Morphological differences of the neuroretinal rim between temporally tilted and non-tilted optic discs in healthy eyes.

This study aimed to compare morphological differences of the neuroretinal rim between the temporally tilted and non-tilted optic discs in healthy eyes. We prospectively enrolled participants aged 20-40 years with temporally tilted or non-tilted optic discs. The optic nerve head parameters were analyzed using spectral domain-optical coherence tomography. The angle between the Bruch's membrane opening (BMO) plane and BMO-minimum rim width (BMO-MRW) was termed "BMO-MRW angle". Peripapillary retinal nerve fiber layer thickness (pRNFLT) and BMO-based parameters were compared between the temporally tilted and non-tilted disc groups. As a result, 55 temporally tilted disc eyes and 38 non-tilted disc eyes were analyzed. Global pRNFLT, global BMO-MRW, and total BMO-minimum rim area (BMO-MRA) were similar between the two groups (p = 0.138, 0.161, and p = 0.410, respectively). In the sectoral analysis, temporally tilted disc group exhibited thicker BMO-MRW in the temporal sector (p = 0.032) and thinner in the nasal superior and nasal sectors (p = 0.025 and p = 0.002, respectively). Temporally tilted disc group showed larger BMO-MRA in the temporal, temporal superior, and temporal inferior sectors (p < 0.001, p < 0.001, and p < 0.016, respectively), alongside a higher BMO-MRW angle in the temporal sector and lower in the nasal superior and nasal sectors. In conclusion, the neuroretinal rim, represented by BMO-MRW and BMO-MRA, showed morphological differences between temporally tilted and non-tilted optic discs in healthy eyes. BMO-MRW and BMO-MRA showed temporalization in the same manner as pRNFLT in the temporally tilted disc eyes. The BMO-MRW angle showed that in temporally tilted disc eyes, optic nerve fibers met the BMO plane steeply in the nasal sector and gently in the temporal sector than in non-tilted disc eyes, suggesting potential stress region of optic nerve fibers in temporally tilted disc eyes.

Multicomponent-Loaded Vesosomal Drug Carrier for Controlled and Sustained Compound Release.

Liposomes have been extensively adopted in drug delivery systems with clinically approved formulations. However, hurdles remain in terms of loading multiple components and precisely controlling their release. Herein, we report a vesosomal carrier composed of liposomes encapsulated inside the core of another liposome for the controlled and sustained release of multiple contents. The inner liposomes are made of lipids with different compositions and are co-encapsulated with a photosensitizer. Upon induction of reactive oxygen species (ROS), the contents of the liposomes are released, with each type of liposome displaying distinct kinetics due to the variance in lipid peroxidation for differential structural deformation. In vitro experiments demonstrated immediate content release from ROS-vulnerable liposomes, followed by sustained release from ROS-nonvulnerable liposomes. Moreover, the release trigger was validated at the organismal level using Caenorhabditis elegans. This study demonstrates a promising platform for more precisely controlling the release of multiple components.

HDAC2 as a target for developing anti-cancer drugs.

Histone deacetylases (HDACs) deacetylate histones H3 and H4. An imbalance between histone acetylation and deacetylation can lead to various diseases. HDAC2 is present in the nucleus. It plays a critical role in modifying chromatin structures and regulates the expression of various genes by functioning as a transcriptional regulator. The roles of HDAC2 in tumorigenesis and anti-cancer drug resistance are discussed in this review. Several reports suggested that HDAC2 is a prognostic marker of various cancers. The roles of microRNAs (miRNAs) that directly regulate the expression of HDAC2 in tumorigenesis are also discussed in this review. This review also presents HDAC2 as a valuable target for developing anti-cancer drugs.

Cryo-EM as a powerful tool for drug discovery: recent structural based studies of SARS-CoV-2.

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has arisen as a global pandemic affecting the respiratory system showing acute respiratory distress syndrome (ARDS). However, there is no targeted therapeutic agent yet and due to the growing cases of infections and the rising death tolls, discovery of the possible drug is the need of the hour. In general, the study for discovering therapeutic agent for SARS-CoV-2 is largely focused on large-scale screening with fragment-based drug discovery (FBDD). With the recent advancement in cryo-electron microscopy (Cryo-EM), it has become one of the widely used tools in structural biology. It is effective in investigating the structure of numerous proteins in high-resolution and also had an intense influence on drug discovery, determining the binding reaction and regulation of known drugs as well as leading the design and development of new drug candidates. Here, we review the application of cryo-EM in a structure-based drug design (SBDD) and in silico screening of the recently acquired FBDD in SARS-CoV-2. Such insights will help deliver better understanding in the procurement of the effective remedial solution for this pandemic.

