Structural analysis of the YqeY proteins from Campylobacter jejuni and Vibrio parahaemolyticus.

YqeY is a functionally and structurally uncharacterized protein that is ubiquitously expressed in bacteria. To gain structural insights into the function of YqeY, we determined the crystal structures of the Campylobacter jejuni and Vibrio parahaemolyticus YqeY proteins (cjYqeY and vpYqeY, respectively) and analyzed the structural and functional roles of conserved residues via a mutational study. Both cjYqeY and vpYqeY were found to adopt a two-domain structure consisting of an N-terminal four-α-helix domain and a C-terminal three-α-helix domain, with a relatively flexible interdomain orientation. The YqeY structure is unique in its linkage of the two α-helix domains although the C-terminal YqeY domain is structurally homologous to the terminal appendages of glutaminyl-tRNA synthetase and tRNA-dependent amidotransferase. We identified six conserved YqeY residues (Y67, R72, E82, Y89, P91, and G119) and evaluated their roles in protein stability via alanine mutation using a thermal shift assay. Residues Y67, R72, Y89, and P91 were shown to be required to maintain the structural integrity of YqeY. In contrast, residues E82 and G119 were not found to be essential for protein stability and are highly likely to contribute to the biological function of YqeY.

A novel G3BP1-GFP reporter human lung cell system enabling real-time monitoring of stress granule dynamics for in vitro lung toxicity assessment.

Under various cellular stress conditions, including exposure to toxic chemicals, RNA-binding proteins (RBPs), including Ras GTPase-activating protein-binding protein 1 (G3BP1), aggregate and form stress granule complexes, which serve as hallmarks of cellular stress. The existing methods for analyzing stress granule assembly have limitations in the rapid detection of dynamic cellular stress and ignore the effects of constitutively overexpressed RBP on cellular stress and stress-related processes. Therefore, to overcome these limitations, we established a G3BP1-GFP reporter in a human lung epithelial cell line using CRISPR/Cas9-based knock-in as an alternative system for stress granule analysis. We showed that the G3BP1-GFP reporter system responds to stress conditions and forms a stress granule complex similar to that of native G3BP1. Furthermore, we validated the stress granule response of an established cell line under exposure to various household chemicals. Overall, this novel G3BP1-GFP reporter human lung cell system is capable of monitoring stress granule dynamics in real time and can be used for assessing the lung toxicity of various substances in vitro.

Unique dimeric structure of the DUF2891 family protein CJ0554 from Campylobacter jejuni.

Campylobacter jejuni is a pathogenic bacterium that causes enteritis and Guillain-Barre syndrome in humans. To identify a protein target for the development of a new therapeutic against C. jejuni infection, each gene product of C. jejuni must be functionally characterized. The cj0554 gene of C. jejuni encodes a DUF2891 family protein with unknown functions. To provide functional insights into CJ0554, we determined and analyzed the crystal structure of the CJ0554 protein. CJ0554 adopts an (α/α)6-barrel structure, which consists of an inner α6 ring and an outer α6 ring. CJ0554 assembles into a dimer in a unique top-to-top orientation that is not observed in its structural homologs, N-acetylglucosamine 2-epimerase superfamily members. Dimer formation was verified by analyzing CJ0554 and its ortholog protein through gel-filtration chromatography. The top of the CJ0554 monomer barrel harbors a cavity, which is connected to that of the second subunit in the dimer structure, generating a larger intersubunit cavity. This elongated cavity accommodates extra nonproteinaceous electron density, presumably as a pseudosubstrate, and is lined with generally catalytically active histidine residues that are invariant in CJ0554 orthologs. Therefore, we propose that the cavity functions as the active site of CJ0554.

Organocatalytic Asymmetric [3 + 2]-Annulations of γ-Sulfonamido/γ-Hydroxy-α,ß-Unsaturated Ketones with Cyclic N-Sulfimines: Synthesis of Chiral Polyheterotricyclic Imidazolidines and Oxazolidines.

The first organocatalytic asymmetric [3 + 2]-annulation of γ-sulfonamido-α,ß-unsaturated ketones with cyclic N-sulfimines has been developed, and enantioenriched functionalized polyheterotricyclic imidazolidines were obtained in good yields and with excellent enantioselectivities. This approach was also extended to the asymmetric [3 + 2]-annulation of γ-hydroxy-α,ß-unsaturated ketones, affording enantioenriched polyheterotricyclic oxazolidines. In addition, base-catalyzed [3 + 2]-annulations of γ-sulfonamido/γ-hydroxy-α,ß-unsaturated ketones with cyclic N-sulfimines were re-investigated under mild reaction conditions for the synthesis of racemic polyheterotricyclic imidazolidines and oxazolidines with excellent diastereoselectivities.

