In Situ Prelithiation by Direct Integration of Lithium Mesh into Battery Cells.

Silicon (Si)-based anodes are promising for next-generation lithium (Li)-ion batteries due to their high theoretical capacity (∼3600 mAh/g). However, they suffer quantities of capacity loss in the first cycle from initial solid electrolyte interphase (SEI) formation. Here, we present an in situ prelithiation method to directly integrate a Li metal mesh into the cell assembly. A series of Li meshes are designed as prelithiation reagents, which are applied to the Si anode in battery fabrication and spontaneously prelithiate Si with electrolyte addition. Various porosities of Li meshes tune prelithiation amounts to control the degree of prelithiation precisely. Besides, the patterned mesh design enhances the uniformity of prelithiation. With an optimized prelithiation amount, the in situ prelithiated Si-based full cell shows a constant >30% capacity improvement in 150 cycles. This work presents a facile prelithiation approach to improve battery performance.

PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing.

Prime editing technology is capable of generating targeted insertions, deletions, and base conversions. However, the process of designing prime editing guide RNAs (pegRNAs), which contain a primer binding site and a reverse-transcription template at the 3' end, is more complex than that for the single guide RNAs used with CRISPR nucleases or base editors. Furthermore, the assessment of high-throughput sequencing data after prime editors (PEs) have been employed should consider the unique feature of PEs; thus, pre-existing assessment tools cannot directly be adopted for PEs. Here, we present two user-friendly web-based tools for PEs, named PE-Designer and PE-Analyzer. PE-Designer, a dedicated tool for pegRNA selection, provides all possible target sequences, pegRNA extension sequences, and nicking guide RNA sequences together with useful information, and displays the results in an interactive image. PE-Analyzer, a dedicated tool for PE outcome analysis, accepts high-throughput sequencing data, summarizes mutation-related information in a table, and provides interactive graphs. PE-Analyzer was mainly written using JavaScript so that it can analyze several data sets without requiring that huge sequencing data (>100MB) be uploaded to the server, reducing analysis time and increasing personal security. PE-Designer and PE-Analyzer are freely available at http://www.rgenome.net/pe-designer/ and http://www.rgenome.net/pe-analyzer/ without a login process.

Electrospun Rubber Nanofiber Web-Based Dry Electrodes for Biopotential Monitoring.

This study aims to find base materials for dry electrode fabrication with high accuracy and without reducing electrode performance for long-term bioelectric potential monitoring after electroless silver plating. Most applications of dry electrodes that have been developed in the past few decades are restricted by low accuracy compared to commercial Ag/AgCl gel electrodes, as in our previous study of PVDF-based dry electrodes. In a recent study, however, nanoweb-based chlorinated polyisoprene (CPI) and poly(styrene-b-butadiene-b-styrene) (SBS) rubber were selected as promising candidates due to their excellent elastic properties, as well as their nanofibril nature, which may improve electrode durability and skin contact. The electroless silver plating technique was employed to coat the nanofiber web with silver, and silver nanoweb(AgNW)-based dry electrodes were fabricated. The key electrode properties (contact impedance, step response, and noise characteristics) for AgNW dry electrodes were investigated thoroughly using agar phantoms. The dry electrodes were subsequently tested on human subjects to establish their realistic performance in terms of ECG, EMG monitoring, and electrical impedance tomography (EIT) measurements. The experimental results demonstrated that the AgNW dry electrodes, particularly the SBS-AgNW dry electrodes, performed similarly to commercial Ag/AgCl gel electrodes and were outperformed in terms of long-term stability.

Current Status and Challenges of DNA Base Editing Tools.

CRISPR-mediated DNA base editors, which include cytosine base editors (CBEs) and adenine base editors (ABEs), are promising tools that can induce point mutations at desired sites in a targeted manner to correct or disrupt gene expression. Their high editing efficiency, coupled with their ability to generate a targeted mutation without generating a DNA double-strand break (DSB) or requiring a donor DNA template, suggests that DNA base editors will be useful for treating genetic diseases, among other applications. However, this hope has recently been challenged by the discovery of DNA base editor shortcomings, including off-target DNA editing, the generation of bystander mutations, and promiscuous deamination effects in both DNA and RNA, which arise from the main DNA base editor constituents, a Cas nuclease variant and a deaminase. In this review, we summarize information about the DNA base editors that have been developed to date, introduce their associated potential challenges, and describe current efforts to minimize or mitigate those issues of DNA base editors.

