Producing high-quantity and high-quality recombinant adeno-associated virus by low-cis triple transfection.

Recombinant adeno-associated virus (rAAV)-based gene therapy is entering clinical and commercial stages at an unprecedented pace. Triple transfection of HEK293 cells is currently the most widely used platform for rAAV manufacturing. Here, we develop low-cis triple transfection that decreases transgene plasmid use by 10- to 100-fold and overcomes several major limitations associated with standard triple transfection. This new method improves packaging of yield-inhibiting transgenes by up to 10-fold, and generates rAAV batches with reduced plasmid backbone contamination that otherwise cannot be eliminated in downstream processing. When tested in mice and compared with rAAV produced by standard triple transfection, low-cis rAAV shows comparable or superior potency and results in diminished plasmid backbone DNA and RNA persistence in tissue. Mechanistically, low-cis triple transfection relies on the extensive replication of transgene cassette (i.e., inverted terminal repeat-flanked vector DNA) in HEK293 cells during production phase. This cost-effective method can be easily implemented and is widely applicable to producing rAAV of high quantity, purity, and potency.

Structural and functional investigation of GajB protein in Gabija anti-phage defense.

Bacteriophages (phages) are viruses that infect bacteria and archaea. To fend off invading phages, the hosts have evolved a variety of anti-phage defense mechanisms. Gabija is one of the most abundant prokaryotic antiviral systems and consists of two proteins, GajA and GajB. GajA has been characterized experimentally as a sequence-specific DNA endonuclease. Although GajB was previously predicted to be a UvrD-like helicase, its function is unclear. Here, we report the results of structural and functional analyses of GajB. The crystal structure of GajB revealed a UvrD-like domain architecture, including two RecA-like core and two accessory subdomains. However, local structural elements that are important for the helicase function of UvrD are not conserved in GajB. In functional assays, GajB did not unwind or bind various types of DNA substrates. We demonstrated that GajB interacts with GajA to form a heterooctameric Gabija complex, but GajB did not exhibit helicase activity when bound to GajA. These results advance our understanding of the molecular mechanism underlying Gabija anti-phage defense and highlight the role of GajB as a component of a multi-subunit antiviral complex in bacteria.

Approaches for 3D Integration Using Plasma-Enhanced Atomic-Layer-Deposited Atomically-Ordered InGaZnO Transistors with Ultra-High Mobility.

As the scale-down and power-saving of silicon-based channel materials approach the limit, oxide semiconductors are being actively researched for applications in 3D back-end-of-line integration. For these applications, it is necessary to develop stable oxide semiconductors with electrical properties similar to those of Si. Herein, a single-crystal-like indium-gallium-zinc-oxide (IGZO) layer (referred to as a pseudo-single-crystal) is synthesized using plasma-enhanced atomic layer deposition and fabricated stable IGZO transistors with an ultra-high mobility of over 100 cm2  Vs-1 . To acquire high-quality atomic layer deposition-processed IGZO layers, the plasma power of the reactant is controlled as an effective processing parameter by evaluating and understanding the effect of the chemical reaction of the precursors on the behavior of the residual hydrogen, carbon, and oxygen in the as-deposited films. Based on these insights, this study found that there is a critical relationship between the optimal plasma reaction energy, superior electrical performance, and device stability.

Selectively Nitrogen Doped ALD-IGZO TFTs with Extremely High Mobility and Reliability.

Achieving high mobility and reliability in atomic layer deposition (ALD)-based IGZO thin-film transistors (TFTs) with an amorphous phase is vital for practical applications in relevant fields. Here, we suggest a method to effectively increase stability while maintaining high mobility by employing the selective application of nitrous oxide plasma reactant during plasma-enhanced ALD (PEALD) at 200 °C process temperature. The nitrogen-doping mechanism is highly dependent on the intrinsic carbon impurities or nature of each cation, as demonstrated by a combination of theoretical and experimental research. The Ga2O3 subgap states are especially dependent on plasma reactants. Based on these insights, we can obtain high-performance indium-rich PEALD-IGZO TFTs (threshold voltage: -0.47 V; field-effect mobility: 106.5 cm2/(V s); subthreshold swing: 113.5 mV/decade; hysteresis: 0.05 V). In addition, the device shows minimal threshold voltage shifts of +0.45 and -0.10 V under harsh positive/negative bias temperature stress environments (field stress: ±2 MV/cm; temperature stress: 95 °C) after 10000 s.

