Butyrate, Valerate, and Niacin Ameliorate Anaphylaxis by Suppressing IgE-Dependent Mast Cell Activation: Roles of GPR109A, PGE2, and Epigenetic Regulation.

Short-chain fatty acids (SCFAs) are produced by the intestinal microbiota during the fermentation of dietary fibers as secondary metabolites. Several recent studies reported that SCFAs modulate the development and function of immune-related cells. However, the molecular mechanisms by which SCFAs regulate mast cells (MCs) remain unclear. In the current study, we analyzed the function and gene expression of mouse MCs in the presence of SCFAs in vitro and in vivo. We found that the oral administration of valerate or butyrate ameliorated passive systemic anaphylaxis and passive cutaneous anaphylaxis in mice. The majority of SCFAs, particularly propionate, butyrate, valerate, and isovalerate, suppressed the IgE-mediated degranulation of bone marrow-derived MCs, which were eliminated by the Gi protein inhibitor pertussis toxin and by the knockdown of Gpr109a. A treatment with the HDAC inhibitor trichostatin A also suppressed IgE-mediated MC activation and reduced the surface expression level of FcεRI on MCs. Acetylsalicylic acid and indomethacin attenuated the suppressive effects of SCFAs on degranulation. The degranulation degree was significantly reduced by PGE2 but not by PGD2. Furthermore, SCFAs enhanced PGE2 release from stimulated MCs. The SCFA-mediated amelioration of anaphylaxis was exacerbated by COX inhibitors and an EP3 antagonist, but not by an EP4 antagonist. The administration of niacin, a ligand of GPR109A, alleviated the symptoms of passive cutaneous anaphylaxis, which was inhibited by cyclooxygenase inhibitors and the EP3 antagonist. We conclude that SCFAs suppress IgE-mediated activation of MCs in vivo and in vitro involving GPR109A, PGE2, and epigenetic regulation.

Fabrication and Characterization of Dissolving Microneedles for Transdermal Drug Delivery of Apomorphine Hydrochloride in Parkinson's Disease.

PURPOSE: We fabricated and characterized polyvinyl alcohol (PVA)-based dissolving microneedles (MNs) for transdermal drug delivery of apomorphine hydrochloride (APO), which is used in treating the wearing-off phenomenon observed in Parkinson's disease. METHODS: We fabricated MN arrays with 11 × 11 needles of four different lengths (300, 600, 900, and 1200 µm) by micromolding. The APO-loaded dissolving MNs were characterized in terms of their physicochemical and functional properties. We also compared the pharmacokinetic parameters after drug administration using MNs with those after subcutaneous injection by analyzing the blood concentration of APO in rats. RESULTS: PVA-based dissolving MNs longer than 600 µm could effectively puncture the stratum corneum of the rat skin with penetrability of approximately one-third of the needle length. Although APO is known to have chemical stability issues in aqueous solutions, the drug content in APO-loaded MNs was retained at 25°C for 12 weeks. The concentration of APO after the administration of APO-loaded 600-µm MNs that dissolved completely in skin within 60 min was 81%. The absorption of 200-µg APO delivered by MNs showed a Tmax of 20 min, Cmax of 76 ng/mL, and AUC0-120 min of 2,829 ng・min/mL, compared with a Tmax of 5 min, Cmax of 126 ng/mL, and AUC0-120 min of 3,224 ng・min/mL for subcutaneous injection. The bioavailability in terms of AUC0-120 min of APO delivered by MNs was 88%. CONCLUSION: APO-loaded dissolving MNs can deliver APO via skin into the systemic circulation with rapid absorption and high bioavailability.

Characterization of a TNFR2-Selective Agonistic TNF-α Mutant and Its Derivatives as an Optimal Regulatory T Cell Expander.

