Applications and Limitations of Equilibrium Density Gradient Analytical Ultracentrifugation for the Quantitative Characterization of Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are produced as a mixture of the desired particle (full particle, FP), which is filled with the designed DNA, product-related impurities such as particle without DNA (empty particle, EP), and aggregates. Cesium chloride or iodixanol equilibrium density gradient ultracentrifugation (DGE-UC) has been used for the purification of AAV vectors. DGE-UC can separate FP from impurities based on the difference in their buoyant densities. Here, we report the applications and limitations of equilibrium density gradient analytical ultracentrifugation (DGE-AUC) using a modern AUC instrument that employs DGE-UC principles for the characterization and quantitation of AAV vectors. We evaluated the quantitative ability of DGE-AUC in comparison with sedimentation velocity AUC (SV-AUC) or band sedimentation AUC (BS-AUC) using AAVs with different DNA lengths and different serotypes. DGE-AUC enabled the accurate quantification of the ratio of FP to EP when the AAV vector primarily contains these particles. Furthermore, we developed a new workflow to identify the components of separated peaks in addition to FP and EP. Ultraviolet absorption spectra obtained by multiwavelength detection can also support peak assignment following component identification. DGE-AUC experiments for AAV vectors have limitations with regard to minor components with low absorption at the detected wavelength or those with a density similar to that of major components of AAV vectors. DGE-AUC is the only analytical method that can evaluate particle density heterogeneity; therefore, SV-AUC or BS-AUC and DGE-AUC are complementary methods for reliable assessment of the purity of AAV vectors.

Longitudinal Analysis of Mitochondrial Function in a Choline-Deficient L-Amino Acid-Defined High-Fat Diet-Induced Metabolic Dysfunction-Associated Steatohepatitis Mouse Model.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is one of the most common chronic liver diseases worldwide. Some patients with MAFLD develop metabolic dysfunction-associated steatohepatitis (MASH), which can lead to severe liver fibrosis. However, the molecular mechanisms underlying this progression remain unknown, and no effective treatment for MASH has been developed so far. In this study, we performed a longitudinal detailed analysis of mitochondria in the livers of choline-deficient, methionine-defined, high-fat-diet (CDAHFD)-fed mice, which exhibited a MASH-like pathology. We found that FoF1-ATPase activity began to decrease in the mitochondria of CDAHFD-fed mice prior to alterations in the activity of mitochondrial respiratory chain complex, almost at the time of onset of liver fibrosis. In addition, the decrease in FoF1-ATPase activity coincided with the accelerated opening of the mitochondrial permeability transition pore (PTP), for which FoF1-ATPase might be a major component or regulator. As fibrosis progressed, mitochondrial permeability transition (PT) induced in CDAHFD-fed mice became less sensitive to cyclosporine A, a specific PT inhibitor. These results suggest that episodes of fibrosis might be related to the disruption of mitochondrial function via PTP opening, which is triggered by functional changes in FoF1-ATPase. These novel findings could help elucidate the pathogenesis of MASH and lead to the development of new therapeutic strategies.

Natural tetramic acids elicit multiple inhibitory actions against mitochondrial machineries presiding over oxidative phosphorylation.

The mitochondrial machineries presiding over ATP synthesis via oxidative phosphorylation are promising druggable targets. Fusaramin, a 3-acyl tetramic acid isolated from Fusarium concentricum FKI-7550, is an inhibitor of oxidative phosphorylation in Saccharomyces cerevisiae mitochondria, although its target has yet to be identified. Fusaramin significantly interfered with [3H]ADP uptake by yeast mitochondria at the concentration range inhibiting oxidative phosphorylation. A photoreactive fusaramin derivative (pFS-5) specifically labeled voltage-dependent anion channel 1 (VDAC1), which facilitates trafficking of ADP/ATP across the outer mitochondrial membrane. These results strongly suggest that the inhibition of oxidative phosphorylation by fusaramin is predominantly attributable to the impairment of VDAC1 functions. Fusaramin also inhibited FoF1-ATP synthase and ubiquinol-cytochrome c oxidoreductase (complex III) at concentrations higher than those required for the VDAC inhibition. Considering that other tetramic acid derivatives are reported to inhibit FoF1-ATP synthase and complex III, natural tetramic acids were found to elicit multiple inhibitory actions against mitochondrial machineries.

Pentenediol-Type Compounds Specifically Bind to Voltage-Dependent Anion Channel 1 in Saccharomyces cerevisiae Mitochondria.

