Comparable humoral and cellular immunity against Omicron variant BA.4/5 of once-boosted BA.1/2 convalescents and twice-boosted COVID-19-naïve individuals.

The fourth vaccination dose confers additional protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in individuals with no prior coronavirus disease-19 (COVID-19). However, its immunological benefit against currently circulating BA.4/5 is unclear in individuals who have received a booster shot and been infected with Omicron variant BA.1/2. We analyzed immune responses in whom had been boosted once and did not have COVID-19 (n = 16), boosted once and had COVID-19 when BA.1/2 was dominant in Korea (Hybrid-6M group, n = 27), and boosted twice and did not have COVID-19 (Vx4 group, n = 15). Antibody binding activities against RBDo BA.1 and RBDo BA.4/5 , antigen-specific memory CD4+ and CD8+ T-cell responses against BA.4/5, and B-cell responses against SARS-CoV-2 wild-type did not differ statistically between the Hybrid-6M and Vx4 groups. The humoral and cellular immune responses of the Hybrid-6M group against BA.4/5 were comparable to those of the Vx4 group. Individuals who had been boosted and had an Omicron infection in early 2022 may not have high priority for an additional vaccination.

Tracking antigen-specific TCR clonotypes in SARS-CoV-2 infection reveals distinct severity trajectories.

Despite the importance of antigen-specific T cells in infectious disease, characterizing and tracking clonally amplified T cells during the progression of a patient's symptoms remain unclear. Here, we performed a longitudinal, in-depth single-cell multiomics analysis of samples from asymptomatic, mild, usual severe, and delayed severe patients of SARS-CoV-2 infection. Our in-depth analysis revealed that hyperactive or improper T-cell responses were more aggressive in delayed severe patients. Interestingly, tracking of antigen-specific T-cell receptor (TCR) clonotypes along the developmental trajectory indicated an attenuation in functional T cells upon severity. In addition, increased glycolysis and interleukin-6 signaling in the cytotoxic T cells were markedly distinct in delayed severe patients compared to usual severe patients, particularly in the middle and late stages of infection. Tracking B-cell receptor clonotypes also revealed distinct transitions and somatic hypermutations within B cells across different levels of disease severity. Our results suggest that single-cell TCR clonotype tracking can distinguish the severity of patients through immunological hallmarks, leading to a better understanding of the severity differences in and improving the management of infectious diseases by analyzing the dynamics of immune responses over time.

RORα is crucial for attenuated inflammatory response to maintain intestinal homeostasis.

Retinoic acid-related orphan receptor α (RORα) functions as a transcription factor for various biological processes, including circadian rhythm, cancer, and metabolism. Here, we generate intestinal epithelial cell (IEC)-specific RORα-deficient (RORαΔIEC) mice and find that RORα is crucial for maintaining intestinal homeostasis by attenuating nuclear factor κB (NF-κB) transcriptional activity. RORαΔIEC mice exhibit excessive intestinal inflammation and highly activated inflammatory responses in the dextran sulfate sodium (DSS) mouse colitis model. Transcriptome analysis reveals that deletion of RORα leads to up-regulation of NF-κB target genes in IECs. Chromatin immunoprecipitation analysis reveals corecruitment of RORα and histone deacetylase 3 (HDAC3) on NF-κB target promoters and subsequent dismissal of CREB binding protein (CBP) and bromodomain-containing protein 4 (BRD4) for transcriptional repression. Together, we demonstrate that RORα/HDAC3-mediated attenuation of NF-κB signaling controls the balance of inflammatory responses, and therapeutic strategies targeting this epigenetic regulation could be beneficial to the treatment of chronic inflammatory diseases, including inflammatory bowel disease (IBD).

NaCl Ionization-Based Moisture Sensor Prepared by Aerosol Deposition for Monitoring Respiratory Patterns.

A highly polarizable moisture sensor with multimodal sensing capabilities has great advantages for healthcare applications such as human respiration monitoring. We introduce an ionically polarizable moisture sensor based on NaCl/BaTiO3 composite films fabricated using a facile aerosol deposition (AD) process. The proposed sensing model operates based on an enormous NaCl ionization effect in addition to natural moisture polarization, whereas all previous sensors are based only on the latter. We obtained an optimal sensing performance in a 0.5 µm-thick layer containing NaCl-37.5 wt% by manipulating the sensing layer thickness and weight fraction of NaCl. The NaCl/BaTiO3 sensing layer exhibits outstanding sensitivity over a wide humidity range and a fast response/recovery time of 2/2 s; these results were obtained by performing the one-step AD process at room temperature without using any auxiliary methods. Further, we present a human respiration monitoring system using a sensing device that provides favorable and stable electrical signals under diverse respiratory scenarios.

Longitudinal Analysis of Human Memory T-Cell Response According to the Severity of Illness up to 8 Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: Understanding the memory T-cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for assessing the longevity of protective immunity after SARS-CoV-2 infection or coronavirus disease 2019 (COVID-19) vaccination. However, the longitudinal memory T-cell response up to 8 months post-symptom onset (PSO) according to the severity of illness is unknown. METHODS: We analyzed peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with COVID-19 who experienced asymptomatic, mild, or severe illness at 2, 5, and 8 months PSO. SARS-CoV-2 spike, nucleocapsid, and membrane protein-stimