A 28-Day Repeated Oral Administration Study of Mechanically Fibrillated Cellulose Nanofibers According to OECD TG407.

The impact of oral administration of mechanically fibrillated cellulose nanofibers (fib-CNF), a commonly used nanofiber, on toxicity and health remains unclear, despite reports of the safety and beneficial effects of chitin-based nanofibers. Thus, evaluating the oral toxicity of fib-CNF in accordance with OECD Test Guideline 407 (TG407) is essential. This study aimed to assess the safety of orally administered fib-CNF through an acute toxicity study in rats, following the OECD TG407 guidelines for 4 weeks. CNF "BiNFi-s" FMa-10005, derived from mechanically fibrillated pulp cellulose, was administered via gavage to male and female Crl:CD(SD) rats at doses of 50, 150, 500, and 1000 mg/kg/day for 28 days, with a control group receiving water for injection. The study evaluated the toxic effects of repeated administration, and the rats were monitored for an additional 14 days post-administration to assess recovery from any toxic effects. The results showed no mortality in either sex during the administration period, and no toxicological effects related to the test substance were observed in various assessments, including general condition and behavioral function observations, urinalysis, hematological examination, blood biochemical examination, necropsy findings, organ weights, and histopathological examination. Notably, only female rats treated with 1000 mg/kg/day of CNF exhibited a consistent reduction in body weight during the 14-day recovery period after the end of treatment. They also showed a slight decrease in pituitary and liver weights. However, hematological and blood biochemical tests did not reveal significant differences, suggesting a potential weight-suppressive effect of CNF ingestion.

QT Prolongation and Torsade De Pointes After Catheter Ablation for Persistent Atrial Fibrillation in a Patient With Tachycardia-Induced Cardiomyopathy: A Case Report.

Atrial fibrillation (AF) is the most common cause of tachycardia-induced cardiomyopathy (TIC). A 75-year-old woman was referred to our hospital for catheter ablation for persistent AF. On admission, transthoracic echocardiography (TTE) revealed diffuse left ventricular (LV) hypokinesis, which was suspected to be due to TIC. Catheter ablation was performed on the fifth day of hospitalization, and Torsade de Pointes (TdP) appeared on the sixth day. The serum concentration of bepridil and potassium was below the reference level. An electrocardiogram revealed marked QT prolongation, giant-negative T waves, and T-wave alternans on the seventh day of hospitalization. Cardiac magnetic resonance imaging with no contrast indicated diffuse mild LV hypokinesis, mild prolonged native T1, and no evidence of myocardial edema at T2. Coronary angiography revealed normal coronary arteries, and the ergonovine stress test results were negative. The results for five long QT syndrome susceptibility genes, including the three major genes, were negative. Subsequently, QT prolongation, giant-negative T waves, and LV dysfunction improved without treatment. This case report highlights the importance of risk management for AF patients with TIC scheduled for catheter ablation and carefully evaluating the risks of QT prolongation. Moreover, patients with TIC can experience marked QT prolongation and TdP during the perioperative period of catheter ablation. Therefore, caution should be required.

Involvement of dysregulated hippocampal histone H3K9 methylation at the promoter of the BDNF gene in impaired memory extinction.

RATIONALE: Since the precise mechanisms of posttraumatic stress disorder (PTSD) remain unknown, effective treatment interventions have not yet been established. Impaired extinction of fear memory (EFM) is one of the core symptoms of PTSD and is associated with stress-induced epigenetic change in gene expression. OBJECTIVES: In this study, we examined whether the involvement of histone H3 lysine 9 dimethylation (H3K9me2) in EFM is mediated through brain-derived neurotrophic factor (BDNF) expression in the hippocampus, and whether BIX01294, a selective G9a and GLP histone methyltransferase inhibitor, could be treatment for impaired EFM in an animal model of PTSD. METHODS: The single prolonged stress (SPS) paradigm was used to model PTSD. We measured BDNF mRNA levels by RT-PCR, and H3K9me2 levels in the BDNF gene promoters by chromatin immunoprecipitation-qPCR. After undergoing contextual fear conditioning and hippocampal injection of BIX01294, male rats were subjected to extinction training and extinction testing and their freezing times and BDNF mRNA levels were measured. RESULTS: Compared to sham rats, SPS rats showed decreased BDNF mRNA levels 2 h after extinction training, no significant changes in levels of global H3K9me2 prior to extinction training, and increased levels of H3K9me2 in BDNF gene promoter IV, but not in BDNF gene promoter I. Administration of BIX01294 ameliorated the decrease in BDNF mRNA levels 2 h after extinction training and subsequently alleviated impaired EFM in extinction tests in SPS rats. CONCLUSION: We conclude that reduced hippocampal levels of BDNF mRNA due to increase in H3K9me2 levels may play a role in PTSD-associated EFM impairment, and BIX01294 could be a PTSD treatment option.

