Deep Neural Network-Based Method for Detecting Obstructive Meibomian Gland Dysfunction With in Vivo Laser Confocal Microscopy.

PURPOSE: To evaluate the ability of deep learning (DL) models to detect obstructive meibomian gland dysfunction (MGD) using in vivo laser confocal microscopy images. METHODS: For this study, we included 137 images from 137 individuals with obstructive MGD (mean age, 49.9 ± 17.7 years; 44 men and 93 women) and 84 images from 84 individuals with normal meibomian glands (mean age, 53.3 ± 19.6 years; 29 men and 55 women). We constructed and trained 9 different network structures and used single and ensemble DL models and calculated the area under the curve, sensitivity, and specificity to compare the diagnostic abilities of the DL. RESULTS: For the single DL model (the highest model; DenseNet-201), the area under the curve, sensitivity, and specificity for diagnosing obstructive MGD were 0.966%, 94.2%, and 82.1%, respectively, and for the ensemble DL model (the highest ensemble model; VGG16, DenseNet-169, DenseNet-201, and InceptionV3), 0.981%, 92.1%, and 98.8%, respectively. CONCLUSIONS: Our network combining DL and in vivo laser confocal microscopy learned to differentiate between images of healthy meibomian glands and images of obstructive MGD with a high level of accuracy that may allow for automatic obstructive MGD diagnoses in patients in the future.

Evaluation of Q Gene Mycobacteria: A novel and easy nucleic acid chromatography method for mycobacterial species identification.

OBJECTIVES: A simple, rapid, and new diagnostic test for mycobacteria, named Q Gene Mycobacteria, has been developed. It is based on multiplex PCR using primers harbouring DNA tags combined with a dipstick nucleic acid chromatography method, which does not require the denaturation of PCR products for hybridization and can identify five species of mycobacteria including Mycobacterium tuberculosis complex (MTC), Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium gordonae. This study aimed to evaluate Q Gene Mycobacteria for the accurate identification of these five species. METHODS: A total of 340 mycobacterial strains/isolates were tested, of which 159 were type strains (four MTC and 155 non-tuberculosis mycobacteria (NTM) including four subspecies) and 181 were clinical isolates (18 M. tuberculosis, two Mycobacterium bovis Bacillus Calmette et Guérin (BCG), and 161 NTM comprising 16 species) collected from eight laboratories and hospitals in Japan. Species identification of NTM isolates was performed using the DNA-DNA hybridization method and/or direct sequencing of 16S rRNA, hsp65, and rpoB genes. Q Gene Mycobacteria was compared with above conventional methods for identifying the five species. RESULTS: Q Gene Mycobacteria showed excellent concordance for species identification, specifically 99.4% (158/159) for type strains and 99.4% (180/181) for clinical isolates. The two strains that were misidentified as M. gordonae were Mycobacterium paragordonae. As they are genetically close and there is few case reports of M. paragordonae, it might not be a serious critical issue to distinguish M. paragordonae from M. gordonae. CONCLUSIONS: Q Gene Mycobacteria was able to identify frequently isolated mycobacterial species accurately and easily. Therefore, Q Gene Mycobacteria could be a useful tool for the identification of specific mycobacteria in clinical laboratories.

Determination of iris thickness development in children using swept-source anterior-segment optical coherence tomography.

PURPOSE: The uvea comprises the iris, ciliary body, and choroid. However, the development of the anterior part (iris and ciliary body) in children is not yet fully elucidated. We investigated the iris thickness (IT) in children using swept-source anterior-segment optical coherence tomography (ASOCT). METHODS: In this retrospective, clinic-based study, we enrolled 41 children (mean ± standard deviation: 6.8 ± 3.3 years; range: 3-16; 17 males) with normal or mild refractive error. Horizontal scanning images of swept-source ASOCT were analyzed in temporal and nasal angle areas. The ITs at 1 and 2 mm from the pupil edge were measured using swept-source ASOCT. The association between IT and age, sex, and ocular morphological parameters (i.e., axial length, average corneal curvature, central corneal thickness, inter-scleral spur distance, and anterior chamber depth) was assessed using Pearson's correlation coefficient (r) and linear regression analysis. RESULTS: The average IT (temporal and nasal) at 1 and 2 mm were 0.432 ± 0.060 (0.302-0.569 mm) and 0.337 ± 0.045 (0.229-0.414 mm), respectively. There was a significant correlation between age and average IT (r = 0.45, P = 0.002 at 1 mm and r = 0.31, P = 0.042 at 2 mm). Multiple linear regression analysis revealed that age (coefficient: 0.01), axial length (-0.02), average corneal curvature (0.01), and anterior chamber depth (0.01) at 1 mm as well as age (0.00), average corneal curvature (0.09), anterior chamber depth (0.06), and male (-0.02) at 2 mm were identified as predictors of IT. CONCLUSIONS: IT in children increases with age. Additionally, IT was thinner with longer axial length and in males, thicker in eyes with deeper anterior chamber and flatter corneal curvature. Our study may partly explain the development of eyeball structures in children.