ATBS1-INTERACTING FACTOR 2 negatively regulates dark- and brassinosteroid-induced leaf senescence through interactions with INDUCER OF CBF EXPRESSION 1.

ATBS1-INTERACTING FACTOR 2 (AIF2) is a non-DNA-binding basic helix-loop-helix (bHLH) transcription factor. We demonstrated that AIF2 retards dark-triggered and brassinosteroid (BR)-induced leaf senescence in Arabidopsis thaliana. Dark-triggered BR synthesis and the subsequent activation of BRASSINAZOLE RESISTANT 1 (BZR1), a BR signaling positive regulator, result in BZR1 binding to the AIF2 promoter in a dark-dependent manner, reducing AIF2 transcript levels and accelerating senescence. BR-induced down-regulation of AIF2 protein stability partly contributes to the progression of dark-induced leaf senescence. Furthermore, AIF2 interacts with INDUCER OF CBF EXPRESSION 1 (ICE1) via their C-termini. Formation of the AIF2-ICE1 complex and subsequent up-regulation of C-REPEAT BINDING FACTORs (CBFs) negatively regulates dark-triggered, BR-induced leaf senescence. This involves antagonistic down-regulation of PHYTOCHROME INTERACTING FACTOR 4 (PIF4), modulated through AIF2-dependent inhibition of ICE1's binding to the promoter. PIF4-dependent activities respond to dark-induced early senescence and may promote BR synthesis and BZR1 activation to suppress AIF2 and accelerate dark-induced senescence. Taken together, these findings suggest a coordination of AIF2 and ICE1 functions in maintaining stay-green traits.

Brassinosteroid-Induced Transcriptional Repression and Dephosphorylation-Dependent Protein Degradation Negatively Regulate BIN2-Interacting AIF2 (a BR Signaling-Negative Regulator) bHLH Transcription Factor.

Brassinosteroids (BRs) are plant polyhydroxy-steroids that play important roles in plant growth and development via extensive signal integration through direct interactions between regulatory components of different signaling pathways. Recent studies have shown that diverse helix-loop-helix/basic helix-loop-helix (HLH/bHLH) family proteins are actively involved in control of BR signaling pathways and interact with other signaling pathways. In this study, we show that ATBS1-INTERACTING FACTOR 2 (AIF2), a nuclear-localized atypical bHLH transcription factor, specifically interacts with BRASSINOSTEROID-INSENSITIVE 2 (BIN2) among other BR signaling molecules. Overexpression of AIF2 down-regulated transcript expression of growth-promoting genes, thus resulting in retardation of growth. AIF2 renders plants hyposensitive to BR-induced root growth inhibition, but shows little effects on BR-promoted hypocotyl elongation. Notably, AIF2 was dephosphorylated by BR, and the dephosphorylated AIF2 was subject to proteasome-mediated degradation. AIF2 degradation was greatly induced by BR and ABA, but relatively slightly by other hormones such as auxin, gibberellin, cytokinin and ethylene. Moreover, AIF2 transcription was significantly suppressed by a BRI1/BZR1-mediated BR signaling pathway through a direct binding of BRASSINAZOLE RESISTANT 1 (BZR1) to the BR response element (BRRE) region of the AIF2 promoter. In conclusion, our study suggests that BIN2-driven AIF2 phosphorylation could augment the BIN2/AIF2-mediated negative circuit of BR signaling pathways, and the BR-induced transcriptional repression and protein degradation negatively regulate AIF2 transcription factor, reinforcing the BZR1/BES1-mediated positive BR signaling pathway.

Hyperbranched ß-cyclodextrin polymer as an effective multidimensional binder for silicon anodes in lithium rechargeable batteries.

