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Abstract Background Blood–brain barrier (BBB) dysfunction is supposed to be an early event in the development of Alzheimer’s disease (AD). This study aimed to investigate the relationship between BBB alterations and AD progression in terms of amyloid-β peptide (Aβ) accumulation in the brains of humanized amyloid precursor protein knock-in (APP-KI) mice. Methods Brain Aβ accumulation was examined using immunohistochemical analysis. Alterations in differentially expressed proteins were determined using sequential window acquisition of all theoretical fragment ion mass spectroscopy (SWATH-MS)-based quantitative proteomics, and Metascape, STRING, Gene Ontology, and KEGG were used for network analyses of altered biological pathways and processes. Statistical significance was determined using the unpaired two-tailed Student’s t-test and Welch’s t-test for two groups and one-way analysis of variance followed by Tukey’s test for more than two groups. Correlations between two groups were determined using Pearson’s correlation analysis. Results Brain Aβ accumulation in APP-KI mice was detectable at 2 months, increased significantly at 5 months, and remained elevated at 12 months of age. The levels of differentially expressed proteins in isolated brain capillaries were higher in younger mice, whereas those in the brain were higher in older mice. Network analyses indicated changes in basement membrane-associated and ribosomal proteins in the brain capillaries. There were no significant changes in key proteins involved in drug or Aβ transport at the BBB. In contrast, solute carrier transporter levels in astrocytes, microglia, and neurons were altered in the brain of older mice. Moreover, the levels of the lipid transporters Apoe and Apoj were upregulated in both the brain and isolated brain capillaries after Aβ accumulation. Conclusions Our results suggest that changes in the brain occurred after advanced Aβ accumulation, whereas initial Aβ accumulation was sufficient to cause alterations in the BBB. These findings may help elucidate the role of BBB alterations in AD progression and predict the distribution of drugs across the BBB in the brain of patients with AD.

Abstract Background The functions and protein expressions of the blood–brain barrier are changed throughout brain development following birth. This study aimed to develop a method to isolate brain capillaries from a single frozen neonatal mouse brain and elucidate the enrichment of brain capillaries by quantitative proteomic analysis. We further compared the expression profile of proteins between neonatal and adult brain capillary fractions. Methods The brain capillary fraction was prepared by the optimized method from a single frozen mouse neonatal brain on postnatal day 7. The brain capillary fractions and brain lysates were digested by trypsin and analyzed by liquid chromatography-mass spectrometry for quantitative proteomics. Results By optimizing the isolation method, we observed brain capillaries in the fraction prepared from a single neonatal mouse brain (nBC fraction). A protein amount of 31.5 μg, which is enough for proteomic analysis, was recovered from the nBC fraction. By proteomics analysis, the brain capillary selective proteins, including Abcb1a/Mdr1, Slc2a1/Glut1, Claudin-5, and Pecam-1, were found to be concentrated > 13.4-fold more in nBC fractions than in whole brain lysates. The marker proteins for neurons and astrocytes were not concentrated in nBC fractions, while those of pericytes and microglia were concentrated. Compared to adult mouse brain capillary fractions (aBC fractions), the expressions of Abcb1a/Mdr1a, Abcc4/Mrp4, and Slc2a1/Glut1 were significantly lower in nBC fractions than in aBC fractions, whereas those of Slc1a4/Asct1, Slc1a5/Asct2, Slc7a1/Cat1, and Slc16a1/Mct1 were significantly higher. Amino acid transporters, Slc38a5/Snat5, showed the greatest nBC-to-aBC ratio among transporters (9.83-fold). Network analysis of proteins expressed differentially between nBC and aBC fractions revealed that the proteins with terms related to the extracellular matrix were enriched. Conclusions We succeeded in isolating brain capillaries from a single frozen brain of a neonatal mouse at postnatal day 7. Proteomic analysis revealed the differential expression in brain capillaries between neonatal and adult mice. Specifically, amino acid transporters, including Slc1a5/Asct2 and Slc38a5/Snat5, were found to be induced in neonatal brain capillaries. The present isolation method will promote the study of the function and expression of the neonatal blood–brain barrier.

Heteroatom-embedded buckybowl molecules provide a fundamental molecular framework that has potential applications in various research fields. However, selective introduction of heteroatoms into buckybowls is challenging, and thus, studies investigating the effect of introduced heteroatoms have been limited. Here, we report the synthesis of pyridine-fused azacorannulene molecules. The influence of nitrogen atoms introduced into the peripheral positions on the structural, electronic, and optical properties is discussed.