Layered hybrid perovskites with micropores created by alkylammonium functional silsesquioxane interlayers.

Layered organic-inorganic hybrid perovskites that consist of metal halides and organic interlayers are a class of low-dimensional materials. Here, we report the fabrication of layered hybrid perovskites using metal halides and silsesquioxane with a cage-like structure. We used a silsesquioxane as an interlayer to produce a rigid structure and improve the functionality of perovskite layers. Propylammonium-functionalized silsesquioxane and metal halide salts (CuCl2, PdCl2, PbCl2, and MnCl2) were self-assembled to form rigid layered perovskite structures with high crystallinity. The rigid silsesquioxane structure produces micropores between the perovskite layers that can potentially be filled with different molecules to tune the dielectric constants of the interlayers. The obtained silsesquioxane-metal halide hybrid perovskites exhibit some characteristic properties of layered perovskites including magnetic ordering (CuCl4(2-) and MnCl4(2-)) and excitonic absorption/emission (PbCl4(2-)). Our results indicate that inserting silsesquioxane interlayers into hybrid perovskites retains and enhances the low-dimensional properties of the materials.

Acidiphilium iwatense sp. nov., isolated from an acid mine drainage treatment plant, and emendation of the genus Acidiphilium.

Several strains of aerobic, acidophilic, chemo-organotrophic bacteria belonging to the genus Acidiphilium were isolated from an acid mine drainage (AMD) (pH 2.2) treatment plant. 16S rRNA gene sequence comparisons showed that most of the novel isolates formed a phylogenetically coherent group (designated Group Ia) distinguishable from any of the previously established species of the genus Acidiphilium at <98% similarity. This was supported by genomic DNA-DNA hybridization assays. The Group Ia isolates were characterized phenotypically by an oval cell morphology, non-motility, growth in the range pH 2.0-5.5 (optimum pH 3.5), lack of photosynthetic pigment and the presence of C19:0 cyclo ω8c as the main component of the cellular fatty acids and ubiquinone-10 as the major quinone. On the basis of these data, the name Acidiphilium iwatense sp. nov. is proposed to accommodate the Group Ia isolates, and the description of the genus Acidiphilium is emended. The type strain of Acidiphilium iwatense sp. nov. is MS8(T) ( =NBRC 107608(T)=KCTC 23505(T)).

Controlled formation of silica structures using siloxane/block copolymer complexes prepared in various solvent mixtures.

Block copolymers exhibit regularly patterned structures induced by microphase separation. Here we present a method for preparing various particulate silica (SiO2) nanostructures by controlling the microphase separation of block copolymers. In this method, siloxane, a SiO2 precursor, is adsorbed onto poly(4-vinylpyridine) blocks of polystyrene-block-poly(4-vinylpyridine) in solvent mixtures. After siloxane/polymer complexes are coprecipitated via further siloxane polycondensation, the resulting precipitates are heated to remove the polymer. The results of scanning electron microscopy revealed that SiO2 formed various structures including cylindrical, spherical, and lamellar. Different SiO2 nanostructures formed via the microphase separation of siloxane/polymer complexes are prepared simply by varying solvent mixtures without changing the polymer chain. The structural change is interpreted in terms of polymer-solvent interactions and volume fractions in siloxane/polymer complexes.

Preparation of plate-like mesoporous material from layered silicate RUB-15.

