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A pressure-sensitive sensor that reacts to pressure by changing its electrical resistance was used to measure the characteristics of standing mounts of estrous cattle. While an estrous heifer fitted with a sensor on the rump was standing when mounted by a bull (i.e., during a standing mount), the sensor reacted to the addition and removal of pressure against it by the bull. The times of electric contact signals produced by these pressure changes were recorded to pinpoint the time of the mounted response and the duration of the mount. To detect the characteristics of standing mounts accurately, three types of sensors with different sensitivities and various positions on the rump of heifers were examined. When 13.5-cm sensors that operated with a pressure of 1,000 g/cm squared were fitted on the line combining the highest points of the left and right hip bones toward the tail, or 6.5 cm behind this position, on the rump of heifers (200 to 225 kg), mounts by vasectomized bulls (278 to 556 kg) were accurately recorded with the sensors more than 85% of the time. However, the correlation between the observed and automatically recorded lengths of mounting varied with combinations of the mounted and mounting animals or with locations of the sensor (r = .19 to .86). The pressure-sensitive sensor proved to be a promising device for continuously measuring the characteristics of standing mounts, especially the exact time at which a mount occurs.

Monoclonal antibodies are one of the fastest growing class of drugs. Nevertheless, relatively few biologics target multispanning membrane proteins because of technical challenges. To target relatively small extracellular regions of multiple membrane-spanning proteins, synthetic peptides, which are composed of amino acids corresponding to an extracellular region of a membrane protein, are often utilized in antibody discovery. However, antibodies to these peptides often do not recognize parental membrane proteins. In this study, we designed fusion proteins in which an extracellular helix of the membrane protein glucose transporter 1 (Glut1) was grafted onto the scaffold protein Adhiron. In the initial design, the grafted fragment did not form a helical conformation. Molecular dynamics simulations of full-length Glut1 suggested the importance of intramolecular interactions formed by surrounding residues in the formation of the helical conformation. A fusion protein designed to maintain such intramolecular interactions did form the desired helical conformation in the grafted region. We then immunized an alpaca with the designed fusion protein and obtained VHH (variable region of heavy-chain antibodies) using the phage display method. The binding of these VHH antibodies to the recombinant Glut1 protein was evaluated by surface plasmon resonance, and their binding to Glut1 on the cell membrane was further validated by flow cytometry. Furthermore, we also succeeded in the generation of a VHH against another integral membrane protein, glucose transporter 4 (Glut4) with the same strategy. These illustrates that our combined biochemical and computational approach can be applied to designing other novel fusion proteins for generating site-specific antibodies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to investigate the influence of blade width and magnetic field strength on apparent diffusion coefficient values of periodically rotated overlapping parallel lines with enhanced reconstruction (PROPELLER) diffusion-weighted imaging in the head and neck at 1.5 and 3.0 T magnetic resonance imaging., Methods: Fifteen healthy volunteers (eight men and seven women; mean age 36.4 ± 10.1 years) underwent PROPELLER diffusion-weighted imaging using four blade widths at 3.0 T and three blade widths at 1.5 T. For the multigroup comparison of apparent diffusion coefficient values, we performed analysis of variance and Tukey-Kramer tests. The apparent diffusion coefficient values in each organ were compared among the different blade widths and magnetic field strengths by a t test. Inter and intra-observer agreements regarding apparent diffusion coefficient value measurements were evaluated using the intraclass correlation coefficient., Results: No significant differences were observed in apparent diffusion coefficient values of the cerebellum in each blade width at both field strengths. The apparent diffusion coefficient values of the right parotid glands were significantly lower for blade width 32 than for blade width 16 at 3.0 T (1.16 × 10 -3 mm 2 /s vs. 1.01 × 10 -3 mm 2 /s; P  < 0.05). The apparent diffusion coefficient values of bilateral parotid glands were significantly higher at 1.5 T than at 3.0 T. Intraclass correlation coefficients were almost perfect to substantial in the cerebellum, whereas they showed moderate agreement in the parotid glands., Conclusion: The largest blade width 32 at 3.0 T and high magnetic field strength lowered the apparent diffusion coefficient values of parotid glands of PROPELLER diffusion-weighted imaging. The apparent diffusion coefficient values in the parotid glands of PROPELLER diffusion-weighted imaging may be affected for blade width and magnetic field strength.

