Enhanced binding and inhibition of SARS-CoV-2 by a plant-derived ACE2 protein containing a fused mu tailpiece.

Infectious diseases such as Coronavirus disease 2019 (COVID-19) and Middle East respiratory syndrome (MERS) present an increasingly persistent crisis in many parts of the world. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The angiotensin-converting enzyme 2 (ACE2) is a crucial cellular receptor for SARS-CoV-2 infection. Inhibition of the interaction between SARS-CoV-2 and ACE2 has been proposed as a target for the prevention and treatment of COVID-19. We produced four recombinant plant-derived ACE2 isoforms with or without the mu tailpiece (µ-tp) of immunoglobulin M (IgM) and the KDEL endoplasmic reticulum retention motif in a plant expression system. The plant-derived ACE2 isoforms bound whole SARS-CoV-2 virus and the isolated receptor binding domains of SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants. Fusion of µ-tp and KDEL to the ACE2 protein (ACE2 µK) had enhanced binding activity with SARS-CoV-2 in comparison with unmodified ACE2 protein derived from CHO cells. Furthermore, the plant-derived ACE2 µK protein exhibited no cytotoxic effects on Vero E6 cells and effectively inhibited SARS-CoV-2 infection. The efficient and rapid scalability of plant-derived ACE2 µK protein offers potential for the development of preventive and therapeutic agents in the early response to future viral outbreaks.

Strumpellin/WASHC5 regulates the structural plasticity of cortical neurons involved in gait coordination.

Strumpellin/Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex subunit 5 (WASHC5) is a core component of the WASH complex, and its mutations confer pathogenicity for hereditary spastic paraplegia (HSP) type SPG8, a rare neurodegenerative gait disorder. WASH complex activates actin-related protein-2/3-mediated actin polymerization and plays a pivotal role in intracellular membrane trafficking in endosomes. In this study, we examined the role of strumpellin in the regulation of structural plasticity of cortical neurons involved in gait coordination. Administration of a lentivirus containing a strumpellin-targeting short hairpin RNA (shRNA) to cortical motor neurons lead to abnormal motor coordination in mice. Strumpellin knockdown using shRNA attenuated dendritic arborization and synapse formation in cultured cortical neurons, and this effect was rescued by wild-type strumpellin expression. Compared with the wild-type, strumpellin mutants N471D or V626F identified in patients with SPG8 exhibited no differences in rescuing the defects. Moreover, the number of F-actin clusters in neuronal dendrites was decreased by strumpellin knockdown and rescued by strumpellin expression. In conclusion, our results indicate that strumpellin regulates the structural plasticity of cortical neurons via actin polymerization.

High-quality genome assembly and genetic mapping reveal a gene regulating flesh color in watermelon (Citrullus lanatus).

The unique color and type characteristics of watermelon fruits are regulated by many molecular mechanisms. However, it still needs to be combined with more abundant genetic data to fine-tune the positioning. We assembled genomes of two Korean inbred watermelon lines (cv. 242-1 and 159-1) with unique color and fruit-type characteristics and identified 23,921 and 24,451 protein-coding genes in the two genomes, respectively. To obtain more precise results for further study, we resequenced one individual of each parental line and an F2 population composed of 87 individuals. This identified 1,539 single-nucleotide polymorphisms (SNPs) and 80 InDel markers that provided a high-density genetic linkage map with a total length of 3,036.9 cM. Quantitative trait locus mapping identified 15 QTLs for watermelon fruit quality-related traits, including ß-carotene and lycopene content in fruit flesh, fruit shape index, skin thickness, flesh color, and rind color. By investigating the mapping intervals, we identified 33 candidate genes containing variants in the coding sequence. Among them, Cla97C01G008760 was annotated as a phytoene synthase with a single-nucleotide variant (A → G) in the first exon at 9,539,129 bp of chromosome 1 that resulted in the conversion of a lysine to glutamic acid, indicating that this gene might regulate flesh color changes at the protein level. These findings not only prove the importance of a phytoene synthase gene in pigmentation but also explain an important reason for the color change of watermelon flesh.

Effects of External Employment Opportunities, Nursing Professionalism, and Nursing Work Environments on Korean Hospital Nurses' Intent to Stay or Leave.