Sphingomonas cremea sp. nov., isolated from ginseng soil.

A novel Gram-stain-negative, aerobic, rod-shaped, non-motile, cream-coloured strain (G124T) was isolated from ginseng soil collected in Yeongju, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain G124T belongs to a distinct lineage within the genus Sphingomonas (family Sphingomonadaceae, order Sphingomonadales and class Alphaproteobacteria). Strain G124T was closely related to Sphingomonas rhizophila THG-T61T (98.5 % 16S rRNA gene sequence similarity), Sphingomonas mesophila SYSUP0001T (98.3 %), Sphingomonas edaphi DAC4T (97.6 %) and Sphingomonas jaspsi TDMA-16T (97.6 %). The strain contained ubiquinone 10 as the major respiratory quinone. The major polar lipid profile of strain G124T comprised phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and sphingoglycolipids. The predominant cellular fatty acids of strain G124T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c; 33.4 %), summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c; 27.2 %) and C16 : 0 (18.3 %). The genome size of strain G124T was 2 549 305 bp. The genomic DNA G+C content is 62.0 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain G124T and other Sphingomonas species were in the range of 71.2-75.9 % and 18.7-19.9 %, respectively. Based on the polyphasic analysis such as biochemical, phylogenetic and chemotaxonomic characteristics, strain G124T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cremea sp. nov. is proposed. The type strain is G124T (=KACC 21691T=LMG 31729T).

Hymenobacter lucidus sp. nov., and Hymenobacter nitidus sp. nov., isolated from soil.

Two novel bacterial strains BT178T and BT635T were isolated from soil collected in Gyeonggi province, Republic of Korea. Both strains were Gram-negative, rod-shaped circular, and had convex, glistening, pink-colored colonies. Phylogenetic analysis based on 16S rRNA gene sequences showed that each of the strains BT178T and BT635T formed an individual lineage within the genus Hymenobacter (family Hymenobacteraceae, order Cytophagales, class Cytophagia, phylum Bacteroidetes). The 16S rRNA gene sequence similarity between the two strains BT178T and BT635T were 97.7%. Both strains had Q-7 as the major respiratory quinone and phosphatidylethanolamine (PE) as the major polar lipids. The main cellular fatty acids of strain BT178T were iso-C15:0, anteiso-C15:0, and those of strain BT635T were iso-C15:0 and Summed Feature 4 (iso-C17:1 I / anteiso-C17:1 B). Based on the result of polyphasic taxonomic analysis, two novel species, Hymenobacter lucidus BT178T (= KCTC 72333T = NBRC 115442T) and Hymenobacter nitidus BT635T (= KACC 21904T = NBRC 115443T) were proposed.

Fully Immersive Virtual Reality Game-Based Training for an Adolescent with Spastic Diplegic Cerebral Palsy: A Case Report.