Regulatory T cells (Tregs) are a subpopulation of lymphocytes that play a role in suppressing and regulating immune responses. Recently, it was suggested that controlling the functions and activities of Tregs might be applicable to the treatment of human diseases such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease. TNF receptor type 2 (TNFR2) is a target molecule that modulates Treg functions. In this study, we investigated the role of TNFR2 signaling in the differentiation and activation of mouse Tregs. We previously reported the generation of a TNFR2-selective agonist TNF mutant, termed R2agoTNF, by using our unique cytokine modification method based on phage display. R2agoTNF activates cell signaling via mouse TNFR2. In this study, we evaluated the efficacy of R2agoTNF for the proliferation and activation of Tregs in mice. R2agoTNF expanded and activated mouse CD4+CD25+ Tregs ex vivo. The structural optimization of R2agoTNF by internal cross-linking or IgG-Fc fusion selectively and effectively enhanced Treg expansion in vivo. Furthermore, the IgG-Fc fusion protein suppressed skin-contact hypersensitivity reactions in mice. TNFR2 agonists are expected to be new Treg expanders.

Evaluation of cholesterol crystals in carotid plaque by dual energy computed tomography.

Cholesterol crystals (CCs) in carotid plaques might be an indicator of vulnerability, although they have not been fully investigated and non-invasive methods of assessment have not been established. This study examines the validity of assessing CCs using dual-energy computed tomography (DECT) that uses X-rays with different tube voltages for imaging, allowing material discrimination. We retrospectively evaluated patients who had undergone preoperative cervical computed tomography angiography and carotid endarterectomy between December 2019 and July 2020. We developed CC-based material decomposition images (MDIs) by scanning CCs crystallized in the laboratory using DECT. We compared the percentage of CCs in stained slides defined by cholesterol clefts with the percentage of CCs displayed by CC-based MDIs. Thirty-seven pathological sections were obtained from 12 patients. Thirty-two sections had CCs; of these, 30 had CCs on CC-based MDIs. CC-based MDIs and pathological specimens showed a strong correlation. Thus, DECT allows the evaluation of CCs in carotid artery plaques.

Kaempferol Suppresses the Activation of Mast Cells by Modulating the Expression of FcεRI and SHIP1.

In the present study, we evaluated the effects of kaempferol on bone marrow-derived mast cells (BMMCs). Kaempferol treatment significantly and dose-dependently inhibited IgE-induced degranulation, and cytokine production of BMMCs under the condition that cell viability was maintained. Kaempferol downregulated the surface expression levels of FcεRI on BMMCs, but the mRNA levels of FcεRIα, ß, and γ-chains were not changed by kaempferol treatment. Furthermore, the kaempferol-mediated downregulation of surface FcεRI on BMMCs was still observed when protein synthesis or protein transporter was inhibited. We also found that kaempferol inhibited both LPS- and IL-33-induced IL-6 production from BMMCs, without affecting the expression levels of their receptors, TLR4 and ST2. Although kaempferol treatment increased the protein amount of NF-E2-related factor 2 (NRF2)-a master transcription factor of antioxidant stress-in BMMCs, the inhibition of NRF2 did not alter the suppressive effect of kaempferol on degranulation. Finally, we found that kaempferol treatment increased the levels of mRNA and protein of a phosphatase SHIP1 in BMMCs. The kaempferol-induced upregulation of SHIP1 was also observed in peritoneal MCs. The knockdown of SHIP1 by siRNA significantly enhanced IgE-induced degranulation of BMMCs. A Western blotting analysis showed that IgE-induced phosphorylation of PLCγ was suppressed in kaempferol-treated BMMCs. These results indicate that kaempferol inhibited the IgE-induced activation of BMMCs by downregulating FcεRI and upregulating SHIP1, and the SHIP1 increase is involved in the suppression of various signaling-mediated stimulations of BMMCs, such as those associated with TLR4 and ST2.

Salicylaldehyde Suppresses IgE-Mediated Activation of Mast Cells and Ameliorates Anaphylaxis in Mice.

Mast cells (MCs) play key roles in IgE-mediated immunoresponses, including in the protection against parasitic infections and the onset and/or symptoms of allergic diseases. IgE-mediated activation induces MCs to release mediators, including histamine and leukotriene, as an early response, and to produce cytokines as a late phase response. Attempts have been made to identify novel antiallergic compounds from natural materials such as Chinese medicines and food ingredients. We herein screened approximately 60 compounds and identified salicylaldehyde, an aromatic aldehyde isolated from plant essential oils, as an inhibitor of the IgE-mediated activation of MCs. A degranulation assay, flow cytometric analyses, and enzyme-linked immunosorbent assays revealed that salicylaldehyde inhibited the IgE-mediated degranulation and cytokine expression of bone-marrow-derived MCs (BMMCs). The salicylaldehyde treatment reduced the surface expression level of FcεRI, the high affinity receptor for IgE, on BMMCs, and suppressed the IgE-induced phosphorylation of tyrosine residues in intercellular proteins, possibly Lyn, Syk, and Fyn, in BMMCs. We also examined the effects of salicylaldehyde in vivo using passive anaphylaxis mouse models and found that salicylaldehyde administration significantly enhanced the recovery of a reduced body temperature due to systemic anaphylaxis and markedly suppressed ear swelling, footpad swelling, and vascular permeability in cutaneous anaphylaxis.