Load and Display: Engineering Encapsulin as a Modular Nanoplatform for Protein-Cargo Encapsulation and Protein-Ligand Decoration Using Split Intein and SpyTag/SpyCatcher.

Protein cage nanoparticles have a unique spherical hollow structure that provides a modifiable interior space and an exterior surface. For full application, it is desirable to utilize both the interior space and the exterior surface simultaneously with two different functionalities in a well-combined way. Here, we genetically engineered encapsulin protein cage nanoparticles (Encap) as modular nanoplatforms by introducing a split-C-intein (IntC) fragment and SpyTag into the interior and exterior surfaces, respectively. A complementary split-N-intein (IntN) was fused to various protein cargoes, such as NanoLuc luciferase (Nluc), enhanced green fluorescent protein (eGFP), and Nluc-miniSOG, individually, which led to their successful encapsulation into Encaps to form Cargo@Encap through split intein-mediated protein ligation during protein coexpression and cage assembly in bacteria. Conversely, the SpyCatcher protein was fused to various protein ligands, such as a glutathione binder (GST-SC), dimerizing ligands (FKBP12-SC and FRB-SC), and a cancer-targeting affibody (SC-EGFRAfb); subsequently, they were displayed on Cargo@Encaps through SpyTag/SpyCatcher ligation to form Cargo@Encap/Ligands in a mix-and-match manner. Nluc@Encap/glutathione-S-transferase (GST) was effectively immobilized on glutathione (GSH)-coated solid supports exhibiting repetitive and long-term usage of the encapsulated luciferases. We also established luciferase-embedded layer-by-layer (LbL) nanostructures by alternately depositing Nluc@Encap/FKBP12 and Nluc@Encap/FRB in the presence of rapamycin and applied enhanced green fluorescent protein (eGFP)@Encap/EGFRAfb as a target-specific fluorescent imaging probe to visualize specific cancer cells selectively. Modular functionalization of the interior space and the exterior surface of a protein cage nanoparticle may offer the opportunity to develop new protein-based nanostructured devices and nanomedical tools.

Long-term HbA1c variability and the development and progression of diabetic retinopathy in subjects with type 2 diabetes.

This study aimed to investigate whether long-term HbA1c variability is associated with the development and progression of diabetic retinopathy (DR) in subjects with type 2 diabetes. We retrospectively reviewed 434 type 2 diabetes subjects without DR who underwent regular DR screening. We reviewed fundus findings, collected HbA1c levels, and calculated the coefficient of variation (CV) and average real variability (ARV) of each subject's HbA1c level. DR was developed in 55 subjects and progressed to moderate nonproliferative DR or worse DR in 23 subjects. On Cox proportional hazards regression analysis, HbA1c ARV, but not HbA1c CV, was significantly associated with DR development. However, the association between HbA1c variability and the DR progression rate to moderate nonproliferative DR or worse DR was not significant. The inter-visit HbA1c difference value on consecutive examination predicted DR development well and more careful screening for DR is needed for those with an absolute value change of 2.05%, an absolute increase of 1.75%, and an absolute decrease of 1.45% in HbA1c levels on consecutive examination. These results indicate that long-term glucose variability measured by HbA1c ARV might be an independent risk factor for DR development in addition to the mean HbA1c level in early diabetic subjects.

New record of the genus Leucinodella Strand from Laos (Lepidoptera, Crambidae, Spilomelinae), with description of a new species.

The genus Leucinodella Strand (1918) is reported from Laos for the first time, with a new species, Leucinodella banthaensis n. sp., and one newly recorded species, Leucinodella leucostola (Hampson, 1896). A key to Leucinodella species in Laos is provided, with illustrations of adults and genitalia.

Dual Binding to Orthosteric and Allosteric Sites Enhances the Anticancer Activity of a TRAP1-Targeting Drug.