BACKGROUND: Recently, virtual reality-based training (VR-based training) is receiving attention as greater emphasis is placed on the importance of interest and motivation in participation. However, studies investigating the effects of fully immersive VR-based training are insufficient. CASE PRESENTATION: We report a case of using a fully immersive VR game-based training in a patient with cerebral palsy. A 15-year-old girl was diagnosed with spastic diplegia cerebral palsy Gross Motor Function Classification System level II. A six-week intervention (18 sessions) phase was performed with one fully immersive VR game using PlayStation®VR in three sessions per week. After 18 sessions of training, the scores on the gross motor function measure-88 (Gross Motor Function Measure-88-GMFM-88), pediatric balance scale (PBS), timed up and go test (TUG), functional gait assessment (FGA), and 10 m walking test (10MWT) were improved: GMFM-88, 91.56 points (9.31 points increase); PBS, 45 points (6 points increase); TUG, 8.23 s (6.9 s decrease); FGA, 11 points (3 points increase); the 10 MWT, 5.27 s (6.59 s decrease). CONCLUSIONS: This study found that a fully immersive VR game-based training using PlayStation®VR may be an effective intervention for GMFCS level II adolescent, leading to some improvement of motor function, balance and gait skills in adolescents with cerebral palsy.

Enhanced performance and stability in InGaZnO NIR phototransistors with alumina-infilled quantum dot solid.

The optimized ALD infilling process for depositing Al2O3 in the vertical direction of PbS QDs enhances the photoresponsivity, relaxation rate and the air stability of PbS QDs hybrid IGZO NIR phototransistors. Infilled Al2O3, which is gradually deposited from the top of PbS QDs to the PbS/IGZO interface (1) passivates the trap sites up to the interface of PbS/IGZO without disturbing charge transfer and (2) prevents QDs deterioration caused by outside air. Therefore, an Al2O3 infilled PbS QD/IGZO hybrid phototransistor (AI-PTs) exhibited enhanced photoresponsivity from 96.4 A/W to 1.65 × 102 A/W and a relaxation time decrease from 0.52 to 0.03 s under NIR light (880 nm) compared to hybrid phototransistors without Al2O3 (RF-PTs). In addition, AI-PTs also showed improved shelf stability over 4 months compared to RF-PTs. Finally, all devices we manufactured have the potential to be manufactured in an array, and this ALD technique is a means of fabricating robust QDs/metal oxide hybrids for optoelectronic devices.

Microvirga splendida sp. nov., bacteria isolated from soil.

Two bacterial strains, BT325T and BT690, were isolated from soil samples collected in Korea. Both strains were Gram stain-negative, short rod-shaped, and formed light-pink colored colonies. The 16S rRNA sequence similarity of strains BT325T and BT690 shared a sequence similarity of 99.7%. Both strains shared the highest 16S rRNA gene similarity of 98.6% with Microvirga arabica SV2184PT, followed by Microvirga ossetica V5/3 M T (98.5% and 98.2%, respectively), Microvirga soli R491T (98.3% and 98.2%, respectively), Microvirga aerilata (98.2% and 98.08%, respectively), Microvirga makkahensis (98.08% and 97.8%, respectively). Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain BT325T and BT690 were positioned in a distinct lineage within the family Methylobacteriaceae (order Rhizobiales, class Alphaproteobacteria). The genome size of strain BT325T was 5,200,315 bp and the genomic DNA G + C content was 64.3 mol%. The sole respiratory quinone of strain BT325T was Q-10 and the predominant cellular fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. Polyphasic taxonomic analysis of biochemical, chemotaxonomic, and phylogenetic analyses suggested that strains BT325T represents a novel bacterial species within the genus Microvirga, for which the name Microvirga splendida is proposed. The type strain of Microvirga splendida is BT325T (= KCTC 72406 T = NBRC 114847 T).

Microvirga jeongseonensis sp. nov., isolated from soil in South Korea.

A novel Gram-stain-negative, aerobic, rod-shaped, convex, and light pink-colored strain BT688T was isolated from a soil sample collected in Jeongseon City, South Korea. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain BT688T belongs to a distinct lineage within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alphaproteobacteria, phylum Proteobacteria). The 16S rRNA gene sequence similarity between strain BT688T and Microvirga aerilata 5420S-16T was 98.5%. Strain BT688T had Q-10 as a major respiratory quinone and the major polar lipids were diphosphatidilglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). The major cellular fatty acids of strain BT688T were C18:1 ω7c (76.0%) and summed feature 3 (9.6%). Based on the polyphasic characteristics, strain BT688T represents a novel bacterial species within the genus Microvirga and the proposed name is Microvirga jeongseonensis. The type strain of Microvirga jeongseonensis is BT688T (= KCTC 82701T = NBRC 114857T).