Introduction: Transthyretin amyloid cardiomyopathy (ATTR-CM)-a debilitating, fatal disease resulting from the deposition of transthyretin (TTR) amyloid fibrils-can be hereditary due to mutations in the TTR gene (ATTRm) or wild type (ATTRwt). The global prevalence of ATTR-CM is largely unknown, although likely underestimated, with no formal epidemiological prevalence studies in Japan. This study aimed to estimate the prevalence of ATTR-CM in a large in-hospital database in Japan., Methods: This was a retrospective, observational, cross-sectional study which utilized data from all adult patients (aged ≥ 20 years) in the hospital-based Japan Medical Data Vision (MDV) database from January 2010 to September 2018 to estimate the number of currently diagnosed ATTR-CM patients and describe their demographic and clinical characteristics and diagnostic modalities. ATTR-CM patients (ATTRwt and ATTRm) were identified using a range of diagnosis codes that were applied to create broad and narrow definitions of the disease., Results: Over the 9 years of this study, there were 3255 (155.8 per million adult patients in the MDV database) to 3992 (191.1 per million) diagnoses of ATTRwt and 67 (3.2 per million) to 106 (5.1 per million) diagnoses of ATTRm in the MDV database (based on the narrow and broad definitions, respectively). There were 444 (21.2 per million) diagnoses of amyloid light-chain (AL) amyloidosis. Considering only those patients who were also diagnosed with heart failure, there were 1468 (70.3 per million) to 1798 (86.1 per million) diagnoses of ATTRwt and 50 (2.4 per million) to 61 (2.9 per million) diagnoses of ATTRm. Most ATTRwt patients (~ 90%) did not have a record of endomyocardial or abdominal wall biopsy, or of scintigram., Conclusion: This retrospective study provides an estimate of the number of patients diagnosed with ATTR-CM in a large in-hospital database in Japan over a period of 9 years. Improving awareness of disease prevalence may improve diagnosis and treatment., Funding: Pfizer.

To solve the problem of uncontrolled therapeutic gene integration, which is a critical drawback of retroviral vectors for gene therapy, the integration sites of exogenous genes should be precisely controlled not to perturb endogenous gene expression. To accomplish this, we explored the possibility of site-specific integration using two six-finger artificial zinc-finger proteins (AZPs) tandemly conjugated via a flexible peptide linker (designated "Tandem AZP"). A Tandem AZP in which two AZPs recognize specific 19 bp targets in a donor and acceptor DNA was expected to site-specifically recruit the donor DNA to the acceptor DNA. Thereafter, an exogenously added integrase was expected to integrate the donor DNA into a specific site in the acceptor DNA (as it might be in the human genome). We demonstrated in vitro that in the presence of Tandem AZP, ΦC31 integrase selectively integrated a donor plasmid into a target acceptor plasmid not only at 30 °C (the optimum temperature of the integrase) but also at 37 °C (for future application in humans). We expect that with further improvement of our current system, a combination of Tandem AZP with integrase/recombinase will enable site-specific integration in mammalian cells and provide safer gene therapy technology.

Extreme caution should be taken to avoid uncontrollable bleeding in treating hypervascular tumors via bronchoscope. We report two cases of endobronchial metastasis of renal cell carcinoma treated with bronchial artery embolization (BAE) before endoscopic treatments. The intraluminal lesions were removed swiftly and safely. Although arterial embolization is not always efficacious in cases of tracheal lesions, BAE is effective for tumors located in the carina, bilateral main bronchus or intermediate bronchus. The addition of BAE before endoscopic tumor removal should be considered a treatment option in patients suffering from airway obstructions due to hypervascular tumors such as renal cell carcinoma.