Development of Two Korean IACUC Guidance Documents to Foster Implementation of the Three Rs.

In Korea, Institutional Animal Care and Use Committees (IACUCs) have been legally required to apply the Three Rs principles (i.e. replacement, reduction and refinement) and undertake the ethical review of animal study protocols, since 2008. According to Korean law, each IACUC is required to appoint at least one lay member recommended by a non-governmental animal protection organisation, who participates in the ethical review process as part of this role. Despite the importance of the Three Rs and the ethical review process, limited information and practical resources are available for IACUC members in the Korean language, particularly for lay members who are inexperienced in animal experimentation. In January 2020, the Animal and Plant Quarantine Agency announced the funding for a six-month research project to develop guidance to assist IACUC members in carrying out effective and efficient protocol reviews in line with Korean legislative requirements. This funding was awarded for the production of two IACUC guidance documents - 'Guide for Animal Study Protocols' and 'Guide for the IACUC Lay Member' - which were published in December 2020. These guidance documents aim to foster the implementation of the Three Rs and provide practical resources for IACUC members, researchers and other relevant personnel. This paper describes the framework for animal use in Korea and the overall production of these two IACUC Guidance Documents.

Structural and Biochemical Analysis of the Recombination Mediator Protein RecR from Campylobacter jejuni.

The recombination mediator complex RecFOR, consisting of the RecF, RecO, and RecR proteins, is needed to initiate homologous recombination in bacteria by positioning the recombinase protein RecA on damaged DNA. Bacteria from the phylum Campylobacterota, such as the pathogen Campylobacter jejuni, lack the recF gene and trigger homologous recombination using only RecR and RecO. To elucidate the functional properties of C. jejuni RecR (cjRecR) in recombination initiation that differ from or are similar to those in RecF-expressing bacteria, we determined the crystal structure of cjRecR and performed structure-based binding analyses. cjRecR forms a rectangular ring-like tetrameric structure and coordinates a zinc ion using four cysteine residues, as observed for RecR proteins from RecF-expressing bacteria. However, the loop of RecR that has been shown to recognize RecO and RecF in RecF-expressing bacteria is substantially shorter in cjRecR as a canonical feature of Campylobacterota RecR proteins, indicating that cjRecR lost a part of the loop in evolution due to the lack of RecF and has a low RecO-binding affinity. Furthermore, cjRecR features a larger positive patch and exhibits substantially higher ssDNA-binding affinity than RecR from RecF-expressing bacteria. Our study provides a framework for a deeper understanding of the RecOR-mediated recombination pathway.

Pulmonary fibrosis model using micro-CT analyzable human PSC-derived alveolar organoids containing alveolar macrophage-like cells.

Human lung organoids (hLOs) are useful for disease modelling and drug screening. However, a lack of immune cells in hLOs limits the recapitulation of in vivo cellular physiology. Here, we generated hLOs containing alveolar macrophage (AMφ)-like cells derived from pluripotent stem cells (PSC). To bridge hLOs with advanced human lung high-resolution X-ray computed tomography (CT), we acquired quantitative micro-CT images. Three hLO types were observed during differentiation. Among them, alveolar hLOs highly expressed not only lung epithelial cell markers but also AMφ-specific markers. Furthermore, CD68+ AMφ-like cells were spatially organized on the luminal epithelial surface of alveolar hLOs. Bleomycin-treated alveolar hLOs showed upregulated expression of fibrosis-related markers and extracellular matrix deposits in the alveolar sacs. Alveolar hLOs also showed structural alterations such as excessive tissue fraction under bleomycin treatment. Therefore, we suggest that micro-CT analyzable PSC-derived alveolar hLOs are a promising in vitro model to predict lung toxicity manifestations, including fibrosis.

Polyhexamethylene guanidine phosphate increases stress granule formation in human 3D lung organoids under respiratory syncytial virus infection.