Microvirga pudoricolor sp. nov., and Microvirga alba sp. nov., isolated from soil in South Korea.

Two novel Gram-stain-negative, aerobic, rod-shaped, circular, convex, light-pink and white-colored bacterial strains BT291T and BT350T were isolated from soil collected in Uijeongbu city (37° 44' 55″ N, 127° 2' 20″ E) and Jeju island (33° 22' 48″ N, 126° 31' 48″ E), respectively, South Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that each of the strains BT291T and BT350T belong to a distinct lineages within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alpha Proteobacteria, phylum Proteobacteria, kingdom Bacteria). The 16S rRNA gene sequence similarity between the two strains BT291T and BT350T was 97.4%. The two strains were found to have the same quinone system, with Q-10 as the major respiratory quinone. The major polar lipids of strains BT291T and BT350T were phosphatidylethanolamine (PE), diphosphatydilglycerol (DPG), phosphatidylcholine (PC) and phosphatidylglycerol (PG). The major cellular fatty acids of strain BT291T were C18:1 ω7c (58.2%) and cyclo-C19:0 ω8c (25.7%). The major cellular fatty acids of strain BT350T were C18:1 ω7c (38.5%) and cyclo-C19:0 ω8c (27.7%). Based on the polyphasic analysis (phylogenetic, chemotaxonomic and biochemical), strains BT291T and BT350T can be suggested as two novel bacterial species within the genus Microvirga and the proposed names are Microvirga pudoricolor and Microvirga alba, respectively. The type strain of Microvirga pudoricolor is BT291T (= KCTC 72368T = NBRC 114845T) and the type strain of Microvirga alba is BT350T (= KCTC 72385T = NBRC 114848T).

Hymenobacter puniceus sp. nov., radiation resistant bacteria isolated from soil in South Korea.

Two novel Gram-negative bacterial strains BT190T and BT191 were isolated from soil collected in Uijeongbu city (37°44'55″N, 127°02'20″E), Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains BT190T and BT191 belong to a distinct lineage within the genus Hymenobacter (family Hymenobacteraceae, order Cytophagales, class Cytophagia). The level of 16S rRNA gene sequence similarity between the strains BT190T and BT190 was 99.5%. The strains BT190T and BT191 were closely related to Hymenobacter swuensis DY53T (97.0% 16S rRNA gene similarity), Hymenobacter metallilatus 9PBR-2 T (96.8%), Hymenobacter tibetensis XTM003T (96.8%) and Hymenobacter yonginensis HMD1010T (96.6%). The genome size of strain BT190T was 4,859,864 bp. The DNA G+C content of strain BT190T was 55.3 mol%. Bacterial growth was observed at 4-30 °C (optimum 25 °C) and pH 6.0-9.0 (optimum, pH 6.0-7.0) on R2A agar. Colonies of strains BT190T and BT191 were raised, smooth, circular and red-pink colored. The sole respiratory quinone of strain BT190T was MK-7 and the predominant cellular fatty acids were iso-C15:0, C16:1 ω5c, summed feature 3 (C16:1 ω6c / C16:1 ω7c) and summed feature 4 (iso-C17:1 I / anteiso-C17:1 B). The major polar lipids of strain BT190T were aminophospholipid (APL) and phosphatidylethanolamine (PE). Based on the chemotaxonomic, biochemical, and phylogenetic analysis, strains BT190T and BT191 can be suggested as a novel bacterial species within the genus Hymenobacter, for which the name Hymenobacter puniceus sp. nov is proposed. The type strain of Hymenobacter puniceus is BT190T (= KCTC 72342 T = NBRC 114860 T).