The diagnosis of gastric metastasis from lung cancer is relatively rare in living patients. We describe a case of Type 4 tumor-like metastasis due to primary lung cancer diagnosed with immunohistochemical staining while the patient was alive. A 68-year-old man was admitted to our hospital because of epigastric pain. Gastrointestinal endoscopy revealed a Type 4 tumor and the histological examination showed poorly differentiated adenocarcinoma. His chest X-ray showed mass shadow in the right upper lung field. The resected specimens showed moderately differentiated adenocarcinoma., The diagnosis of gastric metastasis from lung cancer was made by immunohistochemical staining of the lung and gastric tumors which showed positive staining for Thyroid transcriptional factor-1. Diagnosis of gastric metastasis, especially Type 4 metastasis by lung cancer is difficult. However, immunohistochemical staining is very helpful for diagnosis of primary lung cancer metastasis at sites such as the gastrointestinal tract which are not normally prone to metastatis.

Small cell lung cancer (SCLC) is characterized by autocrine mechanisms. Stem cell factor (SCF) and its receptor c-kit can activate Akt and extracellular signal-regulated kinase (Erk) pathways. Imatinib mesylate (STI571) can inhibit c-kit tyrosine kinase activity, but clinical trials have resulted in failure. We investigated the possibility of SCF/c-kit-targeted therapy against SCLC. Using c-kit-positive SCLC cells (H209 and H69 cells) and SCF as a model of the autocrine mechanisms, the effects of SCF, LY294002, PD98059 or STI571 on Akt and Erk were assessed by Western blot analysis. The cell growth inhibitions of cisplatin, etoposide irinotecan and amrubicin (AMR) with or without SCF, LY294002, PD98059 or STI571 were evaluated by MTT assay. Treatment with SCF activated Akt and Erk and the activations were inhibited by STI571 in H209 but not in H69 cells. LY294002 and PD98059 inhibited SCF-induced Akt and Erk activation in H209 cells, respectively. STI571 alone did not exert growth inhibition in the SCF-treated cells. In H209 cells, SCF decreased the cytotoxicity of AMR, but not of other drugs. In H69 cells, SCF did not affect sensitivity to any drugs. LY294002 but not PD98059 restored or enhanced AMR-sensitivity in SCF-treated H209 or untreated H69 cells, respectively. STI571 restored the AMR-sensitivity of SCF-treated H209 cells to the basal level. If the SCF/c-kit contributes to Akt activation in vivo, the combination of STI571 and AMR may be effective against SCLC. Additionally, using a combination of AKT inhibitors and AMR may be a promising treatment in the future.

Background: Leukotriene B4 (LTB4) increases in induced sputum and exhaled breath condensate in people with asthma. Furthermore, the T(H)2-type immune response and airway hyperresponsiveness induced by ovalbumin sensitization is markedly suppressed in LTB4 receptor (BLT) 1 null mice. These studies suggest that LTB4 may contribute to asthma pathophysiology. However, the direct effects of LTB4 on human airway smooth muscle (ASM) have not been studied., Objectives: We sought to determine the expression of LTB4 receptors on human ASM and its functional role in mediating responses of human ASM cells, and the effect of LTB4 on these cells., Methods: Immunohistochemistry, RT-PCR, Western blotting, and flow cytometry were used to determine the expression of LTB4 receptors. To determine the effect of LTB4 on human ASM cells, cell proliferation was assessed by counting cells, and chemokinesis was assessed by gold particle phagokinesis assay., Results: We confirmed expression of both BLT1 and BLT2 in human ASM cells in bronchial tissue and in cell culture. LTB4 markedly induced cyclin D1 expression, proliferation, and chemokinesis of human ASM cells. LTB4 also induced phosphorylation of both p42/p44 mitogen-activated protein kinase (MAPK) and downstream PI3 kinase effector, Akt1. However, we observed no induction of c-Jun N-terminal kinase or p38 MAPK. Notably, LTB4-induced migration and proliferation of ASM cells were inhibited by the BLT1 specific antagonist, U75302, and by inhibitors of p42/p44 MAPK phosphorylation (U1026), and PI3 kinase (LY294002)., Conclusions: These observations are the first to suggest a role for a LTB4-BLT1 signaling axis in ASM responses that may contribute to the pathogenesis of airway remodeling in asthma.