The molecular chaperone TRAP1 is the mitochondrial paralog of Hsp90 and is overexpressed in many cancer cells. The orthosteric ATP-binding site of TRAP1 has been considered the primary inhibitor binding location, but TRAP1 allosteric modulators have not yet been investigated. Here, we generated and characterized the Hsp90 inhibitor PU-H71, conjugated to the mitochondrial delivery vehicle triphenylphosphonium (TPP) with a C10 carbon spacer, named SMTIN-C10, to enable dual binding to orthosteric and allosteric sites. In addition to tight binding with the ATP-binding site through the PU-H71 moiety, SMTIN-C10 interacts with the E115 residue in the N-terminal domain through the TPP moiety and subsequently induces structural transition of TRAP1 to a tightly packed closed form. The data indicate the existence of a druggable allosteric site neighboring the orthosteric ATP pocket that can be exploited to develop potent TRAP1 modulators.

Structural and kinetic insights into flavin-containing monooxygenase and calponin-homology domains in human MICAL3.

MICAL is an oxidoreductase that participates in cytoskeleton reorganization via actin disassembly in the presence of NADPH. Although three MICALs (MICAL1, MICAL2 and MICAL3) have been identified in mammals, only the structure of mouse MICAL1 has been reported. Here, the first crystal structure of human MICAL3, which contains the flavin-containing monooxygenase (FMO) and calponin-homology (CH) domains, is reported. MICAL3 has an FAD/NADP-binding Rossmann-fold domain for mono-oxygenase activity like MICAL1. The FMO and CH domains of both MICAL3 and MICAL1 are highly similar in structure, but superimposition of the two structures shows a different relative position of the CH domain in the asymmetric unit. Based on kinetic analyses, the catalytic efficiency of MICAL3 dramatically increased on adding F-actin only when the CH domain was available. However, this did not occur when two residues, Glu213 and Arg530, were mutated in the FMO and CH domains, respectively. Overall, MICAL3 is structurally highly similar to MICAL1, which suggests that they may adopt the same catalytic mechanism, but the difference in the relative position of the CH domain produces a difference in F-actin substrate specificity.

Structural insights into stressosome assembly.

The stressosome transduces environmental stress signals to SigB to upregulate SigB-dependent transcription, which is required for bacterial viability. The stressosome core is composed of RsbS and at least one of the RsbR paralogs. A previous cryo-electron microscopy (cryo-EM) structure of the RsbRA-RsbS complex determined under a D2 symmetry restraint showed that the stressosome core forms a pseudo-icosahedron consisting of 60 STAS domains of RsbRA and RsbS. However, it is still unclear how RsbS and one of the RsbR paralogs assemble into the stressosome. Here, an assembly model of the stressosome is presented based on the crystal structure of the RsbS icosahedron and cryo-EM structures of the RsbRA-RsbS complex determined under diverse symmetry restraints (nonsymmetric C1, dihedral D2 and icosahedral I envelopes). 60 monomers of the crystal structure of RsbS fitted well into the I-restrained cryo-EM structure determined at 4.1 Šresolution, even though the STAS domains in the I envelope were averaged. This indicates that RsbS and RsbRA share a highly conserved STAS fold. 22 protrusions observed in the C1 envelope, corresponding to dimers of the RsbRA N-domain, allowed the STAS domains of RsbRA and RsbS to be distinguished in the stressosome core. Based on these, the model of the stressosome core was reconstructed. The mutation of RsbRA residues at the binding interface in the model (R189A/Q191A) significantly reduced the interaction between RsbRA and RsbS. These results suggest that nonconserved residues in the conserved STAS folds between RsbS and RsbR paralogs determine stressosome assembly.

Location of Retinal Nerve Fiber Layer Defects in Open-angle Glaucoma and Associated Factors.

PURPOSE: To investigate the location of retinal nerve fiber layer defects (RNFLDs) in open-angle glaucoma and the differences in systemic and ocular factors between superotemporal and inferotemporal RNFLDs. METHODS: We performed a retrospective review of the 2008 to 2012 data from the Korea National Health and Nutrition Examination Survey. Subjects aged ≥19 years with an evaluable fundus photograph of at least one eye were enrolled, and open-angle glaucoma was diagnosed according to modified International Society of Geographical and Epidemiological Ophthalmology criteria. In subjects with open-angle glaucoma, locations of RNFLDs were evaluated, and systemic and ocular factors were compared between the bilateral superotemporal RNFLD group and bilateral inferotemporal RNFLD group. RESULTS: A total of 534 subjects had open-angle glaucoma with RNFLDs. The unilateral inferotemporal region (25.8%) was the most common location for RNFLDs, followed by the unilateral superotemporal region (24.4%). Multivariate analysis revealed that hypertension was more significantly associated (p = 0.048) with the bilateral superotemporal RNFLD group than with the bilateral inferotemporal RNFLD group. CONCLUSIONS: Superotemporal RNFLDs are more related to hypertension than are inferotemporal RNFLDs.