Feasibility of full immersive virtual reality video game on balance and cybersickness of healthy adolescents.

Various virtual reality (VR) games with head-mounted displays (HMDs) can provide immersive experiences, but are often accompanied by negative experiences. We investigated the feasibility of immersive virtual reality game on balance and cybersickness in healthy adolescents. This is cross-over design. This study included 11 healthy adolescents. Balance and cybersickness were measured in the following three conditions. First, as a baseline, only balance was measured while no immersive virtual reality game was being played. Second, balance and cybersickness were measured while the participants played an immersive virtual reality game with a fixed background. Third, balance and cybersickness were measured while the participants played an immersive virtual reality game with a moving background. A force plate was used to measure balance, while the Questionnaire (VRSQ) and Simulator Sickness Questionnaire (SSQ) were used to measure cybersickness. Sway velocity and length significantly increased during the game with a moving background compared to baseline and a fixed background game (p < 0.05). VRSQ and SSQ scores significantly increased during the game with a fixed and moving background compared to baseline (p < 0.05) and were significantly higher with use of the moving versus fixed background (p < 0.05). This study demonstrated that playing an immersive virtual reality game with a moving background could negatively affect balance and cybersickness. These results will help to select game contents that can reduce side effects when applying VR HMD to various fields in the future.

Preparation and Electrochemical Analysis of Ni-Based Metal Organic Frameworks Containing Binary Ligands for Capacitor Electrodes.

As one of the energy storage systems, supercapacitors have quite long charge-discharge cycle life. Among many kinds of electrode materials, metal organic frameworks (MOFs) have unique properties such as high specific surface areas and large pore volume as supercapacitor electrode materials. Nickel-MOFs consist of binary ligand such as 1,3,5-Trimesic acid (H3BTC) and terephthalic acid (TPA) were used as working electrode materials in three electrode cell for capacitor system. When synthesizing MOFs, it is possible to prepare uniform crystals using hydrothermal synthesis. The morphology of composites was analyzed by field emission scanning electron microscopy (FE-SEM). Electrochemical properties were measured by cyclic voltammetry (CV), galvanostatic charge-discharge (GCD) in 6M KOH electrolyte.

Structural and functional evidence of bacterial antiphage protection by Thoeris defense system via NAD+ degradation.

The intense arms race between bacteria and phages has led to the development of diverse antiphage defense systems in bacteria. Unlike well-known restriction-modification and CRISPR-Cas systems, recently discovered systems are poorly characterized. One such system is the Thoeris defense system, which consists of two genes, thsA and thsB. Here, we report structural and functional analyses of ThsA and ThsB. ThsA exhibits robust NAD+ cleavage activity and a two-domain architecture containing sirtuin-like and SLOG-like domains. Mutation analysis suggests that NAD+ cleavage is linked to the antiphage function of Thoeris. ThsB exhibits a structural resemblance to TIR domain proteins such as nucleotide hydrolases and Toll-like receptors, but no enzymatic activity is detected in our in vitro assays. These results further our understanding of the molecular mechanism underlying the Thoeris defense system, highlighting a unique strategy for bacterial antiphage resistance via NAD+ degradation.

Molecular balloon, Pd6L8 cages: recognition of alkyl sulfate surfactants.

The unique molecular balloon system of [Pd6L8](NO3)12 (an inner cavity of 19 × 21 × 25 Å3⇄ 13 × 13 × 13 Å3) was carried out via the anion exchange of nitrate with alkyl sulfates.

SPIN90, an adaptor protein, alters the proximity between Rab5 and Gapex5 and facilitates Rab5 activation during EGF endocytosis.