Purpose: The combination of tegafur-uracil (UFT) with vinorelbine has provided synergistic activity against non-small cell lung cancer (NSCLC) in experimental models. The recommended dose of UFT in combination with vinorelbine in NSCLC was determined in a phase I study. The phase II study evaluated efficacy and tolerability of this combination in elderly patients., Methods: Vinorelbine was infused on days 1 and 8, and UFT was administered twice daily on days 2 to 6 and days 9 to 13 of a 3-week cycle. UFT and vinorelbine were increased during the phase I study from 400 to 600 mg/d and 20 to 25 mg/m(2), respectively, in 12 patients. In the phase II portion, previously untreated elderly patients were treated with 600 mg/d UFT and 20 mg/m(2) vinorelbine., Results: At the dose level of 600 mg/d UFT and 25 mg/m(2) vinorelbine, dose-limiting toxicity of neutropenia or neutropenic fever was observed in two of three patients, determining the recommended dose of 600 mg/d UFT and 20 mg/m(2) vinorelbine. In 30 evaluable elderly patients of the phase II study, the response rate was 27% (8/30). The median survival and progression-free survival time was 11.8 (range 2.7-34.8) and 5.0 (range 0.5-32.5) months, respectively. Grade 3 or grade 4 neutropenia and grade 3 anemia occurred in 40% and 7% of phase II patients, respectively. Gastrointestinal toxicity was frequent but mild. As the most serious toxicity, pneumonitis was observed in three patients., Conclusion: This combination of UFT and vinorelbine is both feasible and active in the treatment of elderly patients with advanced NSCLC.

Small cell lung cancer (SCLC) is one of the intractable malignancies. The goal of this study was to clarify whether Akt activity is involved with chemo-resistance and to improve the sensitivity of SCLC cells to the current standard chemotherapeutic drugs with agents that are expected to suppress Akt activity through tyrosine kinase inhibition. Although Akt activity seemed to be involved with the sensitivity of SCLC cells to chemotherapeutic agents (cisplatin, etoposide, SN38 and amrubicin), in Akt-activated N417 cells, only amrubicin exerted synergistic cell growth inhibition when combined with an Akt inhibitor, LY294002. A non-specific tyrosine kinase inhibitor, genistein, suppressed Akt and showed synergistic interaction in combination with amrubicin in N417 cells. Among tyrosine kinases (insulin-like growth factor I receptor, c-Kit and c-Src), only c-Src was activated in N417 cells compared with Akt-inactive H209 cells. A c-Src-specific inhibitor, PP2, and a clinically available multi-tyrosine kinase inhibitor, dasatinib, suppressed Akt activity in parallel with c-Src inhibition. Both PP2 and dasatinib exerted synergistic growth inhibition of N417 cells in the combination with amrubicin. In immunohistochemical analysis, c-Src was expressed in 17 of 19 of the SCLC tumor tissues. These observations suggested that Akt suppression enhances the cytotoxicity of amrubicin, and for the purpose of Akt suppression, c-Src is a promising target in SCLC.

