment option for people with hemophilia A (PwHA). However, a small proportion of PwHA still experience bleeds even under emicizumab prophylaxis, as observed in the long-term outcomes of clinical studies. A more potent BsAb may be desirable for such patients., Objectives: To identify a potent BsAb to FIXa and FX, NXT007, surpassing emicizumab by in vitro and in vivo evaluation., Methods: New pairs of light chains for emicizumab's heavy chains were screened from phage libraries, and subsequent antibody optimization was performed. For in vitro evaluation, thrombin generation assays were performed with hemophilia A plasma. In vivo hemostatic activity was evaluated in a nonhuman primate model of acquired hemophilia A., Results: NXT007 exhibited an in vitro thrombin generation activity comparable to the international standard activity of FVIII (100 IU/dL), much higher than emicizumab, when triggered by tissue factor. NXT007 also demonstrated a potent in vivo hemostatic activity at approximately 30-fold lower plasma concentrations than emicizumab's historical data. In terms of dose shift between NXT007 and emicizumab, the in vitro and in vivo results were concordant. Regarding pharmacokinetics, NXT007 showed lower in vivo clearance than those shown by typical monoclonal antibodies, suggesting that the Fc engineering to enhance FcRn binding worked well., Conclusion: NXT007, a potent BsAb, was successfully created. Nonclinical results suggest that NXT007 would have a potential to keep a nonhemophilic range of coagulation potential in PwHA or to realize more convenient dosing regimens than emicizumab., Competing Interests: Declaration of competing interests Chugai Pharmaceutical Co, Ltd is developing NXT007 as a clinical compound. Y.T.-I, T.S., H. Koga, Kazuki Y., K.K., K.E., K.A., M.F., M.I., Y.I., S.I., E.J., S.-I.K., A. Muto, K.N., M.O., H. Sanada, M.S., N.S., T.W., Koji Y., A. Matsusaki, Z.S., H. Shiraiwa, H. Konishi, Y.K., K.H., T.K., and T.I. are employees of Chugai Pharmaceutical Co, Ltd (Chugai); Chugai has filed patent applications related to this work for NXT007. Y.I.-T., T.S., H. Koga, Kazuki Y., K.K., and T.I. are inventors on the patent application (WO/2019/065795) submitted by Chugai that covers the NXT007 molecule. H.Konishi, Y.K., K.H., T.K., and T.I. own Chugai’s stock., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

RNA expression analyses can be used to obtain various information from inside cells, such as physical conditions, the chemical environment, and endogenous signals. For detecting RNA, the system regulating intracellular gene expression has the potential for monitoring RNA expression levels in real time within living cells. Synthetic biology provides powerful tools for detecting and analyzing RNA inside cells. Here, we devised an RNA aptamer-mediated gene activation system, RAMGA, to induce RNA-triggered gene expression activation by employing an inducible complex formation strategy grounded in synthetic biology. This methodology connects DNA-binding domains and transactivators through target RNA using RNA-binding domains, including phage coat proteins. MS2 bacteriophage coat protein fused with a transcriptional activator and PP7 bacteriophage coat protein fused with the tetracycline repressor (tetR) can be bridged by target RNA encoding MS2 and PP7 stem-loops, resulting in transcriptional activation. We generated recombinant CHO cells containing an inducible GFP expression module governed by a minimal promoter with a tetR-responsive element. Cells carrying the trigger RNA exhibited robust reporter gene expression, whereas cells lacking it exhibited no expression. GFP expression was upregulated over 200-fold compared with that in cells without a target RNA expression vector. Moreover, this system can detect the expression of mRNA tagged with aptamer tags and modulate reporter gene expression based on the target mRNA level without affecting the expression of the original mRNA-encoding gene. The RNA-triggered gene expression systems developed in this study have potential as a new platform for establishing gene circuits, evaluating endogenous gene expression, and developing novel RNA detectors.

Biopharmaceuticals, including therapeutic antibodies, are rapidly growing products in the pharmaceutical market. Mammalian cells, such as Chinese hamster ovary (CHO) cells, are widely used as production hosts because recombinant antibodies require complex three-dimensional structures modified with sugar chains. Recombinant protein production using mammalian cells is generally performed with cell growth. In this study, we developed a technology that controls cell growth and recombinant protein production to induce recombinant protein production with predetermined timing. Expression of green fluorescent protein (GFP) gene and a single-chain antibody fused with the Fc-region of the human IgG1 (scFv-Fc) gene can be induced and mediated by the estrogen receptor-based artificial transcription factor Gal4-ERT2-VP16 and corresponding inducer drugs. We generated CHO cells using an artificial gene expression system. The addition of various concentrations of inducer drugs to the culture medium allowed control of proliferation and transgene expression of the engineered CHO cells. Use of 4-hydroxytamoxifen, an antagonist of estrogen, as an inducing agent yielded high gene expression at a concentration more than 10-fold lower than that of β-estradiol. When scFv-Fc was produced under inducing conditions, continuous production was possible for more than 2 weeks while maintaining high specific productivity (57 pg cell -1 day -1 ). This artificial gene expression control system that utilizes the estrogen response of estrogen receptors can be an effective method for inducible production of biopharmaceuticals., (© 2023 Wiley-VCH GmbH.)