Polymeric binders play an important role in electrochemical performance of high-capacity silicon (Si) anodes that usually suffer from severe capacity fading due to unparalleled volume change of Si during cycling. In an effort to find efficient polymeric binders that could mitigate such capacity fading, herein, we introduce polymerized ß-cyclodextrin (ß-CDp) binder for Si nanoparticle anodes. Unlike one-dimensional binders, hyperbranched network structure of ß-CDp presents multidimensional hydrogen-bonding interactions with Si particles and therefore offers robust contacts between both components. Even the Si nanoparticles that lost the original contacts with the binder during cycling recover within the multidimensional binder network, thus creating a self-healing effect. Utilizing these advantageous features, ß-CDp-based Si electrode shows markedly improved cycling performance compared to those of other well-known binder cases, especially when combined with linear polymers at an appropriate ratio to form hybrid binders.

Light-Responsive Actuator of Azobenzene-Containing Main-Chain Liquid Crystal Elastomers with Allyl Sulfide Dynamic Exchangeable Linkages.

Herein, a light-responsive and light-induced bond-exchange-reaction (BER)-capable actuator of the monodomain liquid crystal elastomer (xMLCEazo), developed using main-chain mesogenic oligomers containing azobenzene and allyl sulfide linkages, is investigated. Large quantities of the azobenzene and allyl dithiol linkages are incorporated into the main-chain mesogenic oligomer prepared via thiol-acrylate Michael addition polymerization (TAMAP). The xMLCEazo film is generated via visible-light-induced BER of the drawn polydomain xLCEazo (xPLCEazo) film prepared via TAMAP of tetrathiol cross-linkers and diacrylate-terminated mesogenic oligomers. The xMLCEazo film exhibits large length actuation (38%) through the photothermal effect, along with excellent self-healing and reprogramming properties, under ultraviolet (UV) light irradiation. UV light induced BER of the xMLCEazo film is used to develop complex-shaped actuators with a bilayer film, containing the xMLCEazo and xPLCEazo films, which are bonded by the UV light induced BER without glue. The individual arm of the complex eight-arm flower is remotely actuated under UV light irradiation, and a circular band is rolled under blue laser light irradiation, demonstrating the local remote-controlled actuation and fuel-free motion of the motile soft robot using light irradiation, respectively. Thus, the xMLCEazo film can be expanded to other interesting applications requiring reprogrammable, self-healing, reprocessable, patternable, and remote-controlled light-triggered elastic, rubber-like actuators.

Neuroprotective effect of trans-cinnamaldehyde on the 6-hydroxydopamine-induced dopaminergic injury.

The anti-inflammatory and neuroprotective effects of trans-cinnamaldehyde (TCA) were investigated on the inflammatory cells and the dopaminergic degeneration in mice. TCA inhibited the up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-induced inflammatory BV2 microglial cells. To investigate the TCA efficacy on the 6-hydroxydopamine (6-OHDA)-induced dopaminergic degeneration in mice, an intracerebroventricular injection of 6-OHDA was given to the mice, and TCA (30 mg/kg) was intraperitoneally administered. At 7 d after the 6-OHDA injection, 6-OHDA led to a severe loss of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the striatum and substantia nigra (SN). On the other hand, TCA dramatically maintained the number of TH-positive dopaminergic neurons in the striatum and SN regions of the 6-OHDA-treated mice, which indicates that TCA is able to inhibit the 6-OHDA-induced reduction of TH expression in the dopaminergic neurons in the striatum and SN regions. TCA also inhibited the induction of iNOS and COX-2 in the 6-OHDA model, similarly as shown in the LPS-induced inflammatory BV2 microglial cells. These results indicate that TCA has a neuroprotective effect on dopaminergic neurons and that this effect may be associated with the inhibition of inflammatory responses. These findings suggest that TCA may be a therapeutic candidate for the prevention of inflammation-mediated neurodegenerative diseases.

Enhancement of Gene Editing and Base Editing with Therapeutic Ribonucleoproteins through In Vivo Delivery Based on Absorptive Silica Nanoconstruct.