During ligand-mediated receptor endocytosis, the small GTPase Rab5 functions in vesicle fusion and trafficking. Rab5 activation is known to require interactions with its guanine nucleotide-exchange factors (GEFs); however, the mechanism regulating Rab5 interactions with GEFs remains unclear. Here, we show that the SH3-adapter protein SPIN90 participates in the activation of Rab5 through the recruitment of both Rab5 and its GEF, Gapex5, to endosomal membranes during epidermal growth factor (EGF)-mediated endocytosis. SPIN90 strongly interacts with the inactive Rab5/GDI2 complex through its C-terminus. In response to EGF signaling, extracellular signal-regulated kinase (ERK)-mediated phosphorylation of SPIN90 at Thr-242 enables SPIN90 to bind Gapex5 through its N-terminal SH3 domain. Gapex5 is a determinant of Rab5 membrane targeting, while SPIN90 mediates the interaction between Gapex5 and Rab5 in a phosphorylation-dependent manner. Collectively, our findings suggest that SPIN90, as an adaptor protein, simultaneously binds inactive Rab5 and Gapex5, thereby altering their spatial proximity and facilitating Rab5 activation.

Novel DNA Aptamers that Bind to Mutant Huntingtin and Modify Its Activity.

The CAG repeat expansion that elongates the polyglutamine tract in huntingtin is the root genetic cause of Huntington's disease (HD), a debilitating neurodegenerative disorder. This seemingly slight change to the primary amino acid sequence alters the physical structure of the mutant protein and alters its activity. We have identified a set of G-quadruplex-forming DNA aptamers (MS1, MS2, MS3, MS4) that bind mutant huntingtin proximal to lysines K2932/K2934 in the C-terminal CTD-II domain. Aptamer binding to mutant huntingtin abrogated the enhanced polycomb repressive complex 2 (PRC2) stimulatory activity conferred by the expanded polyglutamine tract. In HD, but not normal, neuronal progenitor cells (NPCs), MS3 aptamer co-localized with endogenous mutant huntingtin and was associated with significantly decreased PRC2 activity. Furthermore, MS3 transfection protected HD NPCs against starvation-dependent stress with increased ATP. Therefore, DNA aptamers can preferentially target mutant huntingtin and modulate a gain of function endowed by the elongated polyglutamine segment. These mutant huntingtin binding aptamers provide novel molecular tools for delineating the effects of the HD mutation and encourage mutant huntingtin structure-based approaches to therapeutic development.

Main factor causing "faster-is-slower" phenomenon during evacuation: rodent experiment and simulation.

Understanding crowd flow at bottlenecks is important for preventing accidents in emergencies. In this research, a crowd evacuation passing through a narrow exit connected with guide-walls is analysed using the discrete element method based on physical and psychological modelling in parallel with empirical rodent research. Results of rodent experiment and simulation demonstrate the faster-is-slower (FIS) effect, which is a well-known phenomenon in pedestrian dynamics. As the angle of the guide-walls increases, agents rapidly evacuate the room even though they have low velocity. The increase in this angle causes agents to form lanes. It is validated that ordered agents evacuate expeditiously with relatively low velocity despite expectations to the contrary. The extracted experimental and simulation data strongly suggest that the agents' standard deviation of velocity can be a key factor causing the FIS effect. It is found that the FIS effect can be eliminated by controlling the standard deviation.

Isolation and Characterization of Bacillus cereus Bacteriophages from Foods and Soil.

The aim of this study was to isolate and characterize Bacillus cereus bacteriophages of various origins. Twenty-seven bacteriophages against B. cereus were isolated from various Korean traditional fermented foods and soils. Plaque size, transmission electron microscopy, virulence profile, and in vitro lytic activity of bacteriophage isolates were examined. Transmission electron microscopy confirmed B. cereus bacteriophages belonging to the family Siphoviridae. Among B. cereus bacteriophages with broad host range, 18 isolates (66.7%) did not harbor any B. cereus virulence factors. Among them, bacteriophage strain CAU150036, CAU150038, CAU150058, CAU150064, CAU150065, and CAU150066 effectively inhibited B. cereus in vitro within 1 h. Therefore, they are considered potential candidates for controlling the contamination of B. cereus in food or other applications.