The patient is a 70s woman. She underwent cystectomy for bladder cancer 6 years ago and had a ureterocutaneous fistula in the right lower abdomen. After colonoscopy for positive fecal occult blood, a type 1 elevated lesion was found in the ascending colon, which was diagnosed as a well-differentiated adenocarcinoma on biopsy. Surgery was performed with a single hole. The approach from the right lower abdomen, where the ureterocutaneous fistula and ureter are located, was avoided, and the approach from the hepatic flexure of the transverse colon was used first. After the right colon was mobilized, the large mesh adhesions around the ureter were carefully dissected, and the right ureter was identified and preserved, extending from the lateral ascending colon to the abdominal wall. The ileal artery was dissected at the root and after dissection of the D3 lymph node, the intestine was dissected and anastomosed extracorporeally. The operative time was 246 minutes with small amount of blood loss. The patient was discharged on the 6th postoperative day without any postoperative complications. The pathology result was pT3N0M0, pStage Ⅱa, and radical resection had been performed. The patient is currently undergoing recurrence-free follow-up.

We report a case of locally advanced rectal cancer that could not be curatively resected, in which the patient underwent conversion surgery after chemotherapy. The patient is a 70-year-old woman. She came to our hospital with a chief complaint of lower abdominal pain, and a close examination revealed rectal cancer with invasion of the external iliac artery and pelvic wall. She was treated with mFOLFOX6 plus cetuximab for locally advanced rectal cancer that was not amenable to surgical resection. After 11 courses of chemotherapy, significant shrinkage of the tumor was observed, and robot assisted laparoscopic high-anterior resection was performed. The patient didn't relapse at 12 months after surgery without adjuvant chemotherapy.

With the increasing demand for therapeutic antibodies, CHO cells have become the de facto standard as producer host cells for biopharmaceutical production. High production yields are required for antibody production, and developing a high-titer production system is increasingly crucial. This study was established to develop a high-production system using a synthetic biology approach by designing a gene expression system based on an artificial transcription factor that can strongly induce the high expression of target genes in CHO cells. To demonstrate the functionality of this artificial gene expression system and its ability to induce the high expression of target genes in CHO cells, a model antibody (scFv-Fc) was produced using this system. Excellent results were obtained with the plate scale, and when attempting continuous production in semi-continuous cultures using bioreactor tubes with high-cell-density suspension culture using a serum-free medium, high-titer antibody production at the gram-per-liter level was achieved. Shifting the culture temperature to a low temperature of 33 °C achieved scFv-Fc concentrations of up to 5.5 g/L with a specific production rate of 262 pg/(cell∙day). This artificial gene expression system should be a powerful tool for CHO cell engineering aimed at constructing high-yield production systems.

Background: Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid social amoebas acquired diguanylate cyclase (dgcA) by horizontal gene transfer. Dictyostelium discoideum (Ddis) in taxon group 4 uses c-di-GMP as a secreted signal to induce differentiation of stalk cells, the ancestral somatic cell type that supports the propagating spores. We here investigated how this role for c-di-GMP evolved in Dictyostelia by exploring dgcA function in the group 2 species Polysphondylium pallidum (Ppal) and in Polysphondylium violaceum (Pvio), which resides in a small sister clade to group 4., Results: Similar to Ddis, dgcA is upregulated after aggregation in Ppal and Pvio and predominantly expressed in the anterior region and stalks of emerging fruiting bodies. DgcA null mutants in Ppal and Pvio made fruiting bodies with very long and thin stalks and only few spores and showed delayed aggregation and larger aggregates, respectively. Ddis dgcA- cells cannot form stalks at all, but showed no aggregation defects. The long, thin stalks of Ppal and Pvio dgcA- mutants were also observed in acaA- mutants in these species. AcaA encodes adenylate cyclase A, which mediates the effects of c-di-GMP on stalk induction in Ddis. Other factors that promote stalk formation in Ddis are DIF-1, produced by the polyketide synthase StlB, low ammonia, facilitated by the ammonia transporter AmtC, and high oxygen, detected by the oxygen sensor PhyA (prolyl 4-hydroxylase). We deleted the single stlB, amtC and phyA genes in Pvio wild-type and dgcA- cells. Neither of these interventions affected stalk formation in Pvio wild-type and not or very mildly exacerbated the long thin stalk phenotype of Pvio dgcA- cells., Conclusions: The study reveals a novel role for c-di-GMP in aggregation, while the reduced spore number in Pvio and Ppal dgcA- is likely an indirect effect, due to depletion of the cell pool by the extended stalk formation. The results indicate that in addition to c-di-GMP, Dictyostelia ancestrally used an as yet unknown factor for induction of stalk formation. The activation of AcaA by c-di-GMP is likely conserved throughout Dictyostelia., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases., Competing Interests: YF, TI, HI, TS, AM, AI, TG, KM, ET, TK, AK, YK, YF, TF, MI, TM, HM, HM, KW, KI, OO, KN, LP, HT No competing interests declared, MU, DO, TT, TT, YN He previously belonged to the Department of Neurotherapeutics, Osaka University Graduate School of Medicine, that is an endowment department supported by Nihon Medi-Physics Co., AbbVie GK., Otsuka Pharm Co., Kyowakai Med. Co., Fujiikai Med. Co., Yukioka Hosp., Osaka Gyoumeikan Hosp., Kyorin Co., and Tokuyukai Med. Co, (© 2023, Fujino et al.)

Understanding the catalytic mechanism of highly active two-dimensional electrocatalysts is crucial to their rational design. Herein, we reveal the element dependence of the reactivity of two-dimensional metal dichalcogenide sheets for electrocatalytic CO 2 reduction. We found that tin(IV) disulfide (SnS 2 ) and molybdenum(IV) disulfide (MoS 2 ) sheets exhibited Faradaic efficiencies of 63.3% and ∼0%, respectively, for formic acid. Scanning electrochemical cell microscopy and theoretical calculations were used to identify the catalytically active sites of SnS 2 as terraces and edges. Owing to the effective utilization of the entire surface area, SnS 2 can effectively accelerate catalytic reactions. This finding provides a direction for material research in two-dimensional electrocatalysts for energy-efficient chemical production from electrochemical CO 2 reduction, as well as for other energy devices.