This study aims to explore Korean Hospital nurses' intent to stay or leave their working environment, and to identify the difference between the intent to stay and the intent to leave by identifying the relationship between external employment opportunities, professionalism, and work environment. Data were collected via an online survey and analyzed using stepwise multiple regression analysis. As a result of the analysis, the intent to stay among Korean hospital nurses was influenced by the work environment, external employment opportunities, education level, and marital status, whereas the intent to leave was influenced by the nursing work environment, marital status, and total clinical experience. As a result, the reflected variables differed. Thus, it can be concluded that hospital nurses' intent to either stay or leave are not concepts that simply contradict each other in the same context but are, in fact, influenced differently by various factors. Nevertheless, it can also be concluded that nursing managers should make efforts to improve the nursing work environment to lower nurses' intent to leave and increase their intent to stay by improving only the nursing work environment.

Phosphoproteome profiling of hippocampal synaptic plasticity.

The balance between the actions of protein kinases and phosphatases is crucial for neuronal functions, including synaptic plasticity. Although the phosphorylation and dephosphorylation of neuronal proteins are regulated by synaptic plasticity, no systematic analyses of this have yet been conducted. We performed a phosphoproteomic analysis of hippocampal synaptic plasticity using a nano-Acquity/Synapt LC-MS/MS system. Neuronal proteins were extracted from hippocampal tissues and cultured neurons exposed to long-term potentiation (LTP) or long-term depression (LTD). Filter-aided sample preparation (FASP) was performed to remove residual anionic detergents for complete tryptic digestion. Phosphopeptides were then enriched using TiO2 chromatography, followed by immunoaffinity chromatography with an anti-phosphotyrosine antibody. Among the 1500 phosphopeptides identified by LC-MS/MS, 374 phosphopeptides were detected simultaneously in both hippocampal tissues and cultured neurons. Semi-quantification counting the number of spectra of each phosphopeptide showed that 42 of 374 phosphopeptides changed significantly depending on synaptic plasticity. In conclusion, a new proteomic method using sequential enrichment of phosphopeptides and semi-quantification enabled the phosphoproteomic analysis of hippocampal synaptic plasticity.

Label-Free Live-Cell Imaging of Internalized Microplastics and Cytoplasmic Organelles with Multicolor CARS Microscopy.

As the bioaccumulation of microplastics (MPs) is considered as a potential health risk, many efforts have been made to understand the cellular dynamics and cytotoxicity of MPs. Here, we demonstrate that label-free multicolor coherent anti-Stokes Raman scattering (CARS) microscopy enables separate vibrational imaging of internalized MPs and lipid droplets (LDs) with indistinguishable shapes and sizes in live cells. By simultaneously obtaining polystyrene (PS)- and lipid-specific CARS images at two very different frequencies, 1000 and 2850 cm-1, respectively, we successfully identify the local distribution of ingested PS beads and native LDs in Caenorhabditis elegans. We further show that the movements of PS beads and LDs in live cells can be separately tracked in real time, which allows us to characterize their individual intracellular dynamics. We thus anticipate that our multicolor CARS imaging method could be of great use to investigate the cellular transport and cytotoxicity of MPs without additional efforts for pre-labeling to MPs.

Nanoplasmonic immunosensor for the detection of SCG2, a candidate serum biomarker for the early diagnosis of neurodevelopmental disorder.

The neural circuits of the infant brain are rapidly established near 6 months of age, but neurodevelopmental disorders can be diagnosed only at the age of 2-3 years using existing diagnostic methods. Early diagnosis is very important to alleviate life-long disability in patients through appropriate early intervention, and it is imperative to develop new diagnostic methods for early detection of neurodevelopmental disorders. We examined the serum level of secretogranin II (SCG2) in pediatric patients to evaluate its potential role as a biomarker for neurodevelopmental disorders. A plasmonic immunosensor performing an enzyme-linked immunosorbent assay (ELISA) on a gold nanodot array was developed to detect SCG2 in small volumes of serum. This nanoplasmonic immunosensor combined with tyramide signal amplification was highly sensitive to detect SCG2 in only 5 µL serum samples. The analysis using the nanoplasmonic immunosensor revealed higher serum SCG2 levels in pediatric patients with developmental delay than in the control group. Overexpression or knockdown of SCG2 in hippocampal neurons significantly attenuated dendritic arborization and synaptic formation. These results suggest that dysregulated SCG2 expression impairs neural development. In conclusion, we developed a highly sensitive nanoplasmonic immunosensor to detect serum SCG2, a candidate biomarker for the early diagnosis of neurodevelopmental disorders.