Voltage-dependent anion channel 1 (VDAC1) situated in the outer mitochondrial membrane regulates the transfer of various metabolites and is a key player in mitochondria-mediated apoptosis. Although many small chemicals that modulate the functions of VDAC1 have been reported to date, most, if not all, of them cannot be regarded as specific reagents due to their interactions with other transporters or enzymes. By screening our chemical libraries using isolated Saccharomyces cerevisiae mitochondria, we found pentenediol (PTD)-type compounds (e.g., PTD-023) as new specific inhibitors of VDAC1. PTD-023 inhibited overall ADP-uptake/ATP-release reactions in isolated mitochondria at a single digit µM level. To identify the binding position of PTDs in VDAC1 by visualizing PTD-bound peptides, we conducted ligand-directed tosyl (LDT) chemistry using the synthetic LDT reagent t-PTD-023 derived from the parent PTD-023 in combination with mutagenesis experiments. t-PTD-023 made a covalent bond predominantly and subsidiarily with nucleophilic Cys210 and Cys130, respectively, indicating that PTDs bind to the region interactive with both residues. Site-directed mutations of hydrogen bond-acceptable Asp139 and Glu152 to Ala, which were selected as potential interactive partners of the critical pentenediol moiety based on the presumed binding model of PTDs in VDAC1, resulted in a decrease in susceptibility against PTD-023. This result strongly suggests that PTDs bind to VDAC1 through a specific hydrogen bond with the two residues. The present study is the first to demonstrate the binding position of specific inhibitors of VDAC1 at the amino acid level.

Advanced glycation end-products and Porphyromonas gingivalis lipopolysaccharide increase calprotectin expression in human gingival epithelial cells.

Accumulation of advanced glycation end-products (AGEs) in periodontal tissues of patients with diabetes mellitus aggravates periodontitis, but the mechanisms are unknown. Calprotectin, a heterocomplex of S100A8 and S100A9 proteins, is a constitutive cytoplasmic component of healthy gingival epithelial cells. This study aimed at investigating the effects of AGE and Porphyromonas gingivalis lipopolysaccharide (PgLPS) on calprotectin expression in the human gingival epithelial cell line OBA-9. AGE and PgLPS increased the expression of S100A8 and S100A9 mRNAs, and AGE+PgLPS co-stimulation amplified their expression in OBA-9 cells. A higher concentration of calprotectin in cell lysates was also induced by stimulation with AGE and/or PgLPS. S100A8 was mainly translocated from the nucleus to the cytoplasm by AGE stimulation, while cytoplasmic localization of S100A9 was not altered following stimulation with AGE and/or PgLPS. Calprotectin was found in the cytoplasm of BSA-treated cells, but cytoplasmic and nuclear localization was observed following stimulation with AGE and/or PgLPS. AGE-induced S100A8, and S100A9 mRNA expression was partially suppressed by RAGE-specific siRNA. In contrast, PgLPS-induced S100A8 and S100A9 mRNA expression was strongly suppressed by TLR2-specific siRNA. Furthermore, the inhibition of p38, JNK MAPK, and NF-κB attenuated AGE- and PgLPS-induced S100A8 and S100A9 mRNA expression. Taken together, these results demonstrate that AGE acts in synergy with PgLPS to stimulate RAGE and TLR2 expression and activate p38, JNK MAPK, and NF-κB signaling pathways, resulting in increased activation of calprotectin (S100A8/S100A9) in human gingival epithelial cells. Our results suggest that calprotectin may be involved in the pathogenesis of diabetic periodontitis.

Polyethyleneimine renders mitochondrial membranes permeable by interacting with negatively charged phospholipids in them.

Polyethyleneimines (PEIs) are used for transfection of cells with nucleic acids. Meanwhile, the interaction of PEI with mitochondria causes cytochrome c release prior to apoptosis; the mechanisms how PEI causes this permeabilization of mitochondrial membranes and the release of cytochrome c remain unclear. To clarify these mechanisms, we examined the effects of branched-type PEI and linear-type PEI, each of which was 25 kDa in size, on mitochondria. The permeabilization potency of mitochondrial membranes by branched PEI was stronger than that by linear PEI. The permeabilization by PEIs were insensitive to permeability-transition inhibitors, indicating that PEI-induced permeabilization was not attributed to permeability transition. Meanwhile, PEIs caused permeabilization of artificial lipid vesicles; again, the permeabilization potency of branched PEI was stronger than that of linear PEI. Such a difference in this potency was close to that in the case of isolated mitochondria, signifying that the PEI-induced permeabilization of mitochondrial membranes could be attributed to PEI's interaction with the phospholipid phase. Furthermore, this PEI-induced permeabilization of the lipid vesicles was observed only in the case of lipid vesicles including negatively charged phospholipids. These results indicate that PEIs interacted with negatively charged phospholipids in the mitochondrial membranes to directly lead to their permeabilization.