Maternal obesity and malnutrition during gestation and lactation have been recognized to increase the risk of obesity and metabolic disorders in the offspring across their lifespan. However, the gestational period during which malnutrition exerts a decisive effect is unclear. Brown adipose tissue (BAT) plays a critical role in energy metabolism owing to its high efficiency in oxidizing glucose and fatty acids. This study aimed to determine the impact of maternal high-fat diet (HFD) consumption only during pregnancy on BAT and energy metabolism in offspring mice. Dams were fed an HFD or a normal chow diet from embryonic day 2.5. HFD consumption during pregnancy induced glucose intolerance and hypertension in dams. In the offspring of HFD-fed dams, maternal HFD lowered fetal weight without affecting placental weight, whereas HFD consumption after birth exacerbated oxygen consumption and cold-induced thermogenesis at 12 months of age, accompanied by increased lipid droplet size in BAT. These data demonstrate that HFD consumption only during pregnancy exerts a long-lasting effect on BAT. Collectively, these findings indicate the importance of nutrition during pregnancy with respect to the energy metabolism of the offspring, and pregnant women should thus ensure proper nutrition during pregnancy to ensure normal energy metabolism in the offspring.

Group 4 Dictyostelia, like Dictyostelium discoideum, self-organize into aggregates and fruiting bodies using propagating waves of the chemoattractant cAMP, which are produced by a network containing the adenylate cyclase AcaA, cAMP receptors (Cars) and the extracellular cAMP phosphodiesterase PdsA. Additionally, AcaA and the adenylate cyclases AcrA and AcgA produce secreted cAMP for induction of aggregative and prespore gene expression and intracellular cAMP for PKA activation, with PKA triggering initiation of development and spore and stalk maturation. Non-group 4 species also use secreted cAMP to coordinate post-aggregative morphogenesis and prespore induction but use other attractants to aggregate. To understand how cAMP's role in aggregation evolved, we deleted the acaA, carA and pdsA genes of Polysphondylium violaceum, a sister species to group 4. acaA- fruiting bodies had thinner stalks but otherwise developed normally. Deletion of acrA, which was similarly expressed as acaA, reduced aggregation centre initiation and, as also occurred after D. discoideum acrA deletion, caused spore instability. Double acaA-acrA- mutants failed to form stable aggregates, a defect that was overcome by exposure to the PKA agonist 8Br-cAMP, and therefore likely due to reduced intracellular cAMP. The carA- and pdsA- mutants showed normal aggregation and fruiting body development. Together, the data showed that P. violaceum development does not critically require secreted cAMP, while roles of intracellular cAMP in initiation of development and spore maturation are conserved. Apparently, cell-cell communication underwent major taxon-group specific innovation in Dictyostelia., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Neuropathological features of frontotemporal dementia and amyotrophic lateral sclerosis (ALS) due to C9orf72 GGGGCC hexanucleotide repeat expansion include early dipeptide repeats, repeat RNA foci, and subsequent TDP-43 pathologies. Since the discovery of the repeat expansion, extensive studies have elucidated the disease mechanism of how the repeat causes neurodegeneration. In this review, we summarize our current understanding of abnormal repeat RNA metabolism and repeat-associated non-AUG translation in C9orf72 frontotemporal lobar degeneration/ALS. For repeat RNA metabolism, we specifically focus on the role of hnRNPA3, the repeat RNA-binding protein, and the EXOSC10/RNA exosome complex, an intracellular RNA-degrading enzyme. In addition, the mechanism of repeat-associated non-AUG translation inhibition via TMPyP4, a repeat RNA-binding compound, is discussed., Competing Interests: None, (Copyright © Japan Medical Association.)