Reduction in calcium responses to whisker stimulation in the primary somatosensory and motor cortices of the model mouse with trigeminal neuropathic pain.

OBJECTIVE: Chronic constriction injury (CCI) of the infraorbital nerve induces neuropathic pain, such as allodynia and hyperalgesia, in the orofacial area. However, the changes in the local circuits of the central nervous system following CCI remain unclear. This study aimed to identify the changes following CCI in Thy1-GCaMP6s transgenic mice. METHODS: Neural activity in the primary somatosensory cortex (S1) and motor cortex (M1) following whisker stimulation was assessed using in vivo Ca2+ imaging. CCI-induced changes in responses were analyzed. RESULTS: Before CCI, whisker stimulation induced a greater Ca2+ response in the contralateral S1 than in the ipsilateral S1 and contralateral M1. The peak Ca2+ response amplitude in the bilateral S1 and contralateral M1 decreased two days after CCI compared to before CCI. Decreased Ca2+ response amplitude in these regions was observed until four days after CCI. Seven days after CCI, the Ca2+ response amplitude in the contralateral S1 decreased, whereas that in the ipsilateral S1 and contralateral M1 recovered to control levels. CONCLUSION: These results suggest that neural activity in regions receiving excitatory inputs via corticocortical pathways recovers earlier than in regions receiving thalamocortical inputs. (185/250 words).

The randomized study of enteral nutrition with rapid versus conventional administration in acute stroke patients; the protocol of rapid EN trial.

Rationale: Enteral nutrition is beneficial for stroke patients with oral intake difficulties. However, it is time consuming and may interfere with routine medical care. Therefore, there is a clinical benefit if enteral nutrition can be safely administered in a short time. Although our retrospective study showed the safety of rapid administration, it remains unclear whether rapid administration of enteral nutrition is as safe as conventional administration. Aim: The randomized study of Enteral Nutrition with Rapid versus conventional administration in acute stroke patients (Rapid EN trial) aims to clarify the safety of rapid feeding of enteral nutrition compared with conventional feeding. Methods and design: This is an investigator-initiated, multicenter, prospective, randomized, open-label, blinded end-point clinical trial. Eligible criteria include acute stroke patients who have difficulty with oral intake defined as severe altered consciousness (Japan Coma Scale 10-300) or modified water swallowing test <4. The target enrollment is 700 patients, with 350 patients receiving rapid enteral nutrition at a rate of 100 mL in 5 min (Rapid EN group) and 350 patients receiving conventional enteral nutrition at a rate of 100 mL in 30 min (Conventional EN group). Study outcome: The primary outcome is the incidence of one or more complications of vomiting or diarrhea or pneumonia within 7 days would be non-inferior in the rapid EN group compared to the conventional EN group. Secondary outcomes were total time spent on enteral nutrition within 7 days from enteral nutrition, the incidence of vomiting, diarrhea and pneumonia within 3 or 7 days, and the rate of favorable clinical outcome. Discussion: Since no previous reports have focused on the speed of administration, we felt it was necessary to prove the safety of rapid administration. If this study shows positive results, it will not only benefit patients, but also reduce the burden of medical care. We believe this study is novel and will be useful in clinical practice. Clinical trial registration: https://rctportal.niph.go.jp/s/detail/um?trial_id=UMIN000046610 Identifier UMIN000046610.