Abstract Background The outcome of road traffic injury (RTI) is determined by duration of prehospital time, patient’s demographics, and the type of injury and its mechanism. During the emergency medical service (EMS) prehospital time interval, on-scene time should be minimized for early treatment. This study aimed to examine the factors influencing on-scene EMS time among RTI patients. Methods We evaluated 19,141 cases of traffic trauma recorded between April 2014 and March 2020 in the EMS database of the Nara Wide Area Fire Department and the prehospital database of the emergency Medical Alliance for Total Coordination of Healthcare (e-MATCH). To examine the association of the number of EMS phone calls until hospital acceptance, age ≥65 years, high-risk injury, vital signs, holiday, and nighttime (0:00–8:00) with on-scene time, a generalized linear mixed model with random effects for four study regions was conducted. Results EMS phone calls were the biggest factor, accounting for 5.69 minutes per call, and high-risk injury accounted for an additional 2.78 minutes. Holiday, nighttime, and age ≥65 years were also associated with increased on-scene time, but there were no significant vital sign variables for on-scene time, except for the level of consciousness. Regional differences were also noted based on random effects, with a maximum difference of 2 minutes among regions. Conclusions The number of EMS phone calls until hospital acceptance was the most significant influencing factor in reducing on-scene time, and high-risk injury accounted for up to an additional 2.78 minutes. Considering these factors, including regional differences, can help improve the regional EMS policies and outcomes of RTI patients.

Heterofullerenes are an important synthetic target for organic chemists. Here, the authors demonstrate a fully conjugated azacorannulene dimer starting from a bifunctional polycyclic aromatic azomethine ylide, providing a powerful method for the bottom-up synthesis of large multiazafullerene fragments

Collagen is a major structural protein in the human body. It not only provides connective tissues such as ligaments and tendons with toughness and strength but it also constitutes the biomechanical scaffold for cell attachment in the extracellular matrix. Collagen molecules aggregate to form collagen fibrils, which are fibers with a diameter of 10 $\rm {nm}$ to 500 $\rm {nm}$ and a length of up to a centimeter. Tensile tests on individual collagen fibrils reveal a strongly non-linear stress-response to deformation with the tensile modulus reaching the gigapascal range. But not only that, collagen fibrils have been found to be viscoelastic which means collagen fibrils have both elastic and viscous characteristics. However, direct measurement of viscoelastic material properties in tension is only possible with force-controlled tensile tests, which can not be conducted with state-of-the-art methods. In this paper, we report the first force-controlled tensile tests of individual collagen fibrils. To account for the non-linear material characteristics, high responsiveness in the force control and robustness about a property change of the collagen fibril are needed. Therefore, a two-degrees-of-freedom (2-DOF) controller is applied for force control with high responsiveness. The 2-DOF controller is composed of feedforward (FF) and feedback (FB) controllers. In addition, a modeling error compensation is implemented for robustness. The modeling error is calculated from the difference between the actual force response measured by the sensor and the ideal force response calculated from the plant model. The validity of the proposed control method is confirmed from simulation and experimental results.

Background: Some studies have reported that adhesion prevention barriers (APBs) reduce adhesion after abdominal surgery; however, evidence showing that APBs reduce the incidence of postoperative small bowel obstruction (SBO), one of the most serious complications after abdominal surgery, is little. One concern is that APBs are usually applied only under the midline incision, although adhesion can occur at any place in the peritoneum where an incision is made during surgery. INTERCEED® is an APB that reportedly prevents postoperative SBO after surgery. This study aims to assess the clinical utility of INTERCEED® for the prevention of SBO after laparoscopic colorectal cancer surgery and determine whether the application site of INTERCEED® affects the incidence of SBO. Methods/Design: This study is a prospective, multicenter, observational study conducted in Japan. The primary end point is the incidence of postoperative SBO 2 years after laparoscopic colorectal cancer surgery. The secondary end points include whether the site of the application of INTERCEED® affects the incidence of SBO. Each surgeon selects one of the following three procedures: 1) INTERCEED® is placed only under the midline incision; 2) INTERCEED® is placed at the site of bowel mobilization and/or lymph node dissection, but not under the midline incision; and 3) INTERCEED® is placed at both sites. Discussion: This is the first study to assess whether the placement of APBs affects the incidence of SBO. The study results may lead to a subsequent randomized study.

A 68-year-old male was found unconscious in a hot bathtub on a cold winter’s day and was brought to our hospital. On arrival, the patient’s systolic blood pressure was 65 mm hg, heart rate was 134 beats per min, and rectal temperature was 40.5°C. A large volume infusion of acetated Ringer’s solution and vasopressors were administered as an immediate treatment for circulatory failure due to heatstroke. Although the circulatory failure temporarily improved, the blood pressure dropped almost to the point of cardiac arrest. Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) was implemented and the hemodynamics were soon stabilized. Subsequently, VA-ECMO was withdrawn on the third hospital day. The patient recovered enough to feed normally on the 40th hospital day and was transferred to another hospital on the 116th hospital day. Based on this case study, we consider that VA-ECMO implementation before cardiac arrest might be a suitable strategy.