In Parkinson's disease (PD), a decrease in dopamine levels in the striatum causes abnormal circuit activity in the basal ganglia, resulting in increased output via the substantia nigra pars reticulata (SNr). A characteristic feature of glutamatergic synaptic transmission in the basal ganglia circuitry under conditions of dopamine depletion is enhanced synaptic activity of NMDA receptors. However, the cause of this NMDA receptor hyperactivity is not fully understood. We focused on Asc-1 (SLC7A10), an alanine-serine-cysteine transporter, as one of the factors that regulate NMDA receptor activity by modulating D-serine and glycine concentration in synaptic clefts. We generated PD model mice by injection of 6-hydroxydopamine into the unilateral medial forebrain bundle and analyzed the expression level of Asc-1 mRNA in the nuclei of basal ganglia (the external segment of the globus pallidus (GPe), subthalamic nucleus (STN), and SNr) compared to control mice. Each nucleus was dissected using laser microdissection, and RNA was extracted and quantified by quantitative PCR. Asc-1 mRNA expression was significantly higher in the GPe and lower in the SNr under the PD state than that in control naïve mice. The STN showed no change in Asc-1 mRNA expression. We further modeled L-dopa-induced dyskinesia by administering L-dopa continuously for 14 days to the PD model mice and found that Asc-1 mRNA expression in the GPe and SNr became close to that of control mice, regardless of the presence of abnormal involuntary movements. The present study revealed that Asc-1 mRNA expression is differentially regulated in the basal ganglionic nuclei in response to striatal dopamine concentration (depleted or replenished) and suggests that Asc-1 can be a therapeutic target for the amelioration of motor symptoms of PD., Competing Interests: Competing interests The authors declare that they have no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Gold (Au) mining area is known to be one of the major sources of toxic elements; however, the potential risks of toxic elements from abandoned Au mines to the surrounding river basin districts and human exposure pathways to toxic elements need to be clarified. In this study, the distribution and mobilities of nine toxic elements (As, Cd, Cu, Pb, Sb, Zn, Cr, Ni and V) in Kesennuma City, Tohoku Region in Japan, a typical Au-mining district with several river basins, were studied through a geochemical survey (including element total concentrations and water-/acid-leaching concentrations determinations, as well as GB calculations), and environmental assessment on these elements in soil, river sediment, and river water samples from the study area. The contamination evaluation by index of geo-accumulation (I geo ) and enrichment factor (EF) suggested that As, Cu, Ni and Sb enrichments were greatly observed in the vicinity of the abandoned Au mines; moreover, calculated GB upper values for Cu in the river sediment surpass that of Tohoku Region. It has been found in this study that each element has particular mobility, which eventually influences its exposure pathway to humans. For instance, As in soil and sediment poses adverse non-carcinogenic risks and unacceptable carcinogenic risks to especially children mainly through groundwater ingestion. To minimize the potential risks associated with exposure to toxic elements in Au-mining districts, effective risk management measures should be implemented around river system by Au-mining companies even after their long-time closures, based on the consideration of each element's mobility., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Mycoplasma hominis is a commensal pathogen normally found in urogenital tract of humans and has been associated with a wide variety of extra-genitourinary infections, such as mediastinitis, bacteremia, and septic arthritis, particularly in immunocompromised patients. Here, we present a case of a 48-year-old male, who had been treated with fingolimod for relapsing multiple sclerosis and presented with fever and right-sided hip pain following total hip arthroplasty. CT scan revealed localized fluid collection in the right quadriceps femoris muscle adjacent to the joint cavity of right hip. The percutaneously aspirated fluid grew M. hominis, which was also isolated from blood culture. With diagnosis of periprosthetic joint infection, the patient underwent surgical debridement with retained prosthesis and was treated with antimicrobial agents. Infected granulation tissues excised from the hip was observed under an electron microscope, which revealed electron-dense rounded structures contained in neutrophils, consistent with Mycoplasma particles. Fingolimod, an immunomodulatory drug that acts on the sphingosine-1-phosphate receptor and prevents the egress of lymphocytes from lymph nodes, might increase host susceptibility to a systemic M. hominis infection., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: In Dictyostelium discoideum (Ddis), adenylate cyclase A (ACA) critically generates the cAMP oscillations that coordinate aggregation and morphogenesis. Unlike group 4 species like Ddis, other groups do not use extracellular cAMP to aggregate. However, deletion of cAMP receptors (cARs) or extracellular phosphodiesterase (PdsA) in Polyspondylium pallidum (Ppal, group 2) blocks fruiting body formation, suggesting that cAMP oscillations ancestrally control post-aggregative morphogenesis. In group 2, the acaA gene underwent several duplications. We deleted the three Ppal aca genes to identify roles for either gene and tested whether Ppal shows transient cAMP-induced cAMP accumulation, which underpins oscillatory cAMP signalling., Results: In contrast to Ddis, pre-aggregative Ppal cells did not produce a pulse of cAMP upon stimulation with the cAR agonist 2'H-cAMP, but acquired this ability after aggregation. Deletion of Ppal aca1, aca2 and aca3 yielded different phenotypes. aca1- cells showed relatively thin stalks, aca2- showed delayed secondary sorogen formation and aca3- formed less aggregation centers. The aca1-aca2- and aca1-aca3- mutants combined individual defects, while aca2-aca3- and aca1-aca3-aca2- additionally showed > 24 h delay in aggregation, with only few aggregates with fragmenting streams being formed. The fragments developed into small fruiting bodies with stalk and spore cells. Aggregation was restored in aca2-aca3- and aca1-aca3-aca2- by 2.5 mM 8Br-cAMP, a membrane-permeant activator of cAMP-dependent protein kinase (PKA). Like Ddis, Ppal sorogens also express the adenylate cyclases ACR and ACG. We found that prior to aggregation, Ddis aca-/ACG cells produced a pulse of cAMP upon stimulation with 2'H-cAMP, indicating that cAMP oscillations may not be dependent on ACA alone., Conclusions: The three Ppal replicates of acaA perform different roles in stalk morphogenesis, secondary branch formation and aggregation, but act together to enable development by activating PKA. While even an aca1-aca3-aca2- mutant still forms (some) fruiting bodies, suggesting little need for ACA-induced cAMP oscillations in this process, we found that ACG also mediated transient cAMP-induced cAMP accumulation. It, therefore, remains likely that post-aggregative Ppal morphogenesis is organized by cAMP oscillations, favouring a previously proposed model, where cAR-regulated cAMP hydrolysis rather than its synthesis dominates oscillatory behaviour., (© 2022. The Author(s).)

The photoinduced birefringence behaviors of host-guest systems based on heterocyclic thiazole-azo dyes with different substituents, dispersed into PMMA matrix, were investigated under three excitation wavelengths, i.e., 405 nm, 445 nm or 532 nm. The wavelengths fell on the blue side, near the maximum or on the red side of the absorption bands of trans -azo dyes, respectively. We found that photoinduced birefringence was generated at a similar extent in all studied systems, except the system containing a 2-methyl-5-benzothiazolyl as thiazole-azo dye substituent. For this material, the achieved birefringence value was the highest among the whole series, regardless of the excitation wavelength. Moreover, we identified the optimal irradiation wavelength for efficient birefringence generation and showed that large absorption of excitation light by trans isomer does not account for achieving a significant degree of molecular alignment. The obtained results indicate that thiazole-azo dye with a 2-methyl-5-benzothiazolyl substituent shows promising photoinduced birefringence, and can be considered a dye potentially suitable for optical applications.