A plate-like mesoporous material was formed from the lamellar structure of layered silicate RUB-15. RUB-15 was synthesized by a hydrothermal method, as reported previously. TMA (tetramethylammonium) ions exist in the interlayer of RUB-15 were exchanged with C16 TMA (hexadecyl-trimethyl-ammonium) ions, and TEOS (tetraethylorthosilicate) was then intercalated in between the layers. After steaming, the obtained powder was calcined and characterized by XRD, N2 gas adsorption, and scanning electron microscopy (SEM). The XRD patterns and N2 adsorption-desorption isotherms of the finally obtained powders indicated the presence of mesopores in the sample. The morphology of powders was plate-like which originates from the structure of the starting material. Cross-sectional FE-SEM images of the final obtained powders revealed existence of mesopores between the layers. The morphology of the final obtained mesoporous materials was affected by their remaining layered structure due to the starting material RUB-15.

Folliculin regulates cyclin D1 expression through cis-acting elements in the 3' untranslated region of cyclin D1 mRNA.

Birt-Hogg-Dubé syndrome (BHDS) is an autosomal dominantly inherited disease characterized by spontaneous pneumothorax, hair folliculomas and renal tumors. The responsible gene, BHD, is a tumor suppressor and encodes folliculin. Folliculin is an evolutionarily conserved protein (~67 kDa) with no apparent functional motif and its role has not yet been fully elucidated. In this study, we found that knockdown of BHD increased the levels of cyclin D1 in HeLa cells. A reporter assay with the cyclin D1 gene (CCND1) promoter region indicated that this increase was not caused by activation of transcription through known cis-acting elements. We examined the possibility of post-transcriptional mechanism using reporter constructs containing fragments of the cyclin D1 3' untranslated region (3'UTR). Transfection of control cells with a construct carrying a medial 1.3 kb 3'UTR fragment resulted in a significant reduction in luciferase activity. This effect was largely prevented by knockdown of BHD. Our results suggest that the post-transcriptional regulation of the CCND1 expression by BHD may be associated with microRNA(s) or RNA binding protein(s) that bind to the 3'UTR.

Acidipila rosea gen. nov., sp. nov., an acidophilic chemoorganotrophic bacterium belonging to the phylum Acidobacteria.

Two strains of aerobic acidophilic chemoorganotrophic bacteria designed strains AP8(T) and AP9 were isolated from acid mine drainage and acidic soil, respectively. These isolates were gram-negative, nonmotile cocci and coccobacilli measuring 0.5-0.8 µm in diameter. Cells were capsulated. Colonies on solid media were pink colored. The pH range for growth was 3.0-6.0 (optimum pH 4.5). Sugars, gluconate, and some amino acids were good carbon and energy sources for growth. The main components of cellular fatty acids were C(15:0) iso and C(16:1) ω7c. Menaquinone-8 was the major quinone. The G+C content of genomic DNA was 59.5%. Both strains had identical sequences of 16S rRNA genes that were most closely related to that of the type strain of Acidobacterium capsulatum (96% similarity). There were major differences between the isolates and A. capsulatum in cell morphology, carbon nutrition, and fatty acid profiles. Based on these phylogenetic and phenotypic data, we propose the name Acidipila rosea gen. nov., sp. nov. to accommodate the novel isolates. The type strain is AP8(T) (NBRC 107607(T), KCTC 23427(T)).

Cellular distribution of ACT domain repeat protein 9, a nuclear localizing protein, in rice (Oryza sativa).

Regulatory ACT domains serve as amino acid-binding sites in certain amino acid metabolic enzymes and transcriptional regulators in bacteria. The ACT domain repeat protein (ACR) family in plants is primarily composed of four copies of the domain homologous to those of the bacteria Gln sensor GLND. In the current study, to evaluate the possible involvement of the protein OsACR9 in the Gln-sensing system related to nitrogen (N) metabolism in rice (Oryza sativa L.), subcellular localization of OsACR9 and its accumulation and cellular distribution in various rice organs were examined by transient expression analysis and immunological methods using a monospecific antibody, respectively. Transient expression analysis of OsACR9 fused with a synthetic green fluorescent protein in cultured rice cells suggested nuclear localization of OsACR9. In rice roots, OsACR9 protein was distributed in epidermis, exodermis, sclerenchyma and vascular parenchyma cells, and its accumulation markedly increased after supply of NH(+)(4). In rice leaf samples, OsACR9 protein was abundant in the vascular parenchyma and mestome-sheath cells of young leaf blades at the early stage of development and in the vascular parenchyma and phloem-companion cells of mature leaf sheaths. OsACR9 protein also showed a high level of accumulation in vascular parenchyma cells of dorsal vascular bundles and aleurone cells in young rice grains at the early stage of ripening. The possibility of the nuclear protein OsACR9 acting as a Gln sensor in rice is subsequently discussed through comparison of its spatiotemporal expression with that of Gln-responsive N-assimilatory genes.