Despite the high response rates of small cell lung cancer (SCLC) to first-line cisplatin-based chemotherapies, most patients with SCLC will eventually experience disease progression. Accordingly, novel chemotherapeutic regimens are desired. This in vitro study was carried out in order to develop novel chemotherapeutic regimens containing 5-fluorouracil (5-FU) or oral fluoropyrimidine for SCLC. 5-FU was combined with other standard drugs for SCLC (cisplatin, etoposide, an active metabolite of irinotecan and amrubicin) in different schedules. The combination effects were analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and an isobologram method using H69 SCLC cells. Among the examined combinations, synergistic growth inhibition was observed only when H69 cells were treated with 7-ethyl-10-hydroxycamptothecin (SN-38; an active metabolite of irinotecan) followed by 5-FU. The findings of a flow cytometric analysis were consistent with the enhancement of apoptotic cell death by this sequential treatment. This synergism was observed in 4 out of 5 SCLC cell lines tested. The effects of 5-FU and SN-38 on thymidylate synthase (TS) protein expression, an important determinant of 5-FU sensitivity, were assessed by Western blot analysis in H69 cells. Treatment with SN-38 for 24 h suppressed TS protein expression and this low level of TS was maintained for at least 72 h. Pretreatment with SN-38 inhibited the 5-FU-induced increase of TS protein. The synergistic effect induced by the combination of SN-38 and 5-FU may be attributable to the SN-38-induced suppression of TS protein. Furthermore, uracil and 5-chloro-2,4-hydroxypyridine, which are clinically available dihydropyrimidine dehydrogenase inhibitors, enhanced 5-FU-induced growth inhibition. These observations provide evidence supporting the clinical applications of the combination chemotherapy using irinotecan and 5-FU or oral fluoropyrimidines against SCLC.

The combination of trastuzumab with paclitaxel (PTX) is an important option for the treatment of HER2-positive breast cancer. Dexamethasone (Dex) premedication is routinely used in the treatment with PTX. The interactions among Dex, PTX and trastuzumab were evaluated in BT-474 cells. Dex interfered with trastuzumab-induced cell growth inhibition without clear effects on PTX-induced cytotoxicity. Trastuzumab dephosphorylated retinoblastoma protein (pRB). Dex restored this trastuzumab-induced dephosphorylation of pRB and released trastuzumab-induced G1 arrest. Trastuzumab suppressed AKT activity without affecting ERK activity. A specific inhibitor for the phosphatidylinositol 3-kinase/AKT pathway, LY294002, inhibited cell growth and AKT and pRB phosphorylation. Dex restored the trastuzumab-induced suppression of AKT without affecting ERK activity. It was concluded that Dex interferes with trastuzumab-induced cell growth inhibition, at least partially, through the restoration of trastuzumab-induced AKT suppression and subsequent pRB dephosphorylation in BT-474 breast cancer cells. These observations support the development of new chemotherapeutic regimens without glucocorticoid premedication.

Tablets containing both paracetamol (PM) and cloperastine hydrochloride (CLH) in a combination formulation prepared by standard vertical granulation technology were found to have altered pharmaceutical properties. The hardness and disintegration time of tablets containing both PM and CLH gradually increased during storage, and the cross-screw did not operate smoothly during preparation of the mixed powder. The objective of the present study was to investigate the mechanism of formation of eutectic mixtures consisting of PM and CLH. Binary mixtures of PM and CLH in various proportions were prepared as physical mixtures and analyzed by DSC to study their thermal behavior. Phase diagrams obtained from the endothermic peaks due to melting of physical mixtures of PM and CLH demonstrated the formation of eutectic mixtures with eutectic temperatures of 86.9-110.2 degrees C depending on the ratio of constituents. The formation of the eutectic mixture was studied for a 50:50 mol.% ratio of PM and CLH. PXRD analysis revealed that the eutectic mixture of PM and CLH is structurally different from native PM and CLH. The most probable interaction sites between PM and CLH were demonstrated by DSC analysis of a binary mixture of PM and CLH prepared by melt quenching.

Bronchial asthma is a chronic inflammatory disorder of the airways, in which inflammation causes bronchial hyper-responsiveness and flow limitation in the presence of various stimuli. Pulmonary function in asthmatic patients frequently deteriorates between midnight and early morning, which has suggested a role for chronotherapy. Although relationships between bronchial asthma and the function of clock genes remain unclear, some medications given for asthma such as glucocorticoids or beta(2)-adrenoceptor agonists may influence clock genes in vivo. In our studies of clock gene mRNA expressions in human bronchial epithelial cells in vitro and peripheral blood cells in vivo, we demonstrated that glucocorticoid or beta(2)-adrenoceptor agonist treatment strongly induced human Per1 mRNA expression both in vitro and in vivo. Human peripheral blood cells provide a useful indication of peripheral clock gene mRNA expression in vivo.