Genetic factors play critical roles in the onset and progression of obesity. Brown adipose tissue (BAT) activity is also critical for adiposity. The objective of this study was to evaluate the prevalence and effects of BAT gene polymorphisms in pediatric obesity. This case-control study included 270 non-obese and 86 obese children. All participants underwent genotyping for type 2 deiodinase (DIO2) Thr92Ala (rs225014). The prevalence of the homozygous Ala/Ala allele of the DIO2 gene in the obese group was 15.1% versus 6.3% in the non-obese group, resulting in an odds ratio (OR) of 3.393 ( p = 0.003). The results of this study indicate that the homozygous Ala/Ala allele of the DIO2 gene is associated with an increased risk of pediatric obesity and suggest that pediatric obesity might be suitable for assessing the association with gene polymorphisms related to BAT, especially DIO2 Thr92Ala.

Cells in the white matter of the adult brain have a characteristic distribution pattern in which several cells are contiguously connected to each other, making a linear array (LA) resembling pearls-on-a-string parallel to the axon axis. We have been interested in how this pattern of cell distribution changes during aging and remyelination after demyelination. In the present study, with a multiplex staining method, semi-quantitative analysis of the localization of oligodendrocyte lineage cells (oligodendrocyte progenitors, premyelinating oligodendrocytes, and mature oligodendrocytes), astrocytes, and microglia in 8-week-old (young adult) and 32-week-old (aged) corpus callosum showed that young adult cells still include immature oligodendrocytes and that LAs contain a higher proportion of microglia than isolated cells. In aged mice, premyelinating oligodendrocytes were decreased, but microglia continued to be present in the LAs. These results suggest that the presence of microglia is important for the characteristic cell localization pattern of LAs. In a cuprizone-induced demyelination model, we observed re-formation of LAs after completion of cuprizone treatment, concurrent with remyelination. These re-formed LAs again contained more microglia than the isolated cells. This finding supports the hypothesis that microglia contribute to the formation and maintenance of LAs. In addition, regardless of the distribution of cells (LAs or isolated cells), astrocytes were found to be more abundant than in the normal corpus callosum at 24 weeks after cuprizone treatment when remyelination is completed. This suggests that astrocytes are involved in maintaining the functions of remyelinated white matter., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Characteristics of peritubular myoid cells (PMCs) in sexually immature cattle remain largely unknown. Here, we report the character and behavior of peritubular cells expressing alpha smooth muscle actin (αSMA), a marker of PMCs in adult testes, in prepubertal testes procured from 5-months-old bulls. Elastin distribution around αSMA + PMCs was dim and discontinuous in prepubertal testes, but strong and continuous in adult testes. Fibroblast-specific protein 1 (FSP1) was rarely expressed in αSMA + PMCs of prepubertal testes, while in adult testes, majority of αSMA + PMCs were FSP1+. Moreover, αSMA + PMCs in prepubertal testes proliferated more actively than those in adult testes. In vitro culture of isolated seminiferous tubules from prepubertal testes revealed that αSMA + PMCs migrate from peritubular to interstitial area. Hence, in prepubertal bovine testes, (1) PMCs rarely portray fibroblast-like properties, (2) PMCs exhibit heterogeneity in FSP1 expression, (3) PMCs proliferate more actively than those in adult testes, and (4) PMCs have a potential to migrate to the interstitium. Our observations help to understand the maturation of PMCs and their involvement in bovine testicular function., Competing Interests: Declaration of competing interest Authors declare no conflict of interests for this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The industrial use of living organisms for bioproduction of valued substances has been accomplished mostly using microorganisms. To produce high-value bioproducts such as antibodies that require glycosylation modification for better performance, animal cells have been recently gaining attention in bioengineering because microorganisms are unsuitable for producing such substances. Furthermore, animal cells are now classified as products because a large number of cells are required for use in regenerative medicine. In this article, we review animal cell technologies and the use of animal cells, focusing on useable cell generation and large-scale production of animal cells. We review recent advance in mammalian cell line development because this is the first step in the production of recombinant proteins, and it largely affects the efficacy of the production. We next review genetic engineering technology focusing on CRISPR-Cas system as well as surrounding technologies as these methods have been gaining increasing attention in areas that use animal cells. We further review technologies relating to bioreactors used in the context of animal cells because they are essential for the mass production of target products. We also review tissue engineering technology because tissue engineering is one of the main exits for mass-produced cells; in combination with genetic engineering technology, it can prove to be a promising treatment for patients with genetic diseases after the establishment of induced pluripotent stem cell technology. The technologies highlighted in this review cover brief outline of the recent animal cell technologies related to industrial and medical applications., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) can cause a wide variety of infections, ranging from skin and soft tissue infections to life-threatening invasive diseases such as necrotizing pneumonia and infective endocarditis. Here, we present a case of a healthy young female presenting with fever, headache and nausea, who was diagnosed with mitral valve infective endocarditis due to CA-MRSA and whose course was complicated by meningitis and multiple septic emboli. The causative MRSA strain belonged to sequence type 97 and harbored SCCmec Ⅳc but not lukS/F-PV genes. ST97, which is frequently isolated from livestock animals and known as a common lineage of livestock-associated MRSA, may cause invasive infection in the community., (Copyright © 2022 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Researchers have long awaited the technology to develop an in vitro kidney model. Here, we establish a rapid fabricating technique for kidney-like tissues (cysts) using a combination of an organ-derived extracellular matrix (ECM) gel format culture system and a renal stem cell line (CHK-Q cells). CHK-Q cells, which are spontaneously immortalized from the renal stem cells of the Chinese hamster, formed renal cyst-like structures in a type-I collagen gel sandwich culture on day 1 of culture. The cysts fused together and expanded while maintaining three-dimensional structures. The expression of genes related to kidney development and maturation was increased compared with that in a traditional monolayer. Under the kidney-derived ECM (K-ECM) gel format culture system, cyst formation and maturation were induced rapidly. Gene expressions involved in cell polarities, especially for important material transporters (typical markers Slc5a1 and Kcnj1 ), were restored. K-ECM composition was an important trigger for CHK-Q cells to promote kidney-like tissue formation and maturation. We have established a renal cyst model which rapidly expressed mature kidney features via the combination of K-ECM gel format culture system and CHK-Q cells.

