253 J at 0.2†Hz, LD pumped cryogenic helium gas cooled Yb:YAG ceramics laser.

A 253 J with 26 ns at 0.2 Hz laser performance was demonstrated using a LD pumped cryogenically cooled Yb:YAG ceramics laser amplifier. A high energy storage of 344 J was achieved with a stored energy density of 0.58 J/cm3 using a 1 kJ output multidirectional-pumping system. High energy-extraction efficiency of 56.5% was achieved with high energy fluence of 4.63 J /cm2. To the best of our knowledge, this is the highest output energy obtained with a repetitive nanosecond pulse by LD pumped solid-state laser. This paper presented a design of 1 kJ amplifier based on experimentally proven numerical data.

Boron Neutron Capture Therapy (BNCT) Mediated by Maleimide-Functionalized Closo-Dodecaborate Albumin Conjugates (MID:BSA) for Oral Cancer: Biodistribution Studies and In Vivo BNCT in the Hamster Cheek Pouch Oral Cancer Model.

BACKGROUND: BNCT (Boron Neutron Capture Therapy) is a tumor-selective particle radiotherapy that combines preferential boron accumulation in tumors and neutron irradiation. Although p-boronophenylalanine (BPA) has been clinically used, new boron compounds are needed for the advancement of BNCT. Based on previous studies in colon tumor-bearing mice, in this study, we evaluated MID:BSA (maleimide-functionalized closo-dodecaborate conjugated to bovine serum albumin) biodistribution and MID:BSA/BNCT therapeutic effect on tumors and associated radiotoxicity in the hamster cheek pouch oral cancer model. METHODS: Biodistribution studies were performed at 30 mg B/kg and 15 mg B/kg (12 h and 19 h post-administration). MID:BSA/BNCT (15 mg B/kg, 19 h) was performed at three different absorbed doses to precancerous tissue. RESULTS: MID:BSA 30 mg B/kg protocol induced high BSA toxicity. MID:BSA 15 mg B/kg injected at a slow rate was well-tolerated and reached therapeutically useful boron concentration values in the tumor and tumor/normal tissue ratios. The 19 h protocol exhibited significantly lower boron concentration values in blood. MID:BSA/BNCT exhibited a significant tumor response vs. the control group with no significant radiotoxicity. CONCLUSIONS: MID:BSA/BNCT would be therapeutically useful to treat oral cancer. BSA toxicity is a consideration when injecting a compound conjugated to BSA and depends on the animal model studied.

Iodophenyl-conjugated closo-dodecaborate as a promising small boron molecule that binds to serum albumin and accumulates in tumor.

The development of novel boron carriers applicable to various cancers is required for further expansion of boron neutron capture therapy (BNCT). In this study, we took advantage of the fact that serum albumin accumulates in tumors and developed a boron compound that interacts non-covalently with the serum albumin. 4-Iodophenylbutanamide was chosen as an albumin ligand and conjugated with closo-dodecaborate (boron-conjugated 4-iodophenylbutanamide: BC-IP). BC-IP was found to be water soluble with low cytotoxicity. The IC50 values of BC-IP were 475 µM for U87MG cells, 738 µM for HeLa cells, and > 1000 µM for A549 cells. The dissociation constant (Kd) value of BC-IP to HSA was 148 ± 8 µM, while that of disodium closo-dodecaborate (4) was > 1000 µM. Significant tumor accumulation was observed in the U87MG tumor mouse model 3 h after injection. The boron concentration in the tumor reached a maximum of 11 µgB/g at 3 h and gradually decreased to 2.4 and 2.3 µgB/g at 12 and 24 h, respectively.

Boron neutron capture therapy using dodecaborated albumin conjugates with maleimide is effective in a rat glioma model.