Synthetic Ubiquinones Specifically Bind to Mitochondrial Voltage-Dependent Anion Channel 1 (VDAC1) in Saccharomyces cerevisiae Mitochondria.

The role of the voltage-dependent anion channel (VDAC) as a metabolic gate of the mitochondrial outer membrane has been firmly established; however, its involvement in the regulation of mitochondrial permeability transition (PT) remains extremely controversial. Although some low-molecular-weight chemicals have been proposed to modulate the regulatory role of VDAC in the induction of PT, direct binding between these chemicals and VDAC has not yet been demonstrated. In the present study, we investigated whether the ubiquinone molecule directly binds to VDAC in Saccharomyces cerevisiae mitochondria through a photoaffinity labeling technique using two photoreactive ubiquinones (PUQ-1 and PUQ-2). The results of the labeling experiments demonstrated that PUQ-1 and PUQ-2 specifically bind to VDAC1 and that the labeled position is located in the C-terminal region Phe221-Lys234, connecting the 15th and 16th ß-strand sheets. Mutations introduced in this region (R224A, Y225A, D228A, and Y225A/D228A) hardly affected the binding affinity of PUQ-1. PUQ-1 and PUQ-2 both significantly suppressed the Ca2+-induced mitochondrial PT (monitored by mitochondrial swelling) at the one digit µM level. Thus, the results of the present study provided, for the first time to our knowledge, direct evidence indicating that the ubiquinone molecule specifically binds to VDAC1 through its quinone-head ring.

Analysis of the structure and function of EMRE in a yeast expression system.

The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel, and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression system for the mammalian calcium uniporter subunits is useful for investigating their functions. We here established a yeast expression system for the native-form mouse MCU and 4 other subunits. This expression system enabled us to precisely reconstitute the properties of the mammalian MCU complex in yeast mitochondria. Using this expression system, we analyzed the essential MCU regulator (EMRE), which is a key subunit for Ca(2+) uptake but whose functions and structure remain unclear. The topology of EMRE was revealed: its N- and C-termini projected into the matrix and the inter membrane space, respectively. The expression of EMRE alone was insufficient for Ca(2+) uptake; and co-expression of MCU with EMRE was necessary. EMRE was independent of the protein levels of other subunits, indicating that EMRE was not a protein-stabilizing factor. Deletion of acidic amino acids conserved in EMRE did not significantly affect Ca(2+) uptake; thus, EMRE did not have basic properties of ion channels such as ion-selectivity filtration and ion concentration. Meanwhile, EMRE closely interacted with the MCU on both sides of the inner membrane, and this interaction was essential for Ca(2+) uptake. This close interaction suggested that EMRE might be a structural factor for opening of the MCU-forming pore.

Transmission of External Environmental pH Information to the Inside of Liposomes via Pore-Forming Proteins Embedded within the Liposomal Membrane.

Liposomes are closed-membrane vesicles comprised of lipid bilayers, in which the inside of the vesicles is isolated from the external environment. Liposomes are therefore often used as models for biomembranes and as drug delivery carriers. However, materials encapsulated within liposomes often cannot respond to changes in the external environment. The ability of enclosed materials to maintain their responsiveness to changes in the external environment following encapsulation into liposomes would greatly expand the applicability of such systems. We hypothesize that embedding pore-like "access points" into the liposomal membrane could allow for the transmission of information between the internal and external liposomal environments and thus overcome this inherent limitation of conventional liposomes. To investigate this, we evaluated whether a change in the pH of an external solution could be transmitted to the inside of liposomes through the pore-forming protein, yeast voltage-dependent anion channel (VDAC). Transmission of a pH change via VDAC was evaluated using a polyglutamic acid/doxorubicin complex (PGA/Dox) as an internal pH sensor. Upon encapsulation into conventional liposomes, PGA/Dox exhibits no pH sensitivity due to isolation from the external environment. On the other hand, PGA/Dox was found to retain its pH sensitivity upon encapsulation into VDAC-reconstituted liposomes, suggesting that VDAC facilitated the transmission of information on the pH of the external environment to the inside of the liposomes. In conclusion, we successfully demonstrated the transmission of information between the external and internal liposomal environments by a stable pore-like structure embedded into the liposomal membranes, which serve as access points.