The effects of resistance exercise and leucine-enriched essential amino acid supplementation on muscle mass and physical function in post-gastrectomy patients: a pilot randomized controlled trial.

[Purpose] In gastric cancer patients, low muscle mass decreases overall survival and quality of life (QOL). Resistance exercise with leucine-enriched essential amino acid (LEAA) supplementation may prevent muscle mass loss. This study was aimed at determining whether resistance exercise with LEAA supplementation prevents muscle mass loss in post-gastrectomy patients. [Participant and Methods] We conducted a single-center, open-label, randomized controlled pilot trial. Ten participants who underwent gastrectomy were divided into two groups. The intervention group underwent resistance exercise at 70% of one repetition maximum and received a supplement of 3 g of LEAA twice daily for 15 days, while the control group received standard care. We compared changes in muscle mass, physical function (muscle strength and continuous walking distance), and QOL between the groups. [Results] We found good adherence and participation rates in both groups. We failed to detect a significant difference in muscle mass between the groups. The intervention group showed significant improvements in muscle strength and QOL, while the control group showed no significant changes. [Conclusion] We failed to detect a significant difference in muscle mass due to resistance exercise with LEAA supplementation in post-gastrectomy patients. However, resistance exercise with LEAA supplementation might be beneficial for muscle strength recovery and QOL improvements.

Association of fat-to-muscle mass ratio with physical activity and dietary protein, carbohydrate, sodium, and fiber intake in a cross-sectional study.

Higher fat-to-muscle mass ratio (FMR) is reported to be a risk factor for various diseases, including type 2 diabetes and cardiovascular diseases, and mortality. Although this association suggests that reducing FMR may help to prevent certain diseases and mortality, the relationship between FMR and lifestyle factors is unclear. Therefore, we performed a cross-sectional study with the aim to elucidate this relationship. This cross-sectional study included 1518 healthy Japanese adults aged 30 to 64 years. We measured FMR in the whole body, arms, legs, and trunk and assessed various lifestyle factors. Then, we performed forced entry multiple regression analyses for FMR with the following variables: sex, age, physical activity, dietary intake, sleep quality, cigarette smoking, stress levels, and body mass index. As a result, whole-body and regional FMRs were correlated with female sex (ß = 0.71); age (ß = 0.06); physical activity (ß = - 0.07); dietary intake of protein (ß = - 0.12), carbohydrate (ß = 0.04), sodium (ß = 0.13), and fiber (ß = - 0.16); and body mass index (ß = 0.70). The results suggest that in the Japanese middle-aged population, low FMR is associated with certain lifestyle factors, i.e. higher physical activity and a diet with higher protein and fiber and lower carbohydrate and sodium, independent of age, sex, and body mass index.

Intramuscular injection of mesenchymal stem cells augments basal muscle protein synthesis after bouts of resistance exercise in male mice.

Skeletal muscle mass is critical for activities of daily living. Resistance training maintains or increases muscle mass, and various strategies maximize the training adaptation. Mesenchymal stem cells (MSCs) are multipotent cells with differential potency in skeletal muscle cells and the capacity to secrete growth factors. However, little is known regarding the effect of intramuscular injection of MSCs on basal muscle protein synthesis and catabolic systems after resistance training. Here, we measured changes in basal muscle protein synthesis, the ubiquitin-proteasome system, and autophagy-lysosome system-related factors after bouts of resistance exercise by intramuscular injection of MSCs. Mice performed three bouts of resistance exercise (each consisting of 50 maximal isometric contractions elicited by electrical stimulation) on the right gastrocnemius muscle every 48 h, and immediately after the first bout, mice were intramuscularly injected with either MSCs (2.0 × 106 cells) labeled with green fluorescence protein (GFP) or vehicle only placebo. Seventy-two hours after the third exercise bout, GFP was detected only in the muscle injected with MSCs with concomitant elevation of muscle protein synthesis. The injection of MSCs also increased protein ubiquitination. These results suggest that the intramuscular injection of MSCs augmented muscle protein turnover at the basal state after consecutive resistance exercise.