Key to the widespread and secure application of genome editing tools is the safe and effective delivery of multiple components of ribonucleoproteins (RNPs) into single cells, which remains a biological barrier to their clinical application. To overcome this issue, a robust RNP delivery platform based on a biocompatible sponge-like silica nanoconstruct (SN) for storing and directly delivering therapeutic RNPs, including Cas9 nuclease RNP (Cas9-RNP) and base editor RNP (BE-RNP) is designed. Compared with commercialized material such as lipid-based methods, up to 50-fold gene deletion and 10-fold base substitution efficiency is obtained with a low off-target efficiency by targeting various cells and genes. In particular, gene correction is successfully induced by SN-based delivery through intravenous injection in an in vivo solid-tumor model and through subretinal injection in mouse eye. Moreover, because of its low toxicity and high biodegradability, SN has negligible effect on cellular function of organs. As the engineered SN can overcome practical challenges associated with therapeutic RNP application, it is strongly expected this platform to be a modular RNPs delivery system, facilitating in vivo gene deletion and editing.

Adenine base editor engineering reduces editing of bystander cytosines.

Adenine base editors (ABEs) catalyze specific A-to-G conversions at genomic sites of interest. However, ABEs also induce cytosine deamination at the target site. To reduce the cytosine editing activity, we engineered a commonly used adenosine deaminase, TadA7.10, and found that ABE7.10 with a D108Q mutation in TadA7.10 exhibited tenfold reduced cytosine deamination activity. The D108Q mutation also reduces cytosine deamination activity in two recently developed high-activity versions of ABE, ABE8e and ABE8s, and is compatible with V106W, a mutation that reduces off-target RNA editing. ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity and a substantially reduced adenine editing rate, yielding a TC-specific base editing tool for TC-to-TT or TC-to-TG conversions that broadens the utility of base editors.

Respiration monitoring in PACU using ventilation and gas exchange parameters.

The importance of perioperative respiration monitoring is highlighted by high incidences of postoperative respiratory complications unrelated to the original disease. The objectives of this pilot study were to (1) simultaneously acquire respiration rate (RR), tidal volume (TV), minute ventilation (MV), SpO2 and PETCO2 from patients in post-anesthesia care unit (PACU) and (2) identify a practical continuous respiration monitoring method by analyzing the acquired data in terms of their ability and reliability in assessing a patient's respiratory status. Thirteen non-intubated patients completed this observational study. A portable electrical impedance tomography (EIT) device was used to acquire RREIT, TV and MV, while PETCO2, RRCap and SpO2 were measured by a Capnostream35. Hypoventilation and respiratory events, e.g., apnea and hypopnea, could be detected reliably using RREIT, TV and MV. PETCO2 and SpO2 provided the gas exchange information, but were unable to detect hypoventilation in a timely fashion. Although SpO2 was stable, the sidestream capnography using the oronasal cannula was often unstable and produced fluctuating PETCO2 values. The coefficient of determination (R2) value between RREIT and RRCap was 0.65 with a percentage error of 52.5%. Based on our results, we identified RR, TV, MV and SpO2 as a set of respiratory parameters for robust continuous respiration monitoring of non-intubated patients. Such a respiration monitor with both ventilation and gas exchange parameters would be reliable and could be useful not only for respiration monitoring, but in making PACU discharge decisions and adjusting opioid dosage on general hospital floor. Future studies are needed to evaluate the potential clinical utility of such an integrated respiration monitor.

Tissue Characterization Using an Electrical Bioimpedance Spectroscopy-Based Multi-Electrode Probe to Screen for Cervical Intraepithelial Neoplasia.