Structural analysis of the flagellar capping protein FliD from Helicobacter pylori.

Helicobacter pylori is a pathogenic flagellated bacterium that infects the gastroduodenal mucosa and causes peptic ulcers in humans. FliD caps the distal end of the flagellar filament and is essential in filament growth. Moreover, FliD has been studied to diagnose and prevent H. pylori infection. Here, we report structure-based molecular studies of H. pylori FliD (hpFliD). A crystal structure of hpFliD at 2.6 Šresolution presents a four-domain (D2-D5) structure, where the D3 domain forms a central platform surrounded by the other three domains (D2, D4, and D5). hpFliD domains D2 and D3 structurally resemble those of FliD orthologs, whereas the D4 and D5 domains are exclusive to hpFliD. Moreover, our ELISA analysis using anti-H. pylori antibodies demonstrated that the hpFliD-specific D4 and D5 domains are highly antigenic compared to the D2 and D3 domains. Collectively, our structural and serological analyses underscore the structural role of hpFliD domains and provide a molecular basis for vaccine and diagnosis development.

MiR-135-5p-p62 Axis Regulates Autophagic Flux, Tumorigenic Potential, and Cellular Interactions Mediated by Extracellular Vesicles During Allergic Inflammation.

The objective of this study was to investigate the relationship between autophagy and allergic inflammation. In vitro allergic inflammation was accompanied by an increased autophagic flux in rat basophilic leukemia (RBL2H3) cells. 3-MA, an inhibitor of autophagic processes, negatively regulated allergic inflammation both in vitro and in vivo. The role of p62, a selective receptor of autophagy, in allergic inflammation was investigated. P62, increased by antigen stimulation, mediated in vitro allergic inflammation, passive cutaneous anaphylaxis (PCA), and passive systemic anaphylaxis (PSA). P62 mediated cellular interactions during allergic inflammation. It also mediated tumorigenic and metastatic potential of cancer cells enhanced by PSA. TargetScan analysis predicted that miR-135-5p was a negative regulator of p62. Luciferase activity assay showed that miR-135-5p directly regulated p62. MiR-135-5p mimic negatively regulated features of allergic inflammation and inhibited tumorigenic and metastatic potential of cancer cells enhanced by PSA. MiR-135-5p mimic also inhibited cellular interactions during allergic inflammation. Extracellular vesicles mediated allergic inflammation both in vitro and in vivo. Extracellular vesicles were also necessary for cellular interactions during allergic inflammation. Transmission electron microscopy showed p62 within extracellular vesicles of antigen-stimulated rat basophilic leukemia cells (RBL2H3). Extracellular vesicles isolated from antigen-stimulated RBL2H3 cells induced activation of macrophages and enhanced invasion and migration potential of B16F1 mouse melanoma cells in a p62-dependent manner. Extracellular vesicles isolated from PSA-activated BALB/C mouse enhanced invasion and migration potential of B16F1 cells, and induced features of allergic inflammation in RBL2H3 cells. Thus, miR-135-5p-p62 axis might serve as a target for developing anti-allergy drugs.

Development of 17 polymorphic microsatellite loci from Jeju striped field mouse, Apodemus agrarius chejuensis (Rodentia: Muridae), by 454 pyrosequencing.

BACKGROUND: The striped field mouse, Apodemus agrarius, is the most common mammal in Korea. Although microsatellite loci for the species have been identified from populations in southwestern China, amplification of those markers for Korean populations have been unsuccessful. The complicated taxonomy of Korean striped field mouse including populations on Jeju Island (A. a chejuensis) necessitates identification of additional molecular markers. FINDINGS: We applied 454 pyrosequencing systems to develop a suite of microsatellite markers. Muscle tissue was harvested and sequenced from 30 Jeju striped field mouse specimens which yielded 12,165 reads with a mean length per read of 287 bp. From these reads, we identified 17 microsatellite loci for A. a. chejuensis and tested these new markers against samples of both A. a chejuensis and A. a coreae, the mainland taxon. All 17 loci were amplified successfully for both taxa. Of the total 17 loci, one locus failed to amplify for a population on Heuksan Island. The cross-species transferability was also tested with the allied taxon, A. peninsulae and confirmed successful for 12 loci. CONCLUSIONS: These newly developed markers will benefit studies of genetic structure, evolution, and resolving taxonomic problems of striped field mice and allied taxa in Korea.