Comparative Study of Postmortem MRI and Pathological Findings in Malignant Brain Tumors.

This study compared magnetic resonance imaging (MRI) findings of postmortem brain specimens with neuropathological findings to evaluate the value of postmortem MRI. Postmortem MRI was performed on five formalin-fixed whole brains with malignant tumors. Postmortem T2-weighted images detected all neuropathological abnormalities as high-signal regions but also showed histological tumor invasion in areas without edema. Tumor lesions with high necrosis and edema showed high signal intensity on T2-weighted images; in three cases, lesion enlargement was detected on the final prenatal imaging and postmortem MRI. Disease progression immediately before death may have contributed to this difference. In conclusion, the correlation between MRI and neuropathological findings facilitates understanding of the mechanisms responsible for MRI abnormalities. Increased free water due to edema, necrosis, and brain tissue injury can explain the increased signal intensity observed on T2-weighted images. Postmortem MRI may contribute to effective pathology by identifying subtle abnormalities prior to brain dissection.

Neonatal Isolation Increases the Susceptibility to Learned Helplessness through the Aberrant Neuronal Activity in the Ventral Pallidum of Rats.

Objective: : Environmental deprivation, a type of childhood maltreatment, has been reported to constrain the cognitive developmental processes such as associative learning and implicit learning, which may lead to functional and morphological changes in the ventral pallidum (VP) and pessimism, a well-known cognitive feature of major depression. We examined whether neonatal isolation (NI) could influence the incidence of learned helplessness (LH) in a rat model mimicking the pessimism, and the number of vesicular glutamate transporter 2 (VGLUT2)-expressing VP cells and Penk-expressing VP cells. Methods: : The number of escape failures from foot-shocks in the LH test was measured to examine stress-induced depression-like behavior in rats. The number of VGLUT2-expressing VP cells and Penk-expressing VP cells was measured by immunohistochemistry. Results: : In NI rats compared with Sham rats, the incidence of LH in adulthood was increased and VGLUT2-expressing VP cells but not Penk-expressing VP cells in adulthood were decreased. VGLUT2-expressing VP cells were decreased only in the LH group of NI rats and significantly correlated with the escape latency in the LH test. Conclusion: : These findings suggest that the aberrant VP neuronal activity due to environmental deprivation early in life leads to pessimistic associative and implicit learning. Modulating VP neuronal activity could be a novel therapeutic and preventive strategy for the patients with this specific pathophysiology.

Evaluation of Treatment Strategies for Male Prolactin-Secreting Pituitary Neuroendocrine Tumors.

Prolactin-secreting pituitary neuroendocrine tumors (PitNETs) are more common in women. Male patients may also have few symptoms and have macroadenomas extending outside the sella turcica. This study aimed to report the results of cabergoline treatment in male patients with prolactin-secreting PitNET. The study included nine male patients aged 26-65 years (median, 46 years) diagnosed with prolactin-secreting PitNETs. The age at onset, prolactin values, tumor size, symptoms, and treatment were assessed. The mean prolactin value at the initial presentation was 2734.6 ng/mL, and the mean maximum tumor diameter was 40.4 mm. Visual field disturbance was the most common symptom (44.4%), followed by headaches (33.3%), asymptomatic symptoms (11.1%), and galactorrhea (11.1%). Eight patients responded to cabergoline treatment with normalization of prolactin levels and tumor shrinkage. One patient did not respond to the cabergoline treatment and required surgical intervention. There were no cases of cerebrospinal fluid leakage. Cabergoline was found to be an effective treatment for male prolactin-secreting PitNETs.

Controlled Release of Oxygen from Calcium Peroxide in a Weak Acidic Condition by Stabilized Amorphous Calcium Carbonate Nanocoating for Biomedical Applications.