Effects of employment of distinct strategies to capture antibody on antibody delivery into cultured cells.

The characteristics of antibody delivery into cultured HeLa cells were examined using two delivery systems. Both systems used a cell-penetrating peptide as a tool for intrusion of an antibody into the cells, but either a "protein A derivative" or "hydrophobic motif" was employed to capture the antibody. When we examined the uptake of the Alexa Fluor-labeled antibody by the use of these two systems, both systems were found to effectively deliver the antibody into the cultured cells. However, when we compared the amount of antibody delivered by these systems with the amount of transferrin uptake, the former was 10 times smaller than the latter. The lower efficiency of antibody delivery than transferrin uptake seemed to be attributable to the involvement of the antibody delivery reagent, which failed to catch the antibody molecule. This interpretation was validated by an experiment using a larger amount of antibody, and the amount of antibody delivered by the "protein A derivative" system under this condition was determined to be 13 ng proteins/10(5) cells. The antibody delivery achieved by the "protein A derivative" or "hydrophobic motif" showed two differences, i.e., a difference in intracellular distribution of the delivered antibody molecules and a difference in the fluorescence spectrum observed with cellular lysates. Possible reasons for these differences between the two delivery systems are discussed.

Utility of syntenic relationships of VDAC1 pseudogenes for not only an understanding of the phylogenetic divergence history of rodents, but also ascertaining possible pseudogene candidates as genuine pseudogenes.

Rodent and human genomes were screened to identify pseudogenes of the type 1 voltage-dependent anion channel (VDAC1) in mitochondria. In addition to the 16 pseudogenes of rat VDAC1 identified in our recent study, 15 and 13 sequences were identified as pseudogenes of VDAC1 in mouse and human genome, respectively; and 4, 2, and 1 sequences, showing lower similarities with the VDAC1 sequence, were identified as "possible pseudogene candidates" in rat, mouse, and human, respectively. No syntenic combination was observed between rodent and human pseudogenes, but 2 and 1 possible pseudogene candidates of VDAC1 of rat and mouse, respectively, were found to have syntenic counterparts in mouse and rat genome, respectively; and these syntenic counterparts were genuine VDAC1 pseudogenes. Therefore, syntenic combinations of pseudogenes of VDAC1 were useful not only for a better understanding of the phylogenetic divergence history of rodents but also for ascertaining possible pseudogene candidates as genuine pseudogenes.

Embryology of Biebersteinia (Biebersteiniaceae, Sapindales): characteristics and comparisons with related families.

Biebersteinia, a perennial herb of five species distributed from Greece to Central Asia, was long considered to be placed in, or near Geraniaceae. Recent molecular analyses, however, have shown that the genus is the sole member of the family Biebersteiniaceae in Sapindales (not including Geraniaceae). Here, we report the embryological features of Biebersteinia and provide embryological corroboration for the molecular sapindalean affinities of the genus. We compared its embryology with those of eight other families of Sapindales, as well as with those of the related orders Huerteales, Malvales, and Brassicales. Overall comparisons showed that Biebersteinia fits in Sapindales because of the presence of anther tapetal cells with polyploid nuclear mass and non-fibrous exotegmen. Further, the genus is characterized by three-celled pollen grains, tetrasporic 16-nucleate Penaea-type female gametophyte, unitegmic ovules, pseudoporogamy, and the chalaza shifting its position near the concave side in the post-fertilization stage. A considerable number of autapomorphies, combined with the lack of synapomorphies with other sapindalean families, supports placing Biebersteinia in its own family. Biebersteiniaceae appear to represent an early divergent lineage of Sapindales. Previous descriptions of seed coats, which were considered to have developed from "bitegmic" ovules, were revised.

Analysis of the effects of importin α1 on the nuclear translocation of IL-1α in HeLa cells.

Interleukin-1α (IL-1α), a cytokine released by necrotic cells, causes sterile inflammation. On the other hand, IL-1α is present in the nucleus and also regulates the expression of many proteins. A protein substrate containing a classical nuclear localization signal (cNLS) typically forms a substrate/importin α/ß complex, which is subsequently transported to the nucleus. To the best of our knowledge, no study has directly investigated whether IL-1α-which includes cNLS-is imported into the nucleus in an importin α/ß-dependent manner. In this study, we noted that all detected importin α subtypes interacted with IL-1α. In HeLa cells, importin α1-mediated nuclear translocation of IL-1α occurred at steady state and was independent of importin ß1. Importin α1 not only was engaged in IL-1α nuclear transport but also concurrently functioned as a molecule that regulated IL-1α protein level in the cell. Furthermore, we discussed the underlying mechanism of IL-1α nuclear translocation by importin α1 based on our findings.

Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation.

Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.

Regulatory mechanisms of mitochondrial calcium uptake by the calcium uniporter complex.

Mitochondria play an important role in energy conversion as well as in intracellular calcium (Ca2+) storage. Ca2+ uptake from the cytosol to the mitochondria is mediated by the calcium uniporter, which functions as a Ca2+ ion channel. However, the molecular composition of this uniporter has remained unclear until recently. The Ca2+ ion channel consists of seven subunits. The yeast reconstitution technique revealed that the mitochondrial calcium uniporter (MCU) and essential MCU regulatory element (EMRE) are the core subunits of the complex. Furthermore, detailed structure-function analyses of the core subunits (MCU and EMRE) were performed. In this review, the regulatory mechanism of mitochondrial Ca2+ uptake is discussed.

2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Immunogenicity & Risk Assessment of Biotherapeutics and Novel Modalities; NAb Assays Integrated Approach).

The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.

Pseudogenes of rat VDAC1: 16 gene segments in the rat genome show structural similarities with the cDNA encoding rat VDAC1, with 8 slightly expressed in certain tissues.

BLAST analysis of the rat genome revealed the presence of 16 pseudogenes of isoform 1 of the mitochondrial voltage-dependent anion channel (VDAC1). Based on their structural characterization, it was concluded that these pseudogenes were formed by integration of VDAC1 cDNA into the genome, and subsequent rearrangements/mutations. By RT-PCR analysis using carefully designed primers that could not amplify the cDNA of genuine VDAC1, 8 of these 16 pseudogenes showed slight expression in certain tissues, but none of them seemed to encode a functional protein.

Comparison of two expression systems using COS7 cells and yeast cells for expression of heart/muscle-type carnitine palmitoyltransferase 1.

Carnitine palmitoyltransferase 1 (CPT1), catalyzing the transfer of the acyl group from acyl-CoA to carnitine to form acylcarnitine, is located at the outer mitochondrial membrane. Because it is easily inactivated by solubilization, expression systems using living cells are essential for its functional characterization. COS7 cells or yeast cells are often utilized for this purpose; however, the advantages/disadvantages of the use of these cells or the question as to how the CPT1 enzyme expressed by these cells differs are still uncertain. In this study, we characterized the heart/muscle-type isozyme of rat CPT1 (CPT1b) expressed by these two cellular expression systems. The mitochondrial fraction prepared from yeast cells expressing CPT1b showed 25% higher CPT1 activity than that obtained from COS7 cells. However, the expression level of CPT1b in the former was 3.8 times lower than that in the latter; and thus, under the present experimental conditions, the specific activity of CPT1b expressed in yeast cells was estimated to be approximately five times higher than that expressed in COS7 cells. Possible reasons for this difference are discussed.

A hybrid density functional study on the electron and hole trap states in anatase titanium dioxide.

We present a theoretical study on electron and hole trap states in the bulk and (001) surface of anatase titanium dioxide using screened hybrid density functional calculations. In both the bulk and surface, calculations suggest that the neutral and ionized oxygen vacancies are possible electron traps. The doubly ionized oxygen vacancy is the most stable in the bulk, and is a candidate for a shallow donor in colorless anatase crystals. The hole trap states are localized at oxygen anions in both the bulk and surface. The self-trapped electron centered at a titanium cation cannot be produced in the bulk, but can be formed at the surface. The electron trap level at the surface oxygen vacancy is consistent with observations by photoelectron spectroscopy. The optical absorptions and luminescence in UV-irradiated anatase nanoparticles are found to come from the surface self-trapped hole and the surface oxygen vacancy.

Properties of signal intensities observed with individual probes of GeneChip Rat Gene 1.0 ST Array, an affymetric microarray system.

For proper evaluation of the results of microarray experiments, it is important to understand how the signal intensities of individual probes are determined. Our previous studies revealed that signal intensities of individual probes in the Agilent array system (code G4131F) are largely dependent upon the location of the probes in the mRNA. In the present study, we examined the properties of signal intensities of individual probes in an Affymetrix array system (GeneChip Rat Gene 1.0 ST Array), in which a random primer fused to the T7 promoter sequence is employed. Distinct from the Agilent array system, individual probes used in this Affymetrix array system did not show the probe-location effects, but gave relatively diverse signal intensities. However, the diversities of the signal intensities of these individual probes were not due to experimental error.