Formation of cage-like hollow spherical silica via a mesoporous structure by calcination of lysozyme-silica hybrid particles.

Calcination of lysozyme-silica hybrid hollow particles gives novel cage-like hollow spherical silicas with differently patterned through-holes on their shell structure.

Encapsulation of hemoglobin in mesoporous silica (FSM)-enhanced thermal stability and resistance to denaturants.

Hemoblogin (Hb), which is a typical oligomeric protein, was introduced into the pores of mesoporous silica (FSM: folded-sheet mesoporous material) that had a diameter of 7.5 nm. Soret CD spectra of Hb-FSM-7.5 conjugates showed a peak that was identical to that of free Hb. This suggests that Hb retained its highly ordered structure in the mesoporous silica. In addition, the UV-visible absorption spectrum showed that Hb had an increased resistance to heat denaturation in the silica. Even after heat treatment at 85 degrees C, Hb-FSM-7.5 retained its ligand-binding activity. The stability of Hb-FSM-7.5 was examined further by measuring its peroxidase-like activity. Encapsulation of Hb resulted in the retention of activity in the presence of high NaCl or Gdn-HCl levels. This suggests that encapsulation prevented dissociation and denaturing. Thus, it seems that the mesopores created a favorable environment for the oligomeric protein to perform its function, even under harsh conditions.

A novel protein refolding method using a zeolite.

We have succeeded in developing a simple and effective protein refolding method using the inorganic catalyst, beta-zeolite. The method involves the adsorption of proteins solubilized with 6M guanidine hydrochloride from inclusion body (IB) preparations onto the zeolite. The denaturant is then removed, and the proteins in the IBs are released from the zeolite with polyoxyethylene detergent and salt. All of the IBs tested (11 different species) were successfully refolded under these conditions. The refolded proteins are biochemically active, and NMR analysis of one of the proteins (replication protein A 8) supports the conclusion that correct refolding does occur. Based on these results, we discuss the refolding mechanism.

Implementation of the modified-SHIRPA protocol for screening of dominant phenotypes in a large-scale ENU mutagenesis program.

SHIRPA is a three-stage protocol for the comprehensive assessment of primarily mouse behavior. The first stage consists of high-throughput phenotyping of 33 behavioral observations and 7 metabolic or disease observations. We modified this part of the protocol by integrating new morphologic observations into the initial phenotype assay of behavior and dysmorphology. Behavioral observations assessed by this protocol, now referred to as the "modified-SHIRPA," are compatible with the original "SHIRPA" protocol. Using modified-SHIRPA, we screened dominant phenotypes of more than 10,000 G(1) progeny generated by crossing DBA/2J females with ENU-treated C57BL/6J males. To date, we have obtained 136 hereditary-confirmed mutants that exhibit behavioral and morphologic defects. Some independent mutant lines exhibited similar phenotypes, suggesting that they may represent alleles of the same gene or mutations in the same genetic pathway. They could hold great potential for the unraveling of the molecular mechanisms of certain phenotypes.

Synthesis of protein-silica hybrid hollow particles through the combination of protein catalysts and sonochemical treatment.

Hollow spherical particles with protein-silica hybrid shell structures have been synthesized through a combination of the catalytic activity of the protein and sonochemical treatment; the morphologies of the particles were controlled by varying the protein concentration.

Enamelin (Enam) is essential for amelogenesis: ENU-induced mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI).