Introduction Boron neutron capture therapy (BNCT) is a biologically targeted, cell-selective particle irradiation therapy that utilizes the nuclear capture reaction of boron and neutron. Recently, accelerator neutron generators have been used in clinical settings, and expectations for developing new boron compounds are growing. Methods and Results In this study, we focused on serum albumin, a well-known drug delivery system, and developed maleimide-functionalized closo-dodecaborate albumin conjugate (MID-AC) as a boron carrying system for BNCT. Our biodistribution experiment involved F98 glioma-bearing rat brain tumor models systemically administered with MID-AC and demonstrated accumulation and long retention of boron. Our BNCT study with MID-AC observed statistically significant prolongation of the survival rate compared to the control groups, with results comparable to BNCT study with boronophenylalanine (BPA) which is the standard use of in clinical settings. Each median survival time was as follows: untreated control group; 24.5 days, neutron-irradiated control group; 24.5 days, neutron irradiation following 2.5 h after termination of intravenous administration (i.v.) of BPA; 31.5 days, and neutron irradiation following 2.5 or 24 h after termination of i.v. of MID-AC; 33.5 or 33.0 days, respectively. The biological effectiveness factor of MID-AC for F98 rat glioma was estimated based on these survival times and found to be higher to 12. This tendency was confirmed in BNCT 24 h after MID-AC administration. Conclusion MID-AC induces an efficient boron neutron capture reaction because the albumin contained in MID-AC is retained in the tumor and has a considerable potential to become an effective delivery system for BNCT in treating high-grade gliomas.

Carborane as an Alternative Efficient Hydrophobic Tag for Protein Degradation.

Carboranes 1 and 2 were designed and synthesized for hydrophobic tag (HyT)-induced degradation of HaloTag fusion proteins. The levels of the hemagglutinin (HA)-HaloTag2-green fluorescent protein (EGFP) stably expressed in Flp-In 293 cells were significantly reduced by HyT13, HyT55, and carboranes 1 and 2, with expression levels of 49, 79, 43, and 65%, respectively, indicating that carborane is an alternative novel hydrophobic tag (HyT) for protein degradation under an intracellular environment. To clarify the mechanism of HyT-induced proteolysis, bovine serum albumin (BSA) was chosen as an extracellular protein and modified with maleimide-conjugated m-carborane (MIC). The measurement of the ζ-potentials and the lysine residue modification with fluorescein isothiocyanate (FITC) of BSA-MIC conjugates suggested that the conjugation of carborane induced the exposure of lysine residues on BSA, resulting in the degradation via ubiquitin E3 ligase-related proteasome pathways in the cell.

13C-metabolic flux analysis in glycerol-assimilating strains of Saccharomyces cerevisiae.

Glycerol is an attractive raw material for the production of useful chemicals using microbial cells. We previously identified metabolic engineering targets for the improvement of glycerol assimilation ability in Saccharomyces cerevisiae based on adaptive laboratory evolution (ALE) and transcriptome analysis of the evolved cells. We also successfully improved glycerol assimilation ability by the disruption of the RIM15 gene encoding a Greatwall protein kinase together with overexpression of the STL1 gene encoding the glycerol/H+ symporter. To understand glycerol assimilation metabolism in the evolved glycerol-assimilating strains and STL1-overexpressing RIM15 disruptant, we performed metabolic flux analysis using 13C-labeled glycerol. Significant differences in metabolic flux distributions between the strains obtained from the culture after 35 and 85 generations in ALE were not found, indicating that metabolic flux changes might occur in the early phase of ALE (i.e., before 35 generations at least). Similarly, metabolic flux distribution was not significantly changed by RIM15 gene disruption. However, fluxes for the lower part of glycolysis and the TCA cycle were larger and, as a result, flux for the pentose phosphate pathway was smaller in the STL1-overexpressing RIM15 disruptant than in the strain obtained from the culture after 85 generations in ALE. It could be effective to increase flux for the pentose phosphate pathway to improve the glycerol assimilation ability in S. cerevisiae.

Cyclic RGD-Functionalized closo-Dodecaborate Albumin Conjugates as Integrin Targeting Boron Carriers for Neutron Capture Therapy.

Cyclic RGD (cRGD) peptide-conjugated boronated albumin was developed to direct toward integrin αvß3, which overexpresses on many cancer cells. A stepwise conjugation of c[RGDfK(Mal)] and maleimide-conjugated closo-dodecaborate (MID) to bovine serum albumin (BSA) afforded cRGD-MID-BSA, which was noncytotoxic toward both U87MG and A549 cells. As compared with l-BPA, selective antitumor activity of cRGD-MID-BSA toward U87MG cells overexpressing integrin αvß3 was identified after thermal neutron irradiation. In vivo fluorescence live imaging of Cy5-conjugated cRGD-MID-BSA and MID-BSA revealed that both cRGD-MID-BSA and MID-BSA similarly reached the maximum accumulation during 8-12 h after injection. The selective accumulation and retention of Cy5-cRGD-MID-BSA was more pronounced than Cy5-MID-BSA after 24 h. An in vivo boron neutron capture therapy (BNCT) study revealed that the cRGD peptide ligand combination enhanced accumulation of MID-BSA into tumor cells in U87MG xenograft models. The significant tumor growth suppression was observed in U87MG xenograft models at a dose of 7.5 mg [10B]/kg after neutron irradiation.