Paclitaxel is used frequently for the treatment of patients with non-small cell lung cancer. Hypersensitivity reactions remain one of the major adverse events in the clinical use of paclitaxel. Glucocorticoids are used to prevent these adverse events. This study was carried out in order to clarify the effect of glucocorticoids on paclitaxel-induced cytotoxity of cancer cells. Pretreatment with 10 microM of dexamethasone inhibited ERK activation and subsequent retinoblastoma protein (pRB) phosphorylation, and reduced sensitivity to paclitaxel in A549 cells. Then, we utilized ERK (PD98059) and AKT (LY294002) inhibitors. PD98059 and LY294002 effectively suppressed pRB phosphorylation in A549 cells. Dexamethasone (10 microM) suppressed ERK activity as well as PD98059, although it did not affect AKT activity. Furthermore, the combinations of paclitaxel with PD98059 or LY294002 were similarly antagonistic. Our observation in this study raised the possibility that dexamethasone pretreatment antagonizes paclitaxel-induced cytotoxicity through ERK suppression and pRB dephosphorylation. These observations support the development of new generation taxane-based chemotherapy without glucocorticoid premedication.

Noninvasive and nondestructive mobility assessment of dental implants is very important and useful for dental implantation diagnostic-aids. The development of implant movement (IM) checker based on microcontroller is presented in this paper. Data acquisition system and bender-type piezoelectric probe were used to improve measurement quality to the original tooth mobility (TM) tester. The adoption of a microcontroller and the use of a dental drill-sized measuring probe were sufficient in the reproducibility and reliability of the IM checker. When the implant was subjected to a constant force and amplitude, the acceleration of the model was detected using the measuring probe. The data acquisition system controlled for obtaining the appropriate acceleration signals based on the preload detection during measurement. Dental implant models of Molteno and Rigolac were made at different stiffness and were used to verify the reliability and validity of measurements. The values of measurements obtained by the IM checker were reliable and precise. The maximum error for perpendicular measurements was less than 12% measured by a new operator and decreased to 2% by an experienced operator. The IM checker was applied to monitor the stability of dental implantation, which compared the relative IM score of the new 5[see text] implant with the adjacent old 6 [see text] implant that had been used functionally for 3 years.

This study aimed at developing a dental implant movement (IM) checker for assessing quantitative dental implant mobility. The design of the instrument was based on the tooth mobility (TM) tester, which was previously developed by our group. The IM checker consists of a newly developed measuring probe which has the size of a typical dental drill so that it would be easy to measure at all regions of dental implants. The probe has a bimorph ceramics transducer for actuating an implant at constant frequency and force amplitude and for detecting acceleration response. A set of strain gauges were attached to the bimorph ceramics for detecting preload during measurement. A new digital data acquisition system was used to eliminate measurement artifacts mainly due to probe handling. The IM checker could discriminate the artificial dental implant models in the range of clinical tooth mobility M0 with variation less than 6%. The measuring time needed by five operators was less than 15 s. Accordingly, the IM checker has sufficient measuring reliability and therefore it could be introduced in dental clinics.

Two cases of primary signet-ring cell carcinoma of the urinary bladder are presented with histochemical studies. The signet-ring cell carcinoma of the bladder is a rare variant of mucus-producing vesical adenocarcinoma: sixteen cases have been reported in the Occidental and two other cases in Japanese literature. Most of the cases were discovered in the advanced stage and the prognosis was poor: necessity of early detection by cytological examination and treatment with surgery is stressed. The histochemical evidence for the similarity of the neoplasm to colonic epithelium in mucus production is illustrated and the grade of malignancy is discussed from the view of mucus production by the neoplasms.