Functional human hepatocytes have been a pivotal tool in pharmacological studies such as those investigating drug metabolism and hepatotoxicity. However, primary human hepatocytes are difficult to obtain in large quantities and may cause ethical problems, necessitating the development of a new cell source to replace human primary hepatocytes. We previously developed genetically modified murine hepatoma cell lines with inducible enhanced liver functions, in which eight liver-enriched transcription factor (LETF) genes were introduced into hepatoma cells as inducible transgene expression cassettes. Here, we establish a human hepatoma cell line with heat-inducible liver functions using HepG2 cells. The genetically modified hepatoma cells, designated HepG2/8F_HS, actively proliferated under normal culture conditions and, therefore, can be easily prepared in large quantities. When the expression of LETFs was induced by heat treatment at 43 °C for 30 min, cells ceased proliferation and demonstrated enhanced liver functions. Furthermore, three-dimensional spheroid cultures of HepG2/8F_HS cells showed a further increase in liver functions upon heat treatment. Comprehensive transcriptome analysis using DNA microarrays revealed that HepG2/8F_HS cells had enhanced overall expression of many liver function-related genes following heat treatment. HepG2/8F_HS cells could be useful as a new cell source for pharmacological studies and for constructing bioartificial liver systems.

Immortalized kidney cell lines are widely used in basic and applied research such as cell permeability tests and drug screening. Although many cell lines have been established from kidney tissues, the immortalization process has not been clarified in these cell lines. In this study, we analyzed the phenotypic changes that occurred during the immortalization of kidney cells derived from Chinese hamster tissue in terms of karyotype and gene expression profiles. In the newly established cell line, designated as CHK-Q, gene expression profiles at each stage of the immortalization process and during the adaptation to serum-free conditions were analyzed by DNA microarray. Renal stem cell markers CD24 and CD133 were expressed in CHK-Q cells, suggesting that CHK-Q cells were transformed from renal stem cells. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to identify the pathways of upregulated and downregulated genes revealed that the immortalization of CHK-Q cells was associated with increased fluctuations in the expression of specific proto-oncogenes. Karyotype analysis of spontaneously immortalized CHK-Q cells indicated that CHK-Q chromosomes had a typical modal number of 23 but possessed slight chromosomal abnormalities. In this study, we investigated the mechanism of cell environmental adaptation by analyzing gene expression behavior during the immortalization process and serum-free adaptation. CHK-Q cells are applicable to the fields of biotechnology and biomedical science by utilizing their characteristics as kidney-derived cells., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Spinal fusion for children with neuromuscular scoliosis has been known to improve sitting balance and quality of life as well as for high caregiver satisfaction. However, most studies performed were single surveys, and it remains unclear whether high satisfaction levels are maintained. Thus, in this article, we report the short- and medium-term improvements in caregiver standing assessment after neuromuscular scoliosis surgery in children with Gross Motor Function Classification System (GMFCS) level IV or V., Methods: In total, 18 patients with GMFCS levels IV and V were included in this study. The underlying diseases were typical cerebral palsy in 12 cases, chromosomal abnormalities in 5 cases, and congenital myopathy in 1 case. The median age at the time of surgery was 14.5 years. The medians for the first and second follow-up surveys were after 1.4 and 5.9 years, respectively. All the patients had undergone posterior spinal fusion, whereas 12 had undergone pelvic fixation. These patients were assessed using a caregiver questionnaire, in addition to patient demographic data and radiographic assessments., Results: The median BMI was 15.4 kg/m 2 preoperatively, 16.6 kg/m 2 at the first survey, and 17.1 kg/m 2 at the second survey. The main Cobb angles were 97.5°, 36.5°, and 37.0° and the spino-pelvic obliquity angles were 22.5°, 6.0°, and 6.5° preoperatively, at the first survey and at the second survey, respectively. In the questionnaire, most domains were rated similarly in the first and second surveys, but the ratings for the "children's QOL" and "digestion and defecation" domains were noted to increase, while that for the "transfer" and "satisfaction with treatment" domains have decreased., Conclusions: Neuromuscular scoliosis surgery in children has been associated with extremely high treatment satisfaction in the early postoperative period. However, some caregivers showed a decline in the "transfer" and "treatment satisfaction" domains over time., Competing Interests: Conflicts of Interest: The authors declare that there are no relevant conflicts of interest., (Copyright © 2022 by The Japanese Society for Spine Surgery and Related Research.)

