A Transmembrane Single-Polypeptide-Chain (sc) Linker to Connect the Two G-Protein–Coupled Receptors in Tandem and the Design for an In Vivo Analysis of Their Allosteric Receptor- Receptor Interactions

A transmembrane (TM) single-polypeptide-chain (sc) linker can connect two G-protein–coupled receptors (GPCRs) in tandem. The priority of a gene-fusion strategy for any two class A GPCRs has been demonstrated. In the striatal function, dopamine (DA) plays a critical role. In the striatum, how the GPCR for adenosine, subtype A2A (A2AR), contributes to the DA neurotransmission in the “volume transmission”/dual-transmission model has been studied extensively. In addition to the fusion receptor, i.e., the prototype scA2AR/D2R complex (the GPCR for DA, subtype D2), several types were created and tested experimentally. To further elucidate this in vivo, we designed a new molecular tool, namely, the supermolecule scA2AR/D2R. Here, no experiments on its expression were done. However, the TM linker to connect the nonobligate dimer as the transient class A GPCR nanocluster that has not been identified at the cell surface membrane deserves discussion through scA2AR/D2R. Supramolecular designs, are experimentally testable and will be used to confirm in vivo the functions of the two GPCRs interactive in such a low specific signal to the nonspecific noise (S/N) ratio in the neurotransmission in the brain. The sc also has, at last, become straightforward in the field of GPCRs, similar to in the field of antibody

Recent development of the HMM-based speech synthesis system (HTS)

A statistical parametric approach to speech synthesis based on hidden Markov models (HMMs) has grown in popularity over the last few years. In this approach, spectrum, excitation, and duration of speech are simultaneously modeled by context-dependent HMMs, and speech waveforms are generate from the HMMs themselves. Since December 2002, we have publicly released an open-source software toolkit named “HMM-based speech synthesis system (HTS)” to provide a research and development toolkit for statistical parametric speech synthesis. This paper describes recent developments of HTS in detail, as well as future release plans

Lower FEV1 in non-COPD, nonasthmatic subjects: association with smoking, annual decline in FEV1, total IgE levels, and TSLP genotypes

Few studies have investigated the significance of decreased FEV1 in non-COPD, nonasthmatic healthy subjects. We hypothesized that a lower FEV1 in these subjects is a potential marker of an increased susceptibility to obstructive lung disease such as asthma and COPD. This was a cross-sectional analysis of 1505 Japanese adults. We divided the population of healthy adults with no respiratory diseases whose FEV1/FVC ratio was ≥70% (n = 1369) into 2 groups according to their prebronchodilator FEV1 (% predicted) measurements: <80% (n = 217) and ≥80% (n = 1152). We compared clinical data – including gender, age, smoking habits, total IgE levels, and annual decline of FEV1 – between these 2 groups. In addition, as our group recently found that TSLP variants are associated with asthma and reduced lung function, we assessed whether TSLP single nucleotide polymorphisms (SNPs) were associated with baseline lung function in non-COPD, nonasthmatic healthy subjects (n = 1368). Although about half of the subjects with lower FEV1 had never smoked, smoking was the main risk factor for the decreased FEV1 in non-COPD, nonasthmatic subjects. However, the subjects with lower FEV1 had a significantly higher annual decline in FEV1 independent of smoking status. Airflow obstruction was associated with increased levels of total serum IgE (P = 0.029) and with 2 functional TSLP SNPs (corrected P = 0.027–0.058 for FEV1% predicted, corrected P = 0.015–0.033 for FEV1/FVC). This study highlights the importance of early recognition of a decreased FEV1 in healthy subjects without evident pulmonary diseases because it predicts a rapid decline in FEV1 irrespective of smoking status. Our series of studies identified TSLP variants as a potential susceptibility locus to asthma and to lower lung function in non-COPD, nonasthmatic healthy subjects, which may support the contention that genetic determinants of lung function influence susceptibility to asthma

Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers

Resistance of progressive cancers against chemotherapy is a serious clinical problem. In this context, human epidermal growth factor receptor 3 (HER3) can play important roles in drug resistance to HER1- and HER2- targeted therapies. Since clinical testing of anti-HER3 monoclonal antibodies (mAbs) such as patritumab could not show remarkable effect compared with existing drugs, we generated novel mAbs against anti-HER3. Novel rat mAbs reacted with HEK293 cells expressing HER3, but not with cells expressing HER1, HER2 or HER4. Specificity of mAbs was substantiated by the loss of mAb binding with knockdown by siRNA and knockout of CRISPR/Cas9-based genome-editing. Analyses of CDR sequence and germline segment have revealed that seven mAbs are classified to four groups, and the binding of patritumab was inhibited by one of seven mAbs. Seven mAbs have shown reactivity with various human epithelial cancer cells, strong internalization activity of cell-surface HER3, and inhibition of NRG1 binding, NRG1-dependent HER3 phosphorylation and cell growth. Anti-HER3 mAbs were also reactive with in vivo tumor tissues and cancer tissue-originated spheroid. Ab4 inhibited in vivo tumor growth of human colon cancer cells in nude mice. Present mAbs may be superior to existing anti-HER3 mAbs and support existing anti-cancer therapeutic mAbs

Noninvasive Tracking of Donor Cell Homing by Near-Infrared Fluorescence Imaging Shortly after Bone Marrow Transplantation

BACKGROUND: Many diseases associated with bone marrow transplantation (BMT) are caused by transplanted hematopoietic cells, and the onset of these diseases occurs after homing of donor cells in the initial phase after BMT. Noninvasive observation of donor cell homing shortly after transplantation is potentially valuable for improving therapeutic outcomes of BMT by diagnosing the early stages of these diseases. METHODOLOGY/PRINCIPAL FINDINGS: Freshly harvested near-infrared fluorescence-labeled cells were noninvasively observed for 24 h after BMT using a photon counting device to track their homing process. In a congenic BMT model, the homing of Alexa Fluor 750-labeled donor cells in the tibia was detected less than 1 h after BMT. In addition, subsequent cell distribution in an intraBM BMT model was successfully monitored for the first time using this method. In the allogeneic BMT model, T-cell depletion decreased the near-infrared fluorescence (NIRF) signals of the reticuloendothelial system. CONCLUSIONS/SIGNIFICANCE: This approach in several murine BMT models revealed that the transplanted cells homed within 24 h after transplantation. NIRF labeling is useful for tracking transplanted cells in the initial phase after BMT, and this approach can contribute to in vivo studies aimed at improving the therapeutic outcomes of BMT

N 1 -Dansyl-Spermine and N 1 -(n-Octanesulfonyl)-Spermine, Novel Glutamate Receptor Antagonists: Block and Permeation of N-Methyl-D-Aspartate Receptors