Evaluation of Drug Sorption on Laboratory Materials with Abraham Solvation Parameters of Drugs and its Prevention.

PURPOSE: Undesired drug sorption on laboratory material surfaces reduces the performance of analytical methods and results in the generation of unreliable data. Hence, we characterized the sorption of drugs and evaluated the sorption extent using a linear free energy relationship (LFER) model with Abraham solvation parameters of drugs. Furthermore, to prevent sorption, the effects of additives, such as organic solvents and salts, were evaluated. METHODS: The sorption of fifteen model drugs (concentration: 2 µM), with various physicochemical properties, on materials in 0.2% dimethyl sulfoxide aqueous solutions was evaluated. Drug sorption extent on the materials was determined using high-performance liquid chromatography. The obtained results were analyzed using an LFER model with Abraham solvation parameters of the drugs. The effect of additives on the sorption of itraconazole, one of the most hydrophobic drugs among those tested in this study, was investigated. RESULTS: Sorption was dependent on the physicochemical properties of drugs, rather than the type of materials used, and additives altered the rate of drug sorption. Equations were developed to evaluate the sorption extent (nmol) of drugs to glass and polypropylene using the Abraham solvation parameters of the drugs. CONCLUSIONS: LFER modeling with Abraham solvation parameters of drugs enabled us to evaluate drug sorption on materials. All the additives altered the rate of drug sorption, and some organic solvents effectively prevented sorption. The developed LFER model would be useful for assessment of the sorption properties of compounds in in vitro evaluations in drug discovery research and various other biochemical fields.

Advanced Formulation Design for Topical Creams Assisted with Vibrational Spectroscopic Imaging.

Vibrational spectroscopic imaging has become useful analytical tools for quality control of drug products. In this study, we applied microscopic attenuated total reflection (ATR)-IR and confocal Raman microscopy to elucidate microscopic structure of creams and for the formulation design in the development of semi-solid drug products. The model creams were prepared with prednisolone (PRD) and fluconazole (FLC) as active pharmaceutical ingredients and oily solvents such as mineral oil (MO), isopropyl myristate (IPM), benzyl alcohol (BA) and diethyl sebacate (DES). As a result of microscopic ATR-IR imaging, several domains indicating oily internal phase were observed, which had absorption around 1732 and 1734 cm-1 derived from MO, IPM and DES. In addition, domains of BA around 1009 cm-1 were observed at the complemental or similar position in the formulation with MO or DES, respectively. These results suggested that the creams were oil-in-water type and the distribution of domains would reflect the compatibility of the solvents. The contents of PRD and BA were determined quantitatively in each layer after the intentional separation of the creams and the results agreed well with the imaging analysis. Whereas, confocal Raman imaging allowed to visualize the distribution of the components in depth direction as well as two-dimensional plane. In particular, the Raman imaging would ensure the coexistence of FLC and BA as oily phase in the cream. From these results, the feasibility of spectroscopic imaging techniques was successfully demonstrated for the formulation design of semi-solid dosage forms.

Left Atrial Size and Ischemic Events after Ischemic Stroke or Transient Ischemic Attack in Patients with Nonvalvular Atrial Fibrillation.