The successful management of cervical intraepithelial neoplasia (CIN) with proper screening and treatment methods could prevent cervical cancer progression. We propose a bioimpedance spectroscopic measurement device and a multi-electrode probe as an independent screening tool for CIN. To evaluate the performance of this screening method, we enrolled 123 patients, including 69 patients with suspected CIN and 54 control patients without cervical dysplasia who underwent a hysterectomy for benign disease (non-CIN). Following conization, the electrical properties of the excised cervical tissue were characterized using an electrical bioimpedance spectroscopy-based multi-electrode probe. Twenty-eight multifrequency voltages were collected through the two concentric array electrodes via a sensitivity-optimized measurement protocol based on an electrical energy concentration method. The electrical properties of the CIN and non-CIN groups were compared with the results of the pathology reports. Reconstructed resistivity tended to decrease in the CIN and non-CIN groups as frequency increased. Reconstructed resistivity from 625 Hz to 50 kHz differed significantly between the CIN and non-CIN groups (p < 0.001). Using 100 kHz as the reference, the difference between the CIN and non-CIN groups was significant. Based on the difference in reconstructed resistivity between 100 kHz and the other frequencies, this method had a sensitivity of 94.3%, a specificity of 84%, and an accuracy of 90% in CIN screening. The feasibility of noninvasive CIN screening was confirmed through the difference in the frequency spectra evaluated in the excised tissue using the electrical bioimpedance spectroscopy-based multi-electrode screening probe.

Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens.

Coproduction of multiple proteins at high levels in a single human cell line would be extremely useful for basic research and medical applications. Here, a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome is presented. As a proof-of-concept, it is shown that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number. It is confirmed that three different fluorescent proteins, encoded by genes integrated at different loci, can be coexpressed at high levels. Further, a stable cell line is generated, producing antigens from different human coronaviruses: MERS-CoV and HCoV-OC43. Antibodies raised against these antigens, which contain human N-glycosylation, show neutralizing activities against both viruses, suggesting that the coexpression system provides a quick and predictable way to produce multiple coronavirus antigens, such as the recent 2019 novel human coronavirus.

High-purity production and precise editing of DNA base editing ribonucleoproteins.

Ribonucleoprotein (RNP) complex-mediated base editing is expected to be greatly beneficial because of its reduced off-target effects compared to plasmid- or viral vector-mediated gene editing, especially in therapeutic applications. However, production of recombinant cytosine base editors (CBEs) or adenine base editors (ABEs) with ample yield and high purity in bacterial systems is challenging. Here, we obtained highly purified CBE/ABE proteins from a human cell expression system and showed that CBE/ABE RNPs exhibited different editing patterns (i.e., less conversion ratio of multiple bases to single base) compared to plasmid-encoded CBE/ABE, mainly because of the limited life span of RNPs in cells. Furthermore, we found that off-target effects in both DNA and RNA were greatly reduced for ABE RNPs compared to plasmid-encoded ABE. We ultimately applied NG PAM-targetable ABE RNPs to in vivo gene correction in retinal degeneration 12 (rd12) model mice.

[Clinical significance of incidentally detected eosinophilic esophagitis with pathologic review].

BACKGROUND/AIMS: Eosinophilic esophagitis (EE) is a chronic inflammatory disorder characterized by abnormal dense eosinophilic infiltration of esophageal mucosa and results in dysphasia and food impaction. EE is being increasingly recognized in adults. The prevalence is largely unknown. This study was performed to evaluate the detection rate of EE diagnosed based on pathologic criteria and to define the clinical characteristics of EE in Korea. METHODS: We reviewed biopsy specimen of the 1,609 patients who underwent esophageal biopsy from January 2006 till August 2008. The presence of more than 20 eosinophils per high power field in biopsy specimens was considered cases of EE. Clinical information and endoscopic findings were obtained. RESULTS: 7 (0.4%) patients were diagnosed as EE based on pathologic criteria retrospectively. Clinical symptoms were epigastric pain (43%), regurgitation (29%), dyspepsia (14%), and no symptom (14%). Endoscopic findings were whitish exudates or granules (57%), esophageal polyp (29%), and hyperemic change (14%). Two patients received treatment. One patient with bronchial asthma improved after treatment with inhaled corticosteroid, and one patient improved after 8 week proton pump inhibitor therapy. CONCLUSIONS: Eosinophilic esophagitis was found in 0.4% of the total esophageal biopsied cases. Our results suggest that Korean patients with eosinophilic esophagitis showed symptoms mimicking gastroesophageal reflux disease and atypical endoscopic findings. Therefore, regardless of the gross appearance of the mucosa, meticulous diagnostic approaches are needed for patients with swallowing difficulty and lack of response to proton pump inhibitor.

Noninvasive, simultaneous, and continuous measurements of stroke volume and tidal volume using EIT: feasibility study of animal experiments.