Abstract Introduction Primary malignant melanoma of the esophagus is a rare and aggressive disease that tends to have a poor response to chemotherapies. Previous studies have indicated that currently available treatment for primary malignant melanoma of the esophagus is insufficient. Here, we describe a case of recurrent primary malignant melanoma of the esophagus successfully treated with the immune checkpoint inhibitor nivolumab. Case presentation An 81-year-old Japanese female presented with a 3-month history of dysphagia. She was medicated for hypertension and sarcoidosis. The patient had no past history of cutaneous, ocular, or other-site melanomas. An esophagoscopy identified a 30-mm giant tumor in the lower esophagus, at a site 30 cm from the incisors. Enhanced computed tomography revealed wall thickening measuring 30 mm in size at the middle-third of the intrathoracic esophagus, with no significant lymph node infiltration or distant metastasis. Esophageal biopsy showed proliferation of large round tumor cells and melanophages. On the basis of these findings, the patient was diagnosed with esophageal malignant melanoma and underwent esophagectomy and lymph node dissection with gastric tube reconstruction. Although the pathological diagnosis was primary malignant melanoma of the esophagus, the patient presented with multiple lymph node and bone metastases 4 months after surgery. Subsequently, treatment with nivolumab 240 mg every 2 weeks was administered as the first-line treatment. Diffusion-weighted imaging with background body signal suppression following eight courses of nivolumab revealed that the multiple lymph node and bone metastases were markedly reduced. The patient received 30 courses of nivolumab and has maintained the partial response. No severe adverse events related to the immunotherapy were recorded. Conclusion The current study suggests that nivolumab may be a viable option for patients with metastatic primary malignant melanoma of the esophagus. Additional evidence from future clinical trials and research is necessary to fully validate these findings.

Deracemization is a powerful method which allows transformation of racemic mixtures into excess enantiomer, but was applied only to small chiral molecular systems so far. Here the authors report deracemization of a kinetically stable bisporphyrin helicate upon encapsulation of chiral aromatic guests.

Objectives: Temporary ileostomy is used to decrease morbidity from anastomotic leakages (ALs). However, ileostomies are associated with complications (i.e., stoma-related complications; SRCs), ileus due to stenosis, dehydration, and the need for a second operation. Here we retrospectively evaluated the impact of SRCs on the treatment of rectal cancer. Methods: We identified 180 consecutive patients who underwent curative resection for rectal cancer at Juntendo University Hospital between January 2006 and December 2014. We divided the patients into groups with and without defunctioning stoma (DS), and we compared the patient age and gender, tumor location, approach (laparotomy/laparoscopy), surgical procedure, distance of the tumor from the margin of the anus, T factor, stage, duration of postoperative hospital stay, and postoperative complications between these groups. Univariate and multivariate analyses were performed to determine the risk factors for postoperative hospital stay. Results: The symptomatic leakage rate in the DS group (n = 92) was not significantly different from that of the non-DS group (n = 88; p = 0.29). However, Grade 4 AL occurred significantly less frequently in the DS group (0%) than in the non-DS group (5.7%; p = 0.02). SRCs occurred in 14 DS-group patients (15.2%). The multivariate analysis demonstrated that both AL (odds ratio [OR] 9.24; confidence interval [CI] 4.91-19.4) and SRC (OR 1.84; CI 1.03-3.54) were independently predictive of short-term outcomes. Conclusions: The benefit of a DS is balanced against the risk of leakage and SRCs at rectal resection. Surgeons should focus on not only the consequences of AL, but also SRC risk.

Endoscopic unroofing is effective for treating large colonic lipomas. However, additional endoscopic resection is occasionally required when the outcomes of initial unroofing are incomplete. The colonoscopy of an 82-year-old woman with abdominal pain revealed a yellowish lipoma of about 20 mm in the transverse colon. The mass was treated by unroofing, but a follow-up colonoscopy 5 days later revealed residual lipoma. One month later, the regenerated surface had become covered with mucosa, and the status of the lipoma had returned to that before unroofing. The colonoscopy of a 74-year-old man with abdominal pain and melena revealed a 50-mm-wide protruding lipoma in the transverse colon. The mucosa of the upper third of the lipoma was excised using an electric knife and snare, which allowed the immediate partial drainage of adipose tissue. Unroofing proceeded, but 7 days later, the unroofed surface had become coated with a white substance, and the residual lipoma required additional endoscopic resection. Colonic lipomas are often asymptomatic. However, patients with abdominal pain and hemorrhage should be treated in consideration of complete resection, but not by unroofing, which could leave a residual tumor. Drainage should be confirmed after unroofing and any residual lipoma should be treated by additional resection.