BACKGROUND: The present study aimed to clarify the association between left atrial (LA) size and ischemic events after ischemic stroke or transient ischemic attack (TIA) in patients with nonvalvular atrial fibrillation (NVAF). METHODS: Acute ischemic stroke or TIA patients with NVAF were enrolled. LA size was classified into normal LA size, mild LA enlargement (LAE), moderate LAE, and severe LAE. The ischemic event was defined as ischemic stroke, TIA, carotid endarterectomy, carotid artery stenting, acute coronary syndrome or percutaneous coronary intervention, systemic embolism, aortic aneurysm rupture or dissection, peripheral artery disease requiring hospitalization, or venous thromboembolism. RESULTS: A total of 1,043 patients (mean age, 78 years; 450 women) including 1,002 ischemic stroke and 41 TIA were analyzed. Of these, 351 patients (34%) had normal LA size, 298 (29%) had mild LAE, 198 (19%) had moderate LAE, and the remaining 196 (19%) had severe LAE. The median follow-up duration was 2.0 years (interquartile range, 0.9-2.1). During follow-up, 117 patients (11%) developed at least one ischemic event. The incidence rate of total ischemic events increased with increasing LA size. Severe LAE was independently associated with increased risk of ischemic events compared with normal LA size (multivariable-adjusted hazard ratio, 1.75; 95% confidence interval, 1.02-3.00). CONCLUSION: Severe LAE was associated with increased risk of ischemic events after ischemic stroke or TIA in patients with NVAF.

Cold Flow Evaluation in Transdermal Drug Delivery Systems by Measuring the Width of the Oozed Adhesive.

The objective of this study was to develop a simpler and more practical quantitative evaluation method of cold flow (CF) in transdermal drug delivery systems (TDDSs). CF was forcibly induced by loading a weight on a punched-out sample (bisoprolol and tulobuterol tapes). When the extent of CF was analyzed using the area of oozed adhesive as following a previously reported method, the CF profiles were looked different between the samples 12 mm in diameter subjected to a 0.5-kg weight and samples 24 mm in diameter subjected to a 2.0-kg weight despite an equal load per unit area (4.42 g/mm2). The width of oozed adhesive around the original sample was suggested to be an index that properly describes the relationship between the load per unit area and the extent of CF. Further, it was clarified that the average CF width over the entire circumference of the sample was the same whether the samples were round or square as long as the sample area and load were the same. We also observed a linear relationship between the CF width and the aspect ratio of oval and rectangular samples. These results indicated that the CF properties of typical TDDS products lacking CF-proof processing at the edges could be determined by testing samples cut from the product rather than the whole TDDS patch. The proposed width measuring method was simple and useful for optimizing the composition of the adhesive and for testing the quality of the product.

Association of objectively measured physical activity and sedentary behavior with bone stiffness in peripubertal children.

INTRODUCTION: Physical activity (PA) is a key factor of bone mass acquisition in peripubertal children. Sedentary behavior (SB) has been shown to influence bone outcomes. This study aimed to examine the association between objectively measured PA and SB and bone stiffness in Japanese children. MATERIALS AND METHODS: Participants were fifth-grade children aged 10-11 years from Project Koshu. The stiffness index (SI) of the calcaneus was measured by quantitative ultrasound; PA and SB were evaluated by an accelerometer. Each PA parameter was divided into sex-specific tertile or stratified by recommended PA guideline [≥ 60 min/day of moderate-to-vigorous PA (MVPA)]. The SI was compared among PA and SB through analysis of covariance with Bonferroni correction. RESULTS: Of 174 children, complete data were obtained from 134 (60 boys and 74 girls). The SI in boys was higher in the highest tertile of MVPA than that in the other groups. A similar association was found in girls but was not significant. Children who met the PA guideline had higher SI than those who did not, but there was no significant difference. A negative relation was observed in girls, with the SI gradually decreasing along with increasing SB (p for trend = 0.038). This association was not observed among boys. CONCLUSION: This study suggests that MVPA is positively associated with bone stiffness in Japanese schoolchildren in boys and SB is negatively associated with that in girls. Reducing SB might be a brief modifiable factor for preventing lower peak bone mass in girls, in addition to increasing MVPA.

Effect of Subcutaneous Tissue on Changes in Thigh Circumference Following Anterior Cruciate Ligament Reconstruction.