Amelogenesis imperfecta (AI) is a group of commonly inherited defects of dental enamel formation, which exhibits marked genetic and clinical heterogeneity. The genetic basis of this heterogeneity is still poorly understood. Enamelin, the affected gene product in one form of AI (AIH2), is an extracellular matrix protein that is one of the components of enamel. We isolated three ENU-induced dominant mouse mutations, M100395, M100514 and M100521, which caused AI-like phenotypes in the incisors and molars of the affected individuals. Linkage analyses mapped each of the three mutations to a region of chromosome 5 that contained the genes encoding enamelin (Enam) and ameloblastin (Ambn). Sequence analysis revealed that each mutation was a single-base substitution in Enam. M100395 (Enam(Rgsc395)) and M100514 (Enam(Rgsc514)) were putative missense mutations that caused S to I and E to G substitutions at positions 55 and 57 of the translated protein, respectively. Enam(Rgsc395) and Enam(Rgsc514) heterozygotes showed severe breakage of the enamel surface, a phenotype that resembled local hypoplastic AI. The M100521 mutation (Enam(Rgsc521)) was a T to A substitution at the splicing donor site in intron 4. This mutation resulted in a frameshift that gave rise to a premature stop codon. The transcript of the Enam(Rgsc521) mutant allele was degraded, indicating that Enam(Rgsc521) is a loss-of-function mutation. Enam(Rgsc521) heterozygotes showed a hypomaturation-type AI phenotype in the incisors, possibly due to haploinsufficiency of Enam. Enam(Rgsc521) homozygotes showed complete loss of enamel on the incisors and the molars. Thus, we report here that the Enam gene is essential for amelogenesis, and that mice with different point mutations at Enam may provide good animal models to study the different clinical subtypes of AI.

Development-associated myristoylated alanine-rich C kinase substrate phosphorylation in rat brain.

OBJECT: In neuronal cells, myristoylated alanine-rich C kinase substrate (MARCKS), localized to particular areas of the synaptic membrane, is active during brain development. The destination of phosphorylated MARCKS is thought to be the cytoplasm where it is probably inactive. We compared MARCKS phosphorylation in the brains of embryonic, perinatal, and adult rats to determine its possible involvement in neurogenesis. METHODS: We prepared crude and partially purified extracts from various brain regions of rats aged between embryonic day 14 (E14) and 7 weeks after birth and assayed them for MARCKS phosphorylation by immunochemical methods. The isotypes of protein kinase C (PKC) were immunochemically identified in crude brain extracts from embryonic and postnatal rats. Despite negligible MARCKS phosphorylation, E16 brain extracts contained both MARCKS and PKCgamma, delta, epsilon, and lambda. MARCKS and polypeptides were clearly phosphorylated (49 and 45 kDa, respectively) in brain extracts purified on a DE52 column. Embryonic brain extracts manifested a high-molecular-weight activity capable of suppressing polypeptide phosphorylation. This activity was markedly decreased on the day of birth and almost undetectable in the brains of 9-day-old rats. CONCLUSIONS: The embryonic rat brain appears to contain a protein(s) that suppresses the phosphorylation of other proteins including MARCKS. We posit that this inhibitory activity represents a factor(s) that plays a role in the regulation of neurogenesis beginning on the day on which MARCKS appears in the embryonic brain.

Direct Differentiation of the Types of Polarization Responsible for the Electrorheological Effect By a Dielectric Method.

Debye polarization, ionic displacement polarization, and Maxwell-Wagner (interfacial) polarization are discussed in this paper, because they would most likely take place in an electrorheological (ER) suspension. The temperature dependences of the dielectric loss tangent maximums governed by these three types of polarization are theoretically found to be quite different. Given this fact, a method that can directly distinguish the polarization type and clarify which polarization would be mainly responsible for the ER effect is proposed. Two kinds of typical ER suspensions, heterogeneous particle type and homogeneous liquid crystalline polymer type, are studied using our method. It is found that Maxwell-Wagner polarization would be responsible for the ER effect both in a heterogeneous and in a homogeneous ER system. These findings present direct experimental evidence for the previous assumption that the Maxwell-Wagner polarization would dominate in the heterogeneous ER system and also shed light on the ER mechanism in a liquid crystalline polymer-type ER system. Copyright 2001 Academic Press.