Cell-surface proteins that can endocytose into brain microvascular endothelial cells serve as promising candidates for receptor-mediated transcytosis across the blood–brain barrier (BBB). Here, we comprehensively screened endocytic cell-surface proteins in hCMEC/D3 cells, a model of human brain microvascular endothelial cells, using surface biotinylation methodology and sequential window acquisition of all theoretical fragment-ion spectra-mass spectrometry (SWATH-MS)-based quantitative proteomics. Using this method, we identified 125 endocytic cell-surface proteins from hCMEC/D3 cells. Of these, 34 cell-surface proteins were selectively internalized into human brain microvascular endothelial cells, but not into human umbilical vein endothelial cells (HUVECs), a model of human peripheral microvascular endothelial cells. Two cell-surface proteins, intercellular adhesion molecule-1 (ICAM1) and podocalyxin (PODXL), were identified as BBB-localized endocytic cell-surface proteins in humans, using open mRNA and protein databases. Immunohistochemical evaluation confirmed PODXL expression in the plasma membrane of hCMEC/D3 cells and revealed that anti-PODXL antibody-labeled cell-surface PODXL internalized into hCMEC/D3 cells. Immunohistochemistry further revealed that PODXL is localized at the luminal side of human brain microvessels, supporting its potential suitability for translational applications. In conclusion, our findings highlight novel endocytic cell-surface proteins capable of internalizing into human brain microvascular endothelial cells. ICAM1 or PODXL targeted antibody or ligand-labeled biopharmaceuticals and nanocarriers may provide effective targeted delivery to the brain across the BBB for the treatment of central nervous system (CNS) diseases.

We developed an efficient system for delivering short interfering RNA (siRNA) to the liver by using α-tocopherol conjugation. The α-tocopherol–conjugated siRNA was effective and safe for RNA interference–mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid)-DNA gapmer antisense oligonucleotide (ASO) was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5′-end of the ASO sequence by using 5′-α-tocopherol–conjugated 4- to 7-mers of unlocked nucleic acid (UNA) as a “second wing.” Intravenous injection of mice with this α-tocopherol–conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS) molecules are bound to ASO with UNA sequences.

Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues.

BackgroundThe M(5) muscarinic acetylcholine receptor is known to play a crucial role in mediating acetylcholine dependent dilation of cerebral blood vessels. Previously, we reported that male M(5) muscarinic acetylcholine knockout mice (M5R(-/-) mice) suffer from a constitutive constriction of cerebral arteries, reduced cerebral blood flow, dendritic atrophy, and short-term memory loss, without necrosis and/or inflammation in the brain.Methodology/principal findingsWe employed the Magnetic Resonance Angiography to study the area of the basilar artery in male and female M5R(-/-) mice. Here we show that female M5R(-/-) mice did not show the reduction in vascular area observed in male M5R(-/-) mice. However, ovariectomized female M5R(-/-) mice displayed phenotypic changes similar to male M5R(-/-) mice, strongly suggesting that estrogen plays a key role in the observed gender differences. We found that 17beta-estradiol (E2) induced nitric oxide release and ERK activation in a conditional immortalized mouse brain cerebrovascular endothelial cell line. Agonists of ERalpha, ERbeta, and GPR30 promoted ERK activation in this cell line. Moreover, in vivo magnetic resonance imaging studies showed that the cross section of the basilar artery was restored to normal in male M5R(-/-) mice treated with E2. Treatment with E2 also improved the performance of male M5R(-/-) mice in a cognitive test and reduced the atrophy of neural dendrites in the cerebral cortex and hippocampus. M5R(-/-) mice also showed astrocyte swelling in cortex and hippocampus using the three-dimensional reconstruction of electron microscope images. This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and the number of synapses.Conclusions/significanceOur findings indicate that M5R(-/-) mice represent an excellent novel model system to study the beneficial effects of estrogen on cerebrovascular function and cognition. E2 may offer new therapeutic perspectives for the treatment of cerebrovascular insufficiency related memory dysfunction.

This article provides an overview of Taiwan's recent presidential election and its potential impact on global affairs. Vice President Lai Ching-te of the Democratic Progressive Party (DPP) has been elected as Taiwan's next president, which has raised concerns in China. While China has expressed its commitment to peaceful reunification, it continues to exert pressure on the DPP administration. However, the likelihood of China attempting military unification during President Lai's term is considered low. The article discusses the potential for increased pressure from China on Taiwan, including diplomatic, legal, cognitive, and military measures. Economic pressure is also a possibility, with China potentially suspending zero tariffs on certain Taiwanese products. In response, Taiwan aims to strengthen its economic and security systems, enhance military capabilities, and build partnerships with democratic countries. The article emphasizes the importance of maintaining stability in the Taiwan Strait for the global economy and calls for the strengthening of international trade rules and dispute resolution mechanisms. [Extracted from the article]