Circumference measurements have been used to estimate muscle cross-sectional area (CSA) in clinical settings. Measurements of thigh circumference are affected by muscle and subcutaneous fat (SF). In fact, SF could increase over a short period. Therefore, clarifying the relationship between thigh circumference and muscle and SF following ACL reconstruction is important. This study's primary purpose was to examine pre- and post-operative changes in thigh circumference, thigh muscles and SF CSAs in both legs. Secondary, the relationship between thigh circumference and muscle and SF CSAs was examined to demonstrate that circumference measurements could be used to detect atrophy. Quadriceps, hamstrings, and SF CSAs at 15, 10, and 5 cm proximal to the patella were measured by MRI pre- and 4 weeks postoperatively to examine how reconstruction affected those tissues in the thighs. The results showed increases in SF CSA (r=0.72 at 10 cm, r=0.67 at 15 cm) greatly affected thigh circumference in females on the surgical side. In males, increases in SF CSA (r=0.83) at 15- and 5-cm and decreases in quadriceps muscle CSA (r=0.73) at 5 cm affected thigh circumference on the surgical side. Thigh circumference measurements might not reflect actual muscle CSA in ACL patients.

A trimeric structural fusion of an antagonistic tumor necrosis factor-α mutant enhances molecular stability and enables facile modification.

Tumor necrosis factor-α (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (TNFR1-selective antagonistic TNF mutant (R1antTNF)) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality. Here, we aimed to further improve and optimize the activity and behavior of this mutant protein both in vitro and in vivo Specifically, we examined a trimeric structural fusion of R1antTNF, formed via the introduction of short peptide linkers, as a strategy to enhance bioactivity and molecular stability. By comparative analysis with R1antTNF, the trimeric fusion, referred to as single-chain R1antTNF (scR1antTNF), was found to retain in vitro molecular properties of receptor selectivity and antagonistic activity but displayed a marked increase in thermal stability. The residence time of scR1antTNF in vivo was also significantly prolonged. Furthermore, molecular modification using polyethylene glycol (PEG) was easily controlled by limiting the number of reactive sites. Taken together, our findings show that scR1antTNF displays enhanced molecular stability while maintaining biological activity compared with R1antTNF.

Early Achievement of Blood Pressure Lowering and Hematoma Growth in Acute Intracerebral Hemorrhage: Stroke Acute Management with Urgent Risk-Factor Assessment and Improvement-Intracerebral Hemorrhage Study.

BACKGROUND: Previous studies have revealed that hematoma growth mainly occurs during the first 6 h after the onset of spontaneous intracerebral hemorrhage (ICH). Early lowering of blood pressure (BP) may be beneficial for preventing hematoma growth. However, relationships between timing of BP lowering and hematoma growth in ICH remain unclear. We investigated associations between timing of BP lowering and hematoma growth for ICH. METHODS: The Stroke Acute Management with Urgent Risk-factor Assessment and Improvement (SAMURAI)-ICH Study was a multicenter, prospective, observational study investigating the safety and feasibility of early (within 3 h from onset) reduction of systolic BP (SBP) to < 160 mm Hg with intravenous nicardipine for acute hypertension in cases of spontaneous ICH. The present study was a post hoc analysis of the SAMURAI-ICH study. We examined relationships between time from onset, imaging, and initiation of treatment to target SBP achievement and hematoma growth (absolute growth ≥6 mL) in ICH patients. Target SBP achievement was defined as the time at which SBP first became < 160 mm Hg. RESULTS: Among 211 patients, hematoma growth was seen in 31 patients (14.7%). The time from imaging to target SBP and time from treatment to target SBP were significantly shorter in patients without hematoma growth than in those with (p = 0.043 and p = 0.032 respectively), whereas no significant difference was seen in time from onset to SBP < 160 mm Hg between groups (p = 0.177). Patients in the lower quartiles of time from imaging to target SBP and time from treatment to target SBP showed lower incidences of hematoma growth (p trend = 0.023 and 0.037 respectively). The lowest quartile of time from imaging to target SBP (< 38 min) was negatively associated with hematoma growth on multivariable logistic regression (OR 0.182; 95% CI 0.038-0.867; p = 0.032). CONCLUSIONS: Early achievement of target SBP < 160 mm Hg is associated with a lower risk of hematoma growth in ICH.

Two-Year Outcomes of Anticoagulation for Acute Ischemic Stroke With Nonvalvular Atrial Fibrillation　- SAMURAI-NVAF Study.