The discharged state affects the charge transfer resistance of lithium-ion secondary batteries (LIBs), which is referred to as the depth of discharge (DOD). To understand the intrinsic charge/discharge property of LIBs, the DOD-dependent charge transfer resistance at the solid-liquid interface is required. However, in a general composite electrode, the conductive additive and organic polymeric binder are unevenly distributed, resulting in a complicated electron conduction/ion conduction path. As a result, estimating the DOD-dependent rate-determining factor of LIBs is difficult. In contrast, in micro/nanoscale electrochemical measurements, the primary or secondary particle is evaluated without using a conductive additive and providing an ideal mass transport condition. To control the DOD state of a single LiFePO 4 active material and evaluate the DOD-dependent charge transfer kinetic parameters, we use scanning electrochemical cell microscopy (SECCM), which uses a micropipette to form an electrochemical cell on a sample surface. The difference in charge transfer resistance at the solid-liquid interface depending on the DOD state and electrolyte solution could be confirmed using SECCM.

This clinical report describes a ceramic complete-mouth rehabilitation with screw-retained implant abutment crowns, optimizing esthetics by combining the properties of ceramic materials. The abutments connected a titanium insert to a computer-aided design and computer-aided manufacturing (CAD-CAM) monolithic zirconia framework, offering improved esthetics and biologic response without negatively affecting the implant abutment interface. Lithium disilicate crowns were cemented extraorally on the abutments, resulting in a screw-retained restoration., (Copyright © 2020 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.)

The use of Chlamydomonas for biofuel and biopharmaceutical production has been anticipated. However, the genetic engineering technology for Chlamydomonas is not as advanced as that for other organisms. Here, we established transgenic Chlamydomonas strains capable of high and stable transgene expression. The established cells exhibited stable reporter gene expression at a high level throughout long-term culture (∼60 days), even in the absence of drug pressure. The transgene insertion sites in the cell genome that may be suitable for exogenous gene expression were identified. Because the transgene contains a loxP site, the cells can be used as founders for retargeting other transgenes using the Cre-loxP system to generate transgenic Chlamydomonas producing useful substances. As a model biopharmaceutical gene, an interferon expression cassette was integrated into the genomic locus of the cells using Cre recombinase. The transgenic cells stably produced interferon protein in medium for 12 passages under non-selective conditions. These results indicate that the Chlamydomonas cells established in this study can serve as valuable and powerful tools not only for basic research on microalgae but also for the rapid establishment of cell lines expressing exogenous genes., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

GGGGCC (G 4 C 2 ) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through repeat-associated non-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G 4 C 2 repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G 4 C 2 repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G 4 C 2 repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G 4 C 2 repeat RNA. Urea-resistant interaction between G 4 C 2 repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G 4 C 2 repeat translation elongation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Constructing three-dimensional (3D) tissues is an important process to improve cellular functions in tissue engineering. When transplanting artificially constructed tissues, a poor vascular network restricts oxygen and nutrient supplies to the tissue cells, which leads to cell death and reduced rates of tissue engraftment. Therefore, it is necessary to develop a system that builds a vascular network within 3D tissues. Here, we developed a hypoxia-responsive gene expression system for production of an angiogenic factor, vascular endothelial growth factor (VEGF), to improve hypoxia and nutrition deficiencies inside artificial 3D tissues. We demonstrated that cells into which the hypoxia-responsive VEGF gene expression system had been introduced autonomously controlled VEGF expression in a hypoxic stress-dependent manner. Next, we confirmed that VEGF expression within a 3D cell sheet was induced in response to a hypoxic environment in vitro. The genetically modified cell sheet was subcutaneously transplanted into mice to evaluate the feasibility of the hypoxia-responsive VEGF gene expression system in vivo. The results suggest that the hypoxia-responsive VEGF gene expression system is promising to prepare artificial 3D tissues in regenerative medicine., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Duchenne muscular dystrophy (DMD) is a genetic disorder that results from deficiency of the dystrophin protein. In recent years, DMD pathological models have been created using induced pluripotent stem (iPS) cells derived from DMD patients. In addition, gene therapy using CRISPR-Cas9 technology to repair the dystrophin gene has been proposed as a new treatment method for DMD. However, it is not known whether the contractile function of myotubes derived from gene-repaired iPS cells can be restored. We therefore investigated the maturation of myotubes in electrical pulse stimulation culture and examined the effect of gene repair by observing the contractile behaviour of myotubes. The contraction activity of myotubes derived from dystrophin-gene repaired iPS cells was improved by electrical pulse stimulation culture. The iPS cell method used in this study for evaluating muscle contractile activity is a useful technique for analysing the mechanism of hereditary muscular disease pathogenesis and for evaluating the efficacy of new drugs and gene therapy.

Retrotransposons, such as long interspersed element-1 (LINE-1), can copy themselves to other genomic loci via a transposition event (termed retrotransposition). Retrotransposons, therefore, have potential use as an efficient gene delivery tool to integrate multiple copies of a target gene into a host genome. Here, we developed a retrotransposon vector based on LINE-1 that achieves target gene integration of multiple transgene copies. The retrotransposon vector contains a neomycin resistance gene split by an intron as a marker gene, and a gene encoding an antibody single-chain variable fragment (Fv) fused with the constant antibody region (Fc) (scFv-Fc) as a model target gene. G418-resistant Chinese hamster ovary cells were generated using this retrotransposon vector, and scFv-Fc was produced in the culture medium. To regulate retrotransposition, we developed a retrotransposon vector system that separately expressed the two open reading frames (ORF1 and ORF2) of LINE-1. Genomic PCR analysis detected the transgene sequence in almost all tested clones. Compared with clones established using the intact LINE-1 vector, clones generated with the split ORF1 and ORF2 system showed similar specific scFv-Fc productivity and retrotransposition efficiency. This approach of using a retrotransposon-based vector system has the potential to provide a new gene delivery tool for mammalian cells., (© 2021 Wiley-VCH GmbH.)