Extracellular electron transfer mediated by a cytocompatible redox polymer to study the crosstalk among the mammalian circadian clock, cellular metabolism, and cellular redox state.

The circadian clock is an endogenous biological timekeeping system that controls various physiological and cellular processes with a 24 h rhythm. The crosstalk among the circadian clock, cellular metabolism, and cellular redox state has attracted much attention. To elucidate this crosstalk, chemical compounds have been used to perturb cellular metabolism and the redox state. However, an electron mediator that facilitates extracellular electron transfer (EET) has not been used to study the mammalian circadian clock due to potential cytotoxic effects of the mediator. Here, we report evidence that a cytocompatible redox polymer pMFc (2-methacryloyloxyethyl phosphorylcholine-co-vinyl ferrocene) can be used as the mediator to study the mammalian circadian clock. EET mediated by oxidized pMFc (ox-pMFc) extracted intracellular electrons from human U2OS cells, resulting in a longer circadian period. Analyses of the metabolome and intracellular redox species imply that ox-pMFc receives an electron from glutathione, thereby inducing pentose phosphate pathway activation. These results suggest novel crosstalk among the circadian clock, metabolism, and redox state. We anticipate that EET mediated by a redox cytocompatible polymer will provide new insights into the mammalian circadian clock system, which may lead to the development of new treatments for circadian clock disorders.

Amphiphilic triphenylamine-benzothiadiazole dyes: preparation, fluorescence and aggregation behavior, and enzyme fluorescence detection.

Fluorescence change systems that can respond to biological objects have attracted attention for use as biological probes and sensors. In this study, we report emission enhancement in a fluorescent aggregate composed of amphiphilic donor-acceptor dye molecules. The emission efficiency of the aggregate was reduced upon introducing a hydrophilic galactopyranose moiety, because of the decrease in the aggregate stability, which in turn was due to disruption of the hydrophilicity-hydrophobicity balance. In contrast, emission enhancement could be achieved by treatment with ß-galactosidase, as a result of the removal of the galactopyranose moiety. The change in aggregate stabilization based on the hydrophilicity-hydrophobicity balance leads to the emission enhancement into detectable ß-galactosidase activity.

Identification of metabolic engineering targets for improving glycerol assimilation ability of Saccharomyces cerevisiae based on adaptive laboratory evolution and transcriptome analysis.

Glycerol, a by-product of biodiesel production, has been utilized as a raw material for bioproduction. Saccharomyces cerevisiae, which has been used as a host microorganism for bioproduction, possesses the metabolic pathways for glycerol assimilation, but it cannot grow on glycerol as a carbon source. In this study, we identified metabolic engineering targets to improve the glycerol assimilation ability of S. cerevisiae based on adaptive laboratory evolution experiments using serial transfer of culture on glycerol and transcriptome analysis of the evolved cells using RNA-sequencing. The transcriptome data revealed that the upregulation of genes related to the tricarboxylic acid (TCA) cycle and oxidative phosphorylation contributed to the increased specific growth rate on glycerol during adaptive evolution. Furthermore, genes related to the pentose phosphate pathway were downregulated. Based on these observations, we identified metabolic engineering targets for improving glycerol assimilation. Overexpression of HAP4, which encodes one of the subunits of the Hap2p/3p/4p/5p transcription factor complex involved in the upregulation of the TCA cycle genes, or disruption of RIM15, which encodes a protein kinase related to the transcription regulator Gis1p, as well as overexpression of STL1, which encodes the glycerol/H+ symporter, improved the growth of S. cerevisiae on glycerol as the main carbon source. Our results indicate that the engineering targets can be identified based on adaptive laboratory evolution and transcriptome analysis of the evolved cells, and that the glycerol assimilation ability of S. cerevisiae is indeed improved by engineering the identified targets.