Expression of Colorectal Cancer Antigenic Protein Fused to IgM Fc in Chinese Cabbage (Brassica rapa).

The epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen and a potential target for tumor vaccine. The EpCAM is a cell-surface glycoprotein highly expressed in colorectal carcinomas. The objective of the present study is to develop an edible vaccine system through Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa). For the transformation, two plant expression vectors containing genes encoding for the EpCAM recombinant protein along with the fragment crystallizable (Fc) region of immunoglobulin M (IgM) and Joining (J)-chain tagged with the KDEL endoplasmic reticulum retention motif (J-chain K) were constructed. The vectors were successfully transformed and expressed in the Chinese cabbage individually using Agrobacterium. The transgenic Chinese cabbages were screened using genomic polymerase chain reaction (PCR) in T0 transgenic plant lines generated from both transformants. Similarly, the immunoblot analysis revealed the expression of recombinant proteins in the transformants. Further, the T1 transgenic plants were generated by selfing the transgenic plants (T0) carrying EpCAM-IgM Fc and J-chain K proteins, respectively. Subsequently, the T1 plants generated from EpCAM-IgM Fc and J-chain K transformants were crossed to generate F1 plants carrying both transgenes. The presence of both transgenes was validated using PCR in the F1 plants. In addition, the expression of Chinese cabbage-derived EpCAM-IgM Fc × J-chain K was evaluated using immunoblot and ELISA analyses in the F1 plants. The outcomes of the present study can be utilized for the development of a potential anti-cancer vaccine candidate using Chinese cabbage.

Depression-like behaviors induced by defective PTPRT activity through dysregulated synaptic functions and neurogenesis.

PTPRT has been known to regulate synaptic formation and dendritic arborization of hippocampal neurons. PTPRT-/- null and PTPRT-D401A mutant mice displayed enhanced depression-like behaviors compared with wild-type mice. Transient knockdown of PTPRT in the dentate gyrus enhanced the depression-like behaviors of wild-type mice, whereas rescued expression of PTPRT ameliorated the behaviors of PTPRT-null mice. Chronic stress exposure reduced expression of PTPRT in the hippocampus of mice. In PTPRT-deficient mice the expression of GluR2 (also known as GRIA2) was attenuated as a consequence of dysregulated tyrosine phosphorylation, and the long-term potentiation at perforant-dentate gyrus synapses was augmented. The inhibitory synaptic transmission of the dentate gyrus and hippocampal GABA concentration were reduced in PTPRT-deficient mice. In addition, the hippocampal expression of GABA transporter GAT3 (also known as SLC6A11) was decreased, and its tyrosine phosphorylation was increased in PTPRT-deficient mice. PTPRT-deficient mice displayed reduced numbers and neurite length of newborn granule cells in the dentate gyrus and had attenuated neurogenic ability of embryonic hippocampal neural stem cells. In conclusion, our findings show that the physiological roles of PTPRT in hippocampal neurogenesis, as well as synaptic functions, are involved in the pathogenesis of depressive disorder.

Expression of a Large Single-Chain 13F6 Antibody with Binding Activity against Ebola Virus-Like Particles in a Plant System.

Pathogenic animal and human viruses present a growing and persistent threat to humans worldwide. Ebola virus (EBOV) causes zoonosis in humans. Here, two structurally different anti-Ebola 13F6 antibodies, recognizing the heavily glycosylated mucin-like domain (MLD) of the glycoprotein (GP), were expressed in transgenic Nicotiana tabacum plants and designed as inexpensive and effective diagnostic antibodies against Ebola virus disease (EVD). The first was anti-EBOV 13F6 full size antibody with heavy chain (HC) and light chain (LC) (monoclonal antibody, mAb 13F6-FULL), while the second was a large single-chain (LSC) antibody (mAb 13F6-LSC). mAb 13F6-LSC was constructed by linking the 13F6 LC variable region (VL) with the HC of mAb 13F6-FULL using a peptide linker and extended to the C-terminus using the endoplasmic reticulum (ER) retention motif KDEL. Agrobacterium-mediated plant transformation was employed to express the antibodies in N. tabacum. PCR, RT-PCR, and immunoblot analyses confirmed the gene insertion, transcription, and protein expression of these antibodies, respectively. The antibodies tagged with the KDEL motif displayed high-mannose type N-glycan structures and efficient binding to EBOV-like particles (VLPs). Thus, various forms of anti-EBOV plant-derived mAbs 13F6-FULL and LSC with efficient binding affinity to EBOV VLP can be produced in the plant system.