Vertebrate Hox clusters are comprised of multiple Hox genes that control morphology and developmental timing along multiple body axes. Although results of genetic analyses using Hox-knockout mice have been accumulating, genetic studies in other vertebrates have not been sufficient for functional comparisons of vertebrate Hox genes. In this study, we isolated all of the seven hox cluster loss-of-function alleles in zebrafish using the CRISPR-Cas9 system. Comprehensive analysis of the embryonic phenotype and X-ray micro-computed tomography scan analysis of adult fish revealed several species-specific functional contributions of homologous Hox clusters along the appendicular axis, whereas important shared general principles were also confirmed, as exemplified by serial anterior vertebral transformations along the main body axis, observed in fish for the first time. Our results provide insights into discrete sub/neofunctionalization of vertebrate Hox clusters after quadruplication of the ancient Hox cluster. This set of seven complete hox cluster loss-of-function alleles provide a formidable resource for future developmental genetic analysis of the Hox patterning system in zebrafish., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Hepatoma cells are a promising cell source for the construction of bioartificial liver (BAL) systems owing to their high proliferative capability. However, their low liver function compared with primary hepatocytes is a major problem. In a previous study, we established a genetically modified hepatoma cell line, Hepa/8F5, in which eight liver-enriched transcription factor (LETF) genes were transduced into mouse hepatoma Hepa1-6 cells using a drug-inducible transactivator system. These cells proliferate actively under normal culture conditions, meaning that large quantities can be prepared easily. When the overexpression of the LETFs is induced by the addition of an inducer drug, cell growth stops and cell morphology changes with concomitant high expression of liver functions. However, the liver functions largely depend on the presence of the inducer drug, which must be continuously added to maintain these enhanced functions. In the present study, we attempted to modify the method of induction of LETF overexpression in Hepa/8F5 cells to remove the requirement for continual drug addition. To this end, we constructed a system in which the artificial transactivator was transcribed and amplified under the control of a heat-shock protein promoter, and introduced the system into the genome of Hepa/8F5 cells. In our modified cell line, heat-triggered LETF expression was confirmed to induce high liver function. After drug-screening of transfected cells, we established a hepatoma cell line (Hepa/HS), which exhibited high, heat-inducible liver functions. The Hepa/HS cells may represent a new cell source for hepatic studies such as the construction of BAL systems., Supplementary Information: The online version of this article (10.1007/s10616-021-00457-4) contains supplementary material, which is available to authorized users., (© The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021.)

Chloroethenes are common soil and groundwater pollutants. Their dechlorination is impacted by environmental factors, such as the presence of metal ions. We here investigated the effect of ferrous iron on bacterial reductive dechlorination of chloroethenes and on methanogen community. Reductive dechlorination of tetrachloroethene was assayed with a groundwater sample originally containing 6.3 × 103 copies mL-1 of Dehalococcoides 16S rRNA gene and 2 mg L-1 of iron. Supplementation with 28 mg L-1 of ferrous iron enhanced the reductive dechlorination of cis-dichloroethene (cis-DCE) and vinyl chloride in the presence of methanogens. The supplementation shortened the time required for complete dechlorination of 1 mg L-1 of tetrachloroethene to ethene and ethane from 84 to 49 d. Methanogens, such as Candidatus 'Methanogranum', Methanomethylovorans and Methanocorpusculum, were significantly more abundant in iron-supplemented cultures than in non-supplemented cultures (P < 0.01). Upon methanogen growth inhibition by 2-bromoethanesulfonate and in the absence of iron supplementation, cis-DCE was not dechlorinated. Further, iron supplementation induced 71.3% dechlorination of cis-DCE accompanied by an increase in Dehalococcoides 16S rRNA and dehalogenase vcrA gene copies but not dehalogenase tceA gene copies. These observations highlight the cooperative effect of iron and methanogens on the reductive dechlorination of chloroethenes by Dehalococcoides spp., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

River sediments have the effect of aggregating geochemical environmental information, such as that related to geological and artificial pollution resulting from mine closure. This information comprises high-dimensional data and is related to the distribution and quantities of elements in river sediments. However, accessing and interpreting this geochemical information can be difficult. This study employed a data-driven analysis that can be mathematically and statistically reduced in dimension. Using high-dimensional geochemical and environmental information on river sediments, this study evaluated the environmental impact of closed mines. Sample for analysis were collected from three rivers. There are differences in the existence of mines and mine wastewater treatment methods in this river. A total of 33 elements were measured in river sediments. Frequency distribution analysis and Principal component analysis revealed that the elements had unique distribution and frequency characteristics in each river catchment. Four environmental factors could be extracted from the relationship of elements due to lower dimension. PC1 was influenced by the land use in the river area. PC2 captured the geological background. PC3 captured the mixing-diluting effect that occurs in rivers. PC4 effectively captured the effects of domestic wastewater and the effects of closed mines. The effects of the closed mines could be confirmed using the PC4 score for the Okawa River and the Akagawa River. By examining the elemental relationships obtained using these mathematical methods, it is possible to infer the effect of geological features and mines on sediment physiochemistry using existing data on river sediments., Competing Interests: The authors declare no conflict of interest., (© 2021 Published by Elsevier Ltd.)