BACKGROUND: We determined the 2-year long-term risk-benefit profile in patients with stroke or transient ischemic attack (TIA) receiving warfarin or direct oral anticoagulants (DOACs) for nonvalvular atrial fibrillation (NVAF) using a prospective, multicenter, observational registry in Japan.Methods and Results:NVAF patients within 7 days after onset of ischemic stroke/TIA were enrolled in 18 stroke centers. Outcome measures included ischemic and bleeding events and death in the 2-year follow-up period. We enrolled 1,116 patients taking either warfarin (650 patients) or DOACs (466 patients) at acute hospital discharge. DOAC users were younger and had lower National Institutes of Health Stroke Scale, CHADS2and discharge modified Rankin Scale scores than warfarin users (P<0.0001 each). Incidences of stroke/systemic embolism (adjusted hazard ratio, 1.07; 95% CI, 0.66-1.72), all ischemic events (1.13; 0.72-1.75), and ischemic stroke/TIA (1.58; 0.95-2.62) were similar between groups. Risks of intracranial hemorrhage (0.32; 0.09-0.97) and death (0.41; 0.26-0.63) were significantly lower for DOAC users. Infection was the leading cause of death, accounting for 40% of deaths among warfarin users. CONCLUSIONS: Stroke/TIA patients receiving DOACs for secondary prevention were younger and had lower stroke severity and risk indices than those receiving warfarin. Estimated cumulative incidences of stroke and systemic embolism within 2 years were similar between warfarin and DOACs users, but those of death and intracranial hemorrhage were significantly lower among DOAC users.

Sex Differences in Birth Weight and Physical Activity in Japanese Schoolchildren.

BACKGROUND: Lower birth weight (BW) is associated with increased chronic disease risk later in life. Previous studies suggest that this may be mediated principally via physical activity (PA). However, the association between BW and PA in children has not been clarified. The purpose of this study was to examine the association between BW and PA in school-aged children in Japan. METHODS: Participants were children from a prospective birth cohort study (Project Koshu) who were born from 1996 through 2002 in rural Japan. BWs were obtained from the Maternal and Child Health Handbook. Data on PA during childhood were collected using a self-reported questionnaire when participants were 9-15 years of age in July 2011. Analysis of covariance was used to evaluate exercise duration; Poisson regression analysis was used to evaluate if the recommended PA amount was met. RESULTS: Data from 657 children (boys: 54.8%, follow-up rate: 77.6%) were analyzed. Compared with the normal BW group, only girls in the low-BW group had significantly lower PA level (normal BW, 11.4 [standard error, 1.0] hours/week; low BW, 5.8 [standard error, 3.6] hours/week, P = 0.010), and were more likely to not meet the recommended PA level (prevalence ratio 1.57; 95% CI, 1.14-2.16). CONCLUSION: Low BW was associated with a lower PA level in school-aged girls but not boys. Earlier consideration of BW may be an important public health strategy to prevent physical inactivity in school-aged girls.

Inner Blood-Retinal Barrier Dominantly Expresses Breast Cancer Resistance Protein: Comparative Quantitative Targeted Absolute Proteomics Study of CNS Barriers in Pig.

The purpose of this study was to determine absolute protein expression levels of transporters at the porcine inner blood-retinal barrier (BRB) and to compare the transporter protein expression quantitatively among the inner BRB, outer BRB, blood-brain barrier (BBB), and blood-cerebrospinal fluid barrier (BCSFB). Crude membrane fractions of isolated retinal capillaries (inner BRB) and isolated retinal pigment epithelium (RPE, outer BRB) were prepared from porcine eyeballs, while plasma membrane fractions were prepared from isolated porcine brain capillaries (BBB) and isolated choroid plexus (BCSFB). Protein expression levels of 32 molecules, including 16 ATP-binding-cassette (ABC) transporters and 13 solute-carrier (SLC) transporters, were measured using a quantitative targeted absolute proteomic technique. At the inner BRB, five molecules were detected: breast cancer resistance protein (BCRP, ABCG2; 22.8 fmol/µg protein), multidrug resistance protein 1 (MDR1, ABCB1; 8.70 fmol/µg protein), monocarboxylate transporter 1 (MCT1, SLC16A1; 4.83 fmol/µg protein), glucose transporter 1 (GLUT1, SLC2A1; 168 fmol/µg protein), and sodium-potassium adenosine triphosphatase (Na+/K+-ATPase; 53.7 fmol/µg protein). Other proteins were under the limits of quantification. Expression of MCT1 was at least 17.6-, 11.0-, and 19.2-fold greater than those of MCT2, 3, and 4, respectively. The transporter protein expression at the inner BRB was most highly correlated with that at the BBB (R2 = 0.8906), followed by outer BRB (R2 = 0.7988) and BCSFB (R2 = 0.4730). Sodium-dependent multivitamin transporter (SMVT, SLC5A6) and multidrug resistance-associated protein 1 (MRP1, ABCC1) were expressed at the outer BRB (0.378 and 1.03 fmol/µg protein, respectively) but were under the limit of quantification at the inner BRB. These findings may be helpful for understanding differential barrier function.