SUMMARY The effects of several N-sulfonyl-polyamines, including N 1 -dansyl-spermine (N 1 -DnsSpm) and N 1 -(n-octanesulfonyl)-spermine (N 1 -OsSpm), were studied at recombinant N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus laevis oocytes. N 1 -DnsSpm and N 1 -OsSpm inhibited NMDA receptors and were ϳ1000-fold more potent than spermine in oocytes voltage-clamped at Ϫ70 mV. Block by N 1 -DnsSpm and N 1 -OsSpm was strongly voltage dependent, being more pronounced at hyperpolarized membrane potentials. With the Woodhull model of voltage-dependent channel block, the values of K d (0) were 779 M, 882 M, and 7.4 mM and those of z␦ were 2.58, 2.57, and 1.07 for N 1 -DnsSpm, N 1 -OsSpm, and spermine, respectively. This suggests that an increase in the voltage dependence of block together with an increase in affinity contributes to the increased potencies of N 1 -DnsSpm and N 1 -OsSpm compared with spermine. Sensitivity to N 1 -DnsSpm was reduced by mutation NR1(N616Q) and was increased by mutations NR1(N616G) and NR2A(N615G). The NR1(N616G) and NR2A(N615G) mutations decreased the K d (0) value of N 1 -DnsSpm without affecting z␦, whereas the NR1(N616Q) mutation reduced z␦. These mutations may alter the accessibility of part of the polyamine binding site within the channel pore or directly alter the properties of that site. Block by N 1 -DnsSpm (0.3 M) was almost complete at Ϫ100 mV, and there was no relief of block at extreme negative membrane potentials (Ϫ100 to Ϫ200 mV) at wild-type NR1/NR2A channels. In contrast, block by N 1 -DnsSpm was partially relieved at extreme negative potentials at receptors containing NR1(N616G) or NR2A(N615G), suggesting that N 1 -DnsSpm can permeate these mutant channels but not wild-type NR1/NR2A channels. This is hypothesized to be due to an increase in the pore size of channels containing NR1(N616G) or NR2A(N615G), which allows passage of the bulky head group of N 1 -DnsSpm. In contrast to N 1 -DnsSpm, N 1 -OsSpm could easily permeate wildtype NR1/NR2A channels, presumably because the head group of N 1 -OsSpm can pass through the narrowest part of the channel pore. N-Sulfonyl-polyamines such as N 1 -DnsSpm and N 1 -OsSpm represent a new class of polyamine antagonists with which to study glutamate receptor ion channels. The endogenous polyamine spermine has a variety of effects on NMDA and non-NMDA glutamate receptors (1, 2). At NMDA receptors, spermine has both stimulatory and inhibitory effects when applied extracellularly (3-7). Inhibition of NMDA receptors by spermine is strongly voltage dependent and may be caused by an open-channel block and/or screening of surface charges around the mouth or vestibule of the ion channe

MRI for Advanced Gastric Cancer : Especially for Scirrhous Cancer of the Stomach

We conducted MRI examinations in 92 patients with advanced gastric cancer, and evaluated the clinical potential of MRI for diagnosis of scirrhous cancer of the stomach. The feature of scirrhous cancer of stomach by MRI are ; 1) thick-ened gastric wall, 2) shortening of T1 and T2 values ; and 3) clear contrast between the gastric mucosae and cancer areas found in the T1 and T2 weighted images (preservation of the mucosae). MRI for scirrhous cancer of the stomach is thought a useful image diagnosis as an adjunct method to gastric X-ray and gastric endoscopy

Focal Hepatic Tumors Using Inversion Recovery Sequence of 0.1-T MRI - Basic : Clinical Evaluation Gray Scale Vs T1 Values

Optimum conditions for image quality contrast were studied with phantom method by means of MRI system (of constant conduction type ; 0.1-T) in order to detect tumor lesions of liver that show no distinct contrast by usual roentgeno-graphic methods. Signal intensity of liver, fat and muscle were maximally suppressed at 1000 ms of TR and 100 ms of TI by the short inversion time inversion recovery (STIR) method, resulting in distinct visualization of liver tumor with extremely good contrast. Clinical investigation with the usual T1- and T2- weighted images under the same conditions identified hepatocellular carcinoma in 22 out of 31 patients (37 of 58 nodules, 64%), cholangiocellular carcinoma 3 of 5 (3 of 6 nodules, 50%), metastatic liver cancer in 55 out of 68 (111 of 143 nodules, 78%), hepatic hemangioma in 32 out of 36 (41 of 47 nodules, 87%) and liver cyst in 8 out of 8 (100%). In contrast, hepatocellular carcinoma was visualized in 30 out of 31 patients (54 of 58 nodules, 93%), cholangiocellular carcinoma in 5 out of 5 (6 of 6 nodules, 100%), metastatic liver cancer in 66 out of 68 (139 of 143 nod-ules, 97%), hepatic hemangioma in 36 out of 36 (47 of 47 nodules, 100%) and liver cyst in 8 out of 8 (100%). The results suggest that STIR (TR : 1000 ms, TI : 100 ms, TE : 18 ms) is extremely useful in screening tumor lesions of the liver