Calcium peroxide (CaO2) has recently attracted much attention as an oxygen-releasing biomaterial for tissue engineering. CaO2 has also been used in cancer therapies, such as photodynamic therapy. However, the uncontrollability of oxygen release after immersion in water is a challenge. Furthermore, the nanoscale surface chemistry of CaO2 on the oxygen release properties under cell culture conditions has not been taken into account in these applications. Herein, we report the stabilized amorphous calcium carbonate (ACC) nanocoating on CaO2 in a cell culture medium, which suppressed the reaction between CaO2 and water. Stabilized ACC was produced by the reaction between calcium hydroxide (Ca(OH)2) derived from CaO2 and sodium hydrogen carbonate (NaHCO3) including sodium dihydrogen phosphate (NaH2PO4) in a cell culture medium. In contrast, surface modification of CaO2 by calcium carbonate crystals was difficult due to the crystallization process via dissolution-reprecipitation. Strikingly, ACC-CaO2 showed pH-dependent oxygen release in a cell culture medium probably because of the dissolution of ACC under weak acidic condition. Since the environments in ischemic tissue and cancer are weakly acidic, our findings should be important for understanding and designing properties related to biomaterials and drugs using CaO2.

The cell surface is the place to be for brassinosteroid perception and responses.

Adjusting the microenvironment around the cell surface is critical to responding to external cues or endogenous signals and to maintaining cell activities. In plant cells, the plasma membrane is covered by the cell wall and scaffolded with cytoskeletal networks, which altogether compose the cell surface. It has long been known that these structures mutually interact, but the mechanisms that integrate the whole system are still obscure. Here we spotlight the brassinosteroid (BR) plant hormone receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) since it represents an outstanding model for understanding cell surface signalling and regulation. We summarize how BRI1 activity and dynamics are controlled by plasma membrane components and their associated factors to fine-tune signalling. The downstream signals, in turn, manipulate cell surface structures by transcriptional and post-translational mechanisms. Moreover, the changes in these architectures impact BR signalling, resulting in a feedback loop formation. This Review discusses how BRI1 and BR signalling function as central hubs to integrate cell surface regulation.

Assessing the Safety of Mechanically Fibrillated Cellulose Nanofibers (fib-CNF) via Toxicity Tests on Mice: Single Intratracheal Administration and 28 Days' Oral Intake.

Mechanically fibrillated cellulose nanofibers, known as fib-CNF (fiber length: 500 nm; diameter: 45 nm), are used in composites and as a natural thickener in foods. To evaluate their safety, we conducted a 28-day study in mice with inhalation exposure at 0.2 mg/body and oral administration of 400 mg/kg/day. Inhalation exposure to fib-CNF caused transient weight loss, changes in blood cell counts, and increased lung weights. These changes were attributed to adaptive responses. The oral administration of fib-CNF for 28 days resulted in no apparent toxic effects except for a slight decrease in platelet counts. The fib-CNF administration using the protocols studied appears to be safe in mice.

Analysis of Homo- and Heterotriple Helix Formation of Collagen Model Peptides and Evaluation of Their Stability in a Biological Environment.

Self-assembled materials have attracted attention and have been extensively studied because the reversibility of noncovalent interactions allows them to possess various properties, such as stimulus responsiveness and self-healing. Collagen model peptides have an amino acid sequence characteristic of the triple helix region of collagen and exhibit repeatable triple helix formation. Many studies of their applications have used homotrimers, and although some studies on heterotrimers have been reported, few have clarified the details. If the characteristics of heterotrimers can be revealed, they are expected to be applied as new self-assembled materials. In this study, we analyzed the detailed self-assembling properties of hetero- and homohelices formed by (proline-proline-glycine)10 (PPG)10 and (proline-hydroxyproline-glycine)10 (POG)10 to evaluate the potential of the helices for biomedical application. Fluorescein isothiocyanate-labeled (PPG)10 (F(PPG)10) and (POG)10 (F(POG)10) were synthesized to analyze the heterotriple helix formation using concentration quenching based on triple helix formation. When (PPG)10 was added to F(POG)10, the fluorescence intensity did not reach a plateau, while the fluorescence intensity reached about 100% in the other pairs such as (POG)10-F(POG)10, (PPG)10-F(PPG)10, and (POG)10-F(PPG)10. The critical triple helix formation concentration was 7 µM for the heterotrimer prepared under 1:2 mixing conditions of (PPG)10 and (POG)10, 320 µM for [(PPG)10]3, and 4 µM for [(POG)10]3, indicating that the triple helix formation concentration of the heterotrimer is almost half that of [(POG)10]3 but 45 times higher than [(PPG)10]3. Furthermore, the heterotrimer formed at 37 °C was stable after 5 days, which was the same as [(POG)10]3. These results suggest that heterotrimers have different association properties from homotrimers and are expected to be applied in nanotechnology and biomaterials as new self-assembled materials.

Miniaturization of CRISPR/Cas12-Based DNA Sensor Array by Non-Contact Printing.

DNA microarrays have been applied for comprehensive genotyping, but remain a drawback in complicated operations. As a solution, we previously reported the solid-phase collateral cleavage (SPCC) system based on the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 12 (CRISPR/Cas12). Surface-immobilized Cas12-CRISPR RNA (crRNA) can directly hybridize target double-stranded DNA (dsDNA) and subsequently produce a signal via the cleavage of single-stranded DNA (ssDNA) reporter immobilized on the same spot. Therefore, SPCC-based multiplex dsDNA detection can be performed easily. This study reports the miniaturization of SPCC-based spots patterned by a non-contact printer and its performance in comprehensive genotyping on a massively accumulated array. Initially, printing, immobilization, and washing processes of Cas12-crRNA were established to fabricate the non-contact-patterned SPCC-based sensor array. A target dsDNA concentration response was obtained based on the developed sensor array, even with a spot diameter of 0.64 ± 0.05 mm. Also, the limit of detection was 572 pM, 531 pM, and 3.04 nM with 40, 20, and 10 nL-printing of Cas12-crRNA, respectively. Furthermore, the sensor array specifically detected three dsDNA sequences in one-pot multiplexing; therefore, the feasibility of comprehensive genotyping was confirmed. These results demonstrate that our technology can be miniaturized as a CRISPR/Cas12-based microarray by using non-contact printing. In the future, the non-contact-patterned SPCC-based sensor array can be applied as an alternative tool to DNA microarrays.

Deferasirox Targeting Ferroptosis Synergistically Ameliorates Myocardial Ischemia Reperfusion Injury in Conjunction With Cyclosporine A.

BACKGROUND: Ferroptosis, an iron-dependent form of regulated cell death, is a major cell death mode in myocardial ischemia reperfusion (I/R) injury, along with mitochondrial permeability transition-driven necrosis, which is inhibited by cyclosporine A (CsA). However, therapeutics targeting ferroptosis during myocardial I/R injury have not yet been developed. Hence, we aimed to investigate the therapeutic efficacy of deferasirox, an iron chelator, against hypoxia/reoxygenation-induced ferroptosis in cultured cardiomyocytes and myocardial I/R injury. METHODS AND RESULTS: The effects of deferasirox on hypoxia/reoxygenation-induced iron overload in the endoplasmic reticulum, lipid peroxidation, and ferroptosis were examined in cultured cardiomyocytes. In a mouse model of I/R injury, the infarct size and adverse cardiac remodeling were examined after treatment with deferasirox, CsA, or both in combination. Deferasirox suppressed hypoxia- or hypoxia/reoxygenation-induced iron overload in the endoplasmic reticulum, lipid peroxidation, and ferroptosis in cultured cardiomyocytes. Deferasirox treatment reduced iron levels in the endoplasmic reticulum and prevented increases in lipid peroxidation and ferroptosis in the I/R-injured myocardium 24 hours after I/R. Deferasirox and CsA independently reduced the infarct size after I/R injury to a similar degree, and combination therapy with deferasirox and CsA synergistically reduced the infarct size (infarct area/area at risk; control treatment: 64±2%; deferasirox treatment: 48±3%; CsA treatment: 48±4%; deferasirox+CsA treatment: 37±3%), thereby ameliorating adverse cardiac remodeling on day 14 after I/R. CONCLUSIONS: Combination therapy with deferasirox and CsA may be a clinically feasible and effective therapeutic approach for limiting I/R injury and ameliorating adverse cardiac remodeling after myocardial infarction.