Currently, there is no noninvasive method available for simultaneous measurements of tidal volume and stroke volume. Electrical impedance tomography (EIT) has been used for regional lung ventilation imaging. Cardiac EIT imaging, however, has not been successful due to the technical difficulty in extracting weak cardiogenic components. Instead of regional imaging, in this paper, we use the EIT technique to simultaneously measure two global variables of tidal volume and stroke volume. Time-varying patterns of boundary voltage data originating from lung ventilation and cardiac blood flow were extracted from measured boundary voltage data using the principal component analysis (PCA) and independent component analysis (ICA). The source consistency theory was adopted to separately synthesize time-series of boundary voltage data associated with lung ventilation and cardiac blood flow. The respiratory volume signal (RVS) and cardiac volume signal (CVS) were extracted from reconstructed time-difference EIT images of lung ventilation and cardiac blood flow, respectively. After calibrating the volume signals using the mechanical ventilator and the invasive transpulmonary thermodilution (TPTD) method, tidal volume and stroke volume were computed as valley-to-peak values of the RVS and CVS, respectively. The difference in the tidal volume data between EIT and mechanical ventilator was within ± 20 ml from six pigs. The difference in the stroke volume data between EIT and TPTD was within ± 4.7 ml from the same animals. The results show the feasibility of the proposed method as a new noninvasive cardiopulmonary monitoring tool for simultaneous continuous measurements of stroke volume and tidal volume that are two most important vital signs.

Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus via Direct Genome Engineering.

The overproduction and purification of human proteins is a requisite of both basic and medical research. Although many recombinant human proteins have been purified, current protein production methods have several limitations; recombinant proteins are frequently truncated, fail to fold properly, and/or lack appropriate post-translational modifications. In addition, such methods require subcloning of the target gene into relevant plasmids, which can be difficult for long proteins with repeated domains. Here we devised a novel method for target protein production by introduction of a strong promoter for overexpression and an epitope tag for purification in front of the endogenous human gene, in a sense performing molecular cloning directly in the human genome, which does not require cloning of the target gene. As a proof of concept, we successfully purified intact human Reelin protein, which is lengthy (3460 amino acids) and contains repeating domains, and confirmed that it was biologically functional.

Transcription factor EGR-1 transactivates the MMP1 gene promoter in response to TNFα in HaCaT keratinocytes.

Matrix metalloproteinase 1 (MMP-1), a calcium-dependent zinccontaining collagenase, is involved in the initial degradation of native fibrillar collagen. Tissue necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that is rapidly produced by dermal fibroblasts, monocytes/macrophages, and keratinocytes and regulates inflammation and damaged-tissue remodeling. MMP-1 is induced by TNFα and plays a critical role in tissue remodeling and skin aging processes. However, the regulation of the MMP1 gene by TNFα is not fully understood. We aimed to find additional cis-acting elements involved in the regulation of TNFα-induced MMP1 gene transcription in addition to the nuclear factor-kappa B (NF-κB) and activator protein 1 (AP1) sites. Assessments of the 5'-regulatory region of the MMP1 gene, using a series of deletion constructs, revealed the requirement of the early growth response protein 1 (EGR-1)-binding sequence (EBS) in the proximal region for proper transcription by TNFα. Ectopic expression of EGR-1, a zinc-finger transcription factor that binds to G-C rich sequences, stimulated MMP1 promoter activity. The silencing of EGR-1 by RNA interference reduced TNFα-induced MMP-1 expression. EGR-1 directly binds to the proximal region and transactivates the MMP1 gene promoter. Mutation of the EBS within the MMP1 promoter abolished EGR-1-mediated MMP-1 promoter activation. These data suggest that EGR-1 is required for TNFα-induced MMP1 transcriptional activation. In addition, we found that all three MAPKs, ERK1/2, JNK, and p38 kinase, mediate TNFα-induced MMP-1 expression via EGR-1 upregulation. These results suggest that EGR-1 may represent a good target for the development of pharmaceutical agents to reduce inflammation-induced MMP-1 expression. [BMB Reports 2020; 53(6): 323-328].