Fabrication of Nanoreaction Clusters with Dual-Functionalized Protein Cage Nanobuilding Blocks.

Fabrication of functional nanostructures is a prominent issue in nanotechnology, because they often exhibit unique properties that are different from the individual building blocks. Protein cage nanoparticles are attractive nanobuilding blocks for constructing nanostructures due to their well-defined symmetric spherical structures, polyvalent nature, and functional plasticity. Here, a lumazine synthase protein cage nanoparticle is genetically modified to be used as a template to generate functional nanobuilding blocks and covalently display enzymes (ß-lactamase) and protein ligands (FKBP12/FRB) on its surface, making dual-functional nanobuilding blocks. Nanoreaction clusters are subsequently created by ligand-mediated alternate deposition of two complementary building blocks using layer-by-layer (LbL) assemblies. 3D nanoreaction clusters provide enhanced enzymatic activity compared with monolayered building block arrays. The approaches described here may provide new opportunities for fabricating functional nanostructures and nanoreaction clusters, leading to the development of new protein nanoparticle-based nanostructured biosensor devices.

The actin bundling activity of actin bundling protein 34 is inhibited by calcium binding to the EF2.

Actin bundling protein 34 (ABP34) is the one of 11 actin-crosslinking proteins identified in Dictyostelium discoideum, a novel model organism for the study of actin-associated neurodegenerative disorders such as Alzheimer's disease and Huntington's disease. ABP34 localizes at the leading and trailing edges of locomotory cells, i.e., at the cell cortex, filopodia, and pseudopodia. Functionally, it serves to stabilize membrane-associated actin at sites of cell-cell contact. In addition, this small crosslinking protein is involved in actin bundle formation, and its bundling activity is regulated by the concentration of calcium ion. Several studies have sought to determine the mechanism underlying the calcium-regulated actin bundling activity of ABP34, but it remains unclear. Using several mutational and structural analyses, we revealed that calcium binding to the EF2 motif disrupts the inter-domain interaction between the N- and C-domains, thereby inhibiting the actin bundling activity of ABP34. This finding provides clues about the pathogenesis of neurodegenerative disorders related to actin bundling.

Analysis of nano-crystals: Evaluation of heavy metal-embedded biological specimen by high voltage electron microscopy.

Heavy metal compounds are adsorbed onto biological specimen in order to enhance the contrast as well as to preserve the structural features of the specimen against electron beam-induced radiation damage. In particular, in combination with computational image processing, negative staining is widely used for structural analysis of protein complexes to moderate resolutions. Image analysis of negatively stained biological specimen is known to suffer from limited achievable resolution due to dehydration and large grain size of staining molecules although the extent of such effect remains somewhat dubious. Stain molecules exist as grains under electron beam. However, clear observation of the crystalline nature of the grains and their association with biological specimen has not been thoroughly demonstrated. In this study, we attempted high-resolution TEM (HRTEM) using high voltage electron microscopy and electron crystallography analysis for the detailed characterization of negatively stained biological specimen, focusing on physical state and chemical composition of the stain molecules. The electron crystallography analysis allowed for the identification of the crystal constituents of widely used stains, hence revealing the chemical nature and the morphology of the stain molecules at specimen level. This study re-evaluated generally accepted notions on negative staining, and may help correctly interpreting the structural analysis of stained biological specimen.

Locator-Checker-Scaler Object Tracking Using Spatially Ordered and Weighted Patch Descriptor.

In this paper, we propose a simple yet effective object descriptor and a novel tracking algorithm to track a target object accurately. For the object description, we divide the bounding box of a target object into multiple patches and describe them with color and gradient histograms. Then, we determine the foreground weight of each patch to alleviate the impacts of background information in the bounding box. To this end, we perform random walk with restart (RWR) simulation. We then concatenate the weighted patch descriptors to yield the spatially ordered and weighted patch (SOWP) descriptor. For the object tracking, we incorporate the proposed SOWP descriptor into a novel tracking algorithm, which has three components: locator, checker, and scaler (LCS). The locator and the scaler estimate the center location and the size of a target, respectively. The checker determines whether it is safe to adjust the target scale in a current frame. These three components cooperate with one another to achieve robust tracking. Experimental results demonstrate that the proposed LCS tracker achieves excellent performance on recent benchmarks.