Polyhexamethylene guanidine phosphate (PHMG-p), a humidifier disinfectant, is known to cause lung toxicity, including inflammation and pulmonary fibrosis. In this study, we aimed to investigate the effect of PHMG-p on human lung tissue models (2D epithelial cells and 3D organoids) under conditions of oxidative stress and viral infection. The effect of PHMG-p was studied by evaluating the formation of stress granules (SGs), which play a pivotal role in cellular adaptation to various stress conditions. Under oxidative stress and respiratory syncytial virus (RSV) infection, exposure to PHMG-p remarkably increased eIF2α phosphorylation, which is essential for SG-related signalling, and significantly increased SG formation. Furthermore, PHMG-p induced fibrotic gene expression and caused cell death due to severe DNA damage, which was further increased under oxidative stress and RSV infection, indicating that PHMG-p induces severe lung toxicity under stress conditions. Taken together, toxicity evaluation under various stressful conditions is necessary to accurately predict potential lung toxicity of chemicals affecting the respiratory tract.

Neighbor-Aware Non-Orthogonal Multiple Access Scheme for Energy Harvesting Internet of Things.

In a non-orthogonal multiple access (NOMA) environment, an Internet of Things (IoT) device achieves a high data rate by increasing its transmission power. However, excessively high transmission power can cause an energy outage of an IoT device and have a detrimental effect on the signal-to-interference-plus-noise ratio of neighbor IoT devices. In this paper, we propose a neighbor-aware NOMA scheme (NA-NOMA) where each IoT device determines whether to transmit data to the base station and the transmission power at each time epoch in a distributed manner with the consideration of its energy level and other devices' transmission powers. To maximize the aggregated data rate of IoT devices while keeping an acceptable average energy outage probability, a constrained stochastic game model is formulated, and the solution of the model is obtained using a best response dynamics-based algorithm. Evaluation results show that NA-NOMA can increase the average data rate up to 22% compared with a probability-based scheme while providing a sufficiently low energy outage probability (e.g., 0.05).

Squaramide-Catalyzed Asymmetric Intramolecular Oxa-Michael Reaction of α,ß-Unsaturated Carbonyls Containing Benzyl Alcohol: Construction of Chiral 1-Substituted Phthalans.

Organocatalytic enantioselective intramolecular oxa-Michael reactions of benzyl alcohol bearing α,ß-unsaturated carbonyls as Michael acceptors are presented herein. Using cinchona squaramide-based organocatalyst, enones as well as α,ß-unsaturated esters containing benzyl alcohol provided their corresponding 1,3-dihydroisobenzofuranyl-1-methylene ketones and 1,3-dihydroisobenzofuranyl-1-methylene esters in excellent yields with high enantioselectivities. In addition, enantioenriched 1,3-dihydroisobenzofuranyl-1-methylene ketone could be obtained from the Wittig/oxa-Michael reaction cascade of 1,3-dihydro-2-benzofuran-1-ol.

An Energy Efficient UAV-Based Edge Computing System with Reliability Guarantee for Mobile Ground Nodes.

An edge computing system is a distributed computing framework that provides execution resources such as computation and storage for applications involving networking close to the end nodes. An unmanned aerial vehicle (UAV)-aided edge computing system can provide a flexible configuration for mobile ground nodes (MGN). However, edge computing systems still require higher guaranteed reliability for computational task completion and more efficient energy management before their widespread usage. To solve these problems, we propose an energy efficient UAV-based edge computing system with energy harvesting capability. In this system, the MGN makes requests for computing service from multiple UAVs, and geographically proximate UAVs determine whether or not to conduct the data processing in a distributed manner. To minimize the energy consumption of UAVs while maintaining a guaranteed level of reliability for task completion, we propose a stochastic game model with constraints for our proposed system. We apply a best response algorithm to obtain a multi-policy constrained Nash equilibrium. The results show that our system can achieve an improved life cycle compared to the individual computing scheme while maintaining a sufficient successful complete computation probability.

Highly efficient double plasma mirror producing ultrahigh-contrast multi-petawatt laser pulses.

We present a highly efficient double plasma mirror (DPM) that provides ultrahigh-contrast multi-petawatt (PW) laser pulses with a temporal contrast ratio reaching 1017 up to 160 ps and 1012 up to 2 ps before the main pulse. The high reflectivity of 70%, along with the high-contrast enhancement factor of 700,000, was achieved from the DPM installed after the final stage of a 4 PW Ti:sapphire laser. The 4 PW laser was equipped with cross-polarized wave generation and optical parametric chirped-pulse amplification stages for initial high-contrast operation. The DPM operation was undertaken with conditions that did not modify the spatiotemporal profiles of incident multi-PW laser pulses. This highly efficient DPM with the high-contrast enhancement promises the utilization of multiple PMs as a practical rear end for upcoming tens of petawatt lasers to achieve ultrahigh temporal contrast.