Generation and characterization of a bispecific diabody targeting both EPH receptor A10 and CD3.

The EPH receptor A10 (EphA10) is up-regulated in breast cancer but is not normally expressed in healthy tissue, thus it has been suggested that EphA10 may be a useful target for cancer therapy. This study reports a diabody, an antibody derivative binding two different target molecules, EphA10 expressed in tumor cells and CD3 expressed in T cells, which showed T cell dependent-cytotoxicity. The diabody, which has His-tagged and FLAG-tagged chains, was expressed in Escherichia coli and purified in both heterodimer (Db-1) and homodimer (Db-2) formulations by liquid chromatography. Flow cytometry analysis using EphA10-expressing cells showed that binding activity of heterodimers was stronger than that of homodimers. Addition of diabodies to PBMC cultures resulted in T-cell mediated redirected lysis, and the bioactivity was consistent with the stronger binding activity of heterodimeric diabody formulations. Our results indicate that diabodies recognizing both EphA10 and CD3 could have a range of potential applications in cancer therapy, such as breast cancers that express the EPH receptor A10, especially triple negative breast cancer.

High-intensity exercise causes greater irisin response compared with low-intensity exercise under similar energy consumption.

Irisin is mainly released from skeletal muscle (myocytes) and promotes thermogenesis by browning of the white adipose tissue. Although exercise has been shown to increase irisin concentration in blood and myocytes via up-regulation peroxisome proliferator receptor γ coactivator-1α (PGC-1α) expression, the influence of exercise intensity on irisin secretion remains unclear. Therefore, we determined circulating irisin responses following a single bout of running at different intensities. Six sedentary males underwent treadmill running under two different conditions: a low-intensity (40% of VO2max) exercise trial (LIE) or a high-intensity (80% of VO2max) exercise trial (HIE). The exercises in LIE and HIE were lasted for 20 and 40 min, respectively. All subjects underwent the two trials on separate days, and a randomized cross-over design was used. Blood samples were collected before (Pre) and immediately after exercise, at 3, 6, and 19 h after exercise. Energy consumption during exercise did not significantly differ between the two trials. HIE significantly increased blood lactate and serum lactate dehydrogenase levels (P < 0.05). Compared with pre-exercise levels, the irisin concentrations were elevated at 6 h (18% increase) and 19 h (23% increase) after HIE, but significantly decreased after LIE. The relative irisin concentrations (compared with pre-exercise levels) were significantly greater in HIE than in LIE immediately after exercise, and at 6 and 19 h after exercise (P < 0.05). These findings suggest that irisin secretion after acute running exercise is affected by exercise intensity, independent of energy consumption.

Cutting processability of metal-ion-containing cellulose nanofibril films by continuous wave laser.

Understanding the cutting processability of cellulose nanofibril (CNF) films by continuous wave laser is important for precise shape processing that closely follows the design pattern. In this study, laser cutting of films made of surface-carboxylated CNFs with various counterionic species was performed to explore the factors that control the cutting processability. The cut width and the thermally affected width are mainly controlled by the laser irradiation energy per unit length. The processed cross section is tapered and rises above the film thickness. NMR analysis suggests that the pyrolysates contain water-soluble cello-oligosaccharides, the molecular weight of which varies with the type of CNF film. We consequently demonstrated that the COOH-type CNF film is preferable to the COONa-type CNF film for reducing the coloration residue and for processing the film into a shape that best follows the designed processing pattern.