Generation of OCT4-EGFP, NANOG-tdTomato dual reporter human induced pluripotent stem cell line, KKUi001-A, using the CRISPR/Cas9 system.

OCT4 and NANOG are core transcription factor genes in self-renewal, differentiation, and reprogramming. Here, we generated an OCT4-EGFP, NANOG-tdTomato dual reporter hiPSC line, KKUi001-A, on the basis of human induced pluripotent stem cells using CRISPR/Cas9 technology. EGFP and tdTomato reporter were inserted into before the stop codon of OCT4 and NANOG, respectively. Simultaneous expression of EGFP and tdTomato was observed when expression of OCT4 and NANOG was changed during differentiation and reprogramming. KKUi001-A hiPSC line will be a useful tool to find initial time point of OCT4 and NANOG expression during reprogramming process and to screen small molecules that promote reprogramming.

An Efficient Switching-Off of Coherent Anti-Stokes Raman Scattering via Double Stimulated Raman Scattering Processes of Heteromolecular Vibrational Modes.

Recently, we introduced a new switching-off technique applicable to subdiffraction-limited coherent Raman imaging, where a coherent anti-Stokes Raman scattering (CARS) signal can be selectively suppressed via competitive stimulated Raman scattering (SRS) processes between vibrational modes of a single molecular species. Here, we show that such a three-beam CARS suppression can be made via double SRS processes between vibrational modes of heteromolecular species, a mixture of paraffin oil and benzene. We achieve more than 80% suppression of the pump-Stokes-beam CARS signal for the ring-breathing mode (target mode) of benzene when the C-H stretching mode (acceptor mode) of paraffin oil is used to deplete the pump photons via the pump-depletion-beam SRS process. The freedom in the choice of acceptor mode for depletion, which could be a critical advantage of the present switching-off scheme, can be of use for overcoming current challenges of depletion-based super-resolution coherent Raman imaging of biomolecules.

Shot-Noise-Limited Two-Color Stimulated Raman Scattering Microscopy with a Balanced Detection Scheme.

Stimulated Raman scattering (SRS) microscopy has been considered a useful technique for investigating chemical components by selectively targeting the vibration mode of chemical structures. Its practical application to the observation of molecular structures and dynamics in complicated biological environments requires broad spectral coverage with both high resolution and a high signal-to-noise ratio. Here, we demonstrate a two-color SRS microscopy employing a balanced detection scheme and a spectral focusing method. Two different SRS signals are generated with pump and Stokes laser pulse pairs in perpendicular polarization, where each of them acts as an intensity reference for the other, significantly reducing the background noise level close to the shot-noise limit even with a fiber-based femtosecond laser system. The high spectral resolution comparable to that of spontaneous Raman scattering spectroscopy is achieved with the spectral focusing method. The two-color SRS images are obtained for a mixture of polymer beads and for the distributions of lipids and proteins in U2OS cells.

A study on the intent to leave and stay among hospital nurses in Korea: A cross-sectional survey.

AIMS: To investigate the intent to leave or stay among Korean hospital nurses, and to identify what factors influence their intent to leave or stay. BACKGROUND: Previous studies have simply measured nurses' intent to leave or stay; however, this study examines the associations of intent to leave and intent to stay with influential factors among Korean hospital nurses. METHODS: A cross-sectional study was conducted with 267 nurses working at four general hospitals in South Korea. RESULT: The influencing factors on intent to leave were organisational commitment, practice environment and burnout, while intent to stay was influenced by organisational commitment. CONCLUSION: The study demonstrates that, for Korean hospital nurses, intent to leave and intent to stay are not simply contrary concepts, but are different concepts influenced by varying factors. IMPLICATIONS FOR NURSING MANAGEMENT: This study clarifies the difference between the concepts of intent to stay and intent to leave, and demonstrates that the variables affecting Korean nurses' intent to stay and intent to leave differ from each other. Therefore, focusing on improving nurses' intent to either leave or stay would be effective when developing personnel management policies for nurses, thereby contributing to enhancing nursing practice.

Intelligent Image-Based Railway Inspection System Using Deep Learning-Based Object Detection and Weber Contrast-Based Image Comparison.