First Case of Peroxisomal D-bifunctional Protein Deficiency with Novel HSD17B4 Mutations and Progressive Neuropathy in Korea.

Peroxisomal D-bifunctional protein (DBP), encoded by the HSD17B4 gene, catalyzes ß-oxidation of very long chain fatty acids (VLCFAs). The deficiency of this peroxisomal enzyme leads to the accumulation of VLCFAs, causing multisystemic manifestations including the brain, retina, adrenal gland, hearing, and skeletal system. Herein, we report the first Korean neonatal case of peroxisomal DBP deficiency and the clinical prognosis over 2 years. This patient showed craniofacial dysmorphism, club foot, and seizures with cyanosis one day after birth. Elevated VLCFAs levels were indicative of a peroxisomal disorder. Targeted exome sequencing was performed and two missense mutations p.Asp117Val and p.Phe279Ser in the HSD17B4 gene were identified. The patient had type III DBP deficiency; therefore, docosahexaenoic acid and non-soluble vitamins were administered. However, progressive nystagmus, optic nerve atrophy, and bilateral hearing defects were observed and follow-up brain imaging revealed leukodystrophy and brain atrophy. Multiple anti-epileptic drugs were required to control the seizures. Over two years, the patient achieved normal growth with home ventilation and tube feeding. Hereby, the subject's parents had support during the second pregnancy from the proven molecular information. Moreover, targeted exome sequencing is an effective diagnostic approach, considering genetic heterogeneity of Zellweger spectrum disorders.

Success Probability Characterization of Long-Range in Low-Power Wide Area Networks.

In low-power wide area networks (LPWAN), a considerable number of end devices (EDs) communicate with the gateway in a certain area, whereas for transmitted data, a low data rate and high latency are allowed. Long-range (LoRa), as one of the LPWAN technologies, considers pure ALOHA and chirp spread spectrum (CSS) in the media access control (MAC) and physical (PHY) layers such that it can improve the energy efficiency while mitigating inter-cell interference (ICI). This paper investigates the system throughput of LoRa networks under the assumption that the interferences between EDs for exclusive regions are ignored using CSS. In order to establish an analytical model for the performance of LoRa, we introduce the pure ALOHA capture model, which is the power threshold model. For this model, we assume that the interfering power is proportional to the length of the time overlapped. In addition, we discuss LoRa gain by comparing the total throughput of LoRa with that of non-CSS.

Total Biosynthesis for Milligram-Scale Production of Etoposide Intermediates in a Plant Chassis.

Etoposide is a plant-derived drug used clinically to treat several forms of cancer. Recent shortages of etoposide demonstrate the need for a more dependable production method to replace the semisynthetic method currently in place, which relies on extraction of a precursor natural product from Himalayan mayapple. Here we report milligram-scale production of (-)-deoxypodophyllotoxin, a late-stage biosynthetic precursor to the etoposide aglycone, using an engineered biosynthetic pathway in tobacco. Our strategy relies on engineering the supply of coniferyl alcohol, an endogenous tobacco metabolite and monolignol precursor to the etoposide aglycone. We show that transient expression of 16 genes, encoding both coniferyl alcohol and main etoposide aglycone pathway enzymes from mayapple, in tobacco leaves results in the accumulation of up to 4.3 mg/g dry plant weight (-)-deoxypodophyllotoxin, and enables isolation of high-purity (-)-deoxypodophyllotoxin after chromatography at levels up to 0.71 mg/g dry plant weight. Our work reveals that long (>10 step) pathways can be efficiently transferred from difficult-to-cultivate medicinal plants to a tobacco plant production chassis, and demonstrates mg-scale total biosynthesis for access to valuable precursors of the chemotherapeutic etoposide.

Production of Metal-Free Composites Composed of Graphite Oxide and Oxidized Carbon Nitride Nanodots and Their Enhanced Photocatalytic Performances.

A novel metal-free composite (GN) composed of two types of carbon-based nanomaterials, graphite oxide (GO) and 2D oxidized carbon nitride (OCN) nanodots was produced. Chemical and morphological characterizations reveal that GN contains a main component of GO with well-dispersed 2D OCN nanodots. GN shows enhanced photocatalytic performance for degrading an organic pollutant, Rhodamine B, under visible light.

Modular degradable hydrogels based on thiol-reactive oxanorbornadiene linkers.

Oxanorbornadiene dicarboxylate (OND) reagents are potent Michael acceptors, the adducts of which undergo fragmentation by retro-Diels-Alder reaction at rates that vary with the substitution pattern on the OND moiety. Rapid conjugate addition between thiol-terminated tetravalent PEG and multivalent ONDs yielded self-supporting hydrogels within 1 min at physiological temperature and pH. Erosion of representative hydrogel formulations occurred with predictable and pH-independent rates on the order of minutes to weeks. These materials could be made non-degradable by epoxidation of the OND linkers without slowing gelation. Hydrogels prepared with OND linkers of equal valence had comparable physical properties, as determined by equilibrium swelling behavior, indicating similar internal network structure. Diffusion and release of entrained cargo varied with both the rate of degradation of PEG-OND hydrogels and the hydrodynamic radius of the entrained species. These results highlight the utility of OND linkers in the preparation of degradable network materials with potential applications in sustained release.

Oxidized carbon nitrides: water-dispersible, atomically thin carbon nitride-based nanodots and their performances as bioimaging probes.

Three-dimensional (3D) carbon nitride (C3 N4 )-based materials show excellent performance in a wide range of applications because of their suitable band structures. To realize the great promise of two-dimensional (2D) allotropes of various 3D materials, it is highly important to develop routes for the production of 2D C3 N4 materials, which are one-atom thick, in order to understand their intrinsic properties and identify their possible applications. In this work, water-dispersible, atomically thin, and small carbon nitride nanodots were produced using the chemical oxidation of graphitic C3 N4 . Various analyses, including X-ray diffraction, X-ray photoelectron, Fourier-transform infrared spectroscopy, and combustion-based elemental analysis, and thermogravimetric analysis, confirmed the production of 3D oxidized C3 N4 materials. The 2D C3 N4 nanodots were successfully exfoliated as individual single layers; their lateral dimension was several tens of nanometers. They showed strong photoluminescence in the visible region as well as excellent performances as cell-imaging probes in an in vitro study using confocal fluorescence microscopy.

Inducing cells to disperse nickel nanowires via integrin-mediated responses.

We present non-cytotoxic, magnetic, Arg-Gly-Asp (RGD)-functionalized nickel nanowires (RGD-nanowires) that trigger specific cellular responses via integrin transmembrane receptors, resulting in dispersal of the nanowires. Time-lapse fluorescence and phase contrast microscopy showed that dispersal of 3 µm long nanowire increased by a factor of 1.54 with functionalization by RGD, compared to polyethylene glycol (PEG), through integrin-specific binding, internalization and proliferation in osteosarcoma cells. Further, a 35.5% increase in cell density was observed in the presence of RGD-nanowires, compared to an increase of only 15.6% with PEG-nanowires. These results promise to advance applications of magnetic nanoparticles in drug delivery, hyperthermia, and cell separation where uniformity and high efficiency in cell targeting is desirable.

The p.R168Q mutation is associated with the Bw phenotype and a predicted decrease in the stability of the resulting ABO glycosyltransferase.

BACKGROUND: Mutation of ABO glycosyltransferase (GT) can cause protein stability changes that can result in a weak ABO phenotype. To explain the Bw phenotype of a novel ABO*Bw allele, a protein stability of the mutant GT, which enhances the information of the three-dimensional (3D) structural analysis, was calculated. STUDY DESIGN AND METHODS: ABO serology and genotyping were performed on a neonate and her five family members. A 3D structural analysis of the wild-type GTB and enzymes with a variety of mutations at Residue 168, along with predicted protein stability changes (ΔΔG) and flow cytometric analysis of ABO antigen expression on HeLa cells transfected with plasmids containing R168Q, R168L, and R168P mutants was also performed. RESULTS: A novel ABO*Bw allele (c.503G>A, p.R168Q) was discovered. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTB, and the ΔΔG values, which inversely correlated with the mean relative fluorescence intensity of ABO antigen expression, quantitatively explained the reduced ABO antigen expression. CONCLUSIONS: The predicted protein stability change of a mutant GT enzyme might be a useful and convenient approach to objectively and quantitatively explain the reduced ABO antigen expression.