For sustainable operation and maintenance of urban railway infrastructure, intelligent visual inspection of the railway infrastructure attracts increasing attention to avoid unreliable, manual observation by humans at night, while trains do not operate. Although various automatic approaches were proposed using image processing and computer vision techniques, most of them are focused only on railway tracks. In this paper, we present a novel railway inspection system using facility detection based on deep convolutional neural network and computer vision-based image comparison approach. The proposed system aims to automatically detect wears and cracks by comparing a pair of corresponding image sets acquired at different times. We installed line scan camera on the roof of the train. Unlike an area-based camera, the line scan camera quickly acquires images with a wide field of view. The proposed system consists of three main modules: (i) image reconstruction for registration of facility positions, (ii) facility detection using an improved single shot detector, and (iii) deformed region detection using image processing and computer vision techniques. In experiments, we demonstrate that the proposed system accurately finds facilities and detects their potential defects. For that reason, the proposed system can provide various advantages such as cost reduction for maintenance and accident prevention.

Bronchoalveolar lavage affects computed tomographic and radiographic characteristics of the lungs in healthy dogs.

Bronchoalveolar lavage is a common diagnostic test for dogs with suspected pulmonary disease, however there is no published information on whether this procedure could affect the imaging characteristics of the lungs. Aims of this prospective experimental study were to describe computed tomography (CT) and radiographic features of the lungs after bronchoalveolar lavage in a sample of healthy dogs. Thoracic CT and radiographic images of eight healthy Beagles were acquired at the following time points: before bronchoalveolar lavage, immediately following bronchoalveolar lavage, and at 2, 4, 8, 12, and 24 h following bronchoalveolar lavage. Lung consolidation or interstitial patterns were seen in CT and radiographic images immediately after the procedure. Radiographic lung patterns resolved within 2 h and CT patterns resolved within 24 h. Resolution of the CT pulmonary patterns in the ventral areas of the lungs was delayed compared to the dorsal areas. Mean CT imaging scores differed over time (P < 0.001), while mean radiographic imaging scores did not differ over time. This study suggests that thoracic radiography and CT imaging assessments should precede bronchoalveolar lavage procedures if possible, or be performed at least 24 h afterward.

Unveiling the pathway to Z-DNA in the protein-induced B-Z transition.

Left-handed Z-DNA is an extraordinary conformation of DNA, which can form by special sequences under specific biological, chemical or physical conditions. Human ADAR1, prototypic Z-DNA binding protein (ZBP), binds to Z-DNA with high affinity. Utilizing single-molecule FRET assays for Z-DNA forming sequences embedded in a long inactive DNA, we measure thermodynamic populations of ADAR1-bound DNA conformations in both GC and TG repeat sequences. Based on a statistical physics model, we determined quantitatively the affinities of ADAR1 to both Z-form and B-form of these sequences. We also reported what pathways it takes to induce the B-Z transition in those sequences. Due to the high junction energy, an intermediate B* state has to accumulate prior to the B-Z transition. Our study showing the stable B* state supports the active picture for the protein-induced B-Z transition that occurs under a physiological setting.

Effects of nurses' emotional intelligence on their organizational citizenship behavior, with mediating effects of leader trust and value congruence.

AIM: To investigate the causal relationship between nurses' emotional intelligence and their organizational citizenship behavior and the possible mediating effects of leader trust and value congruence. METHODS: The participants were 348 nurses who were working in a general hospital in a metropolitan area. The data were collected from December 16, 2012 to February 20, 2013. The hypothetical model of emotional intelligence, organizational citizenship behavior, leader trust, and value congruence was fitted to the actual data via structural equation modeling. RESULT: The leaders' emotional intelligence had a direct positive effect on leader trust and value congruence; however, the nurses' own emotional intelligence had a negative effect on these two variables. Furthermore, leader trust had a direct positive effect on organizational citizenship behavior; value congruence had no such relationship. The nurses' emotional intelligence had a partial, indirect effect on organizational citizenship behavior via leader trust. CONCLUSION: In a nursing organization, it is necessary to build a system, such as mentoring, to be able to exchange emotions actively among the members in order to enhance emotional intelligence and have the same values between leaders and members throughout open communication. Therefore, nurse managers can contribute greatly to the enhancement of organizational performance by promoting members' organizational citizenship behavior through improving their relationships with them and gaining their trust, while concurrently making efforts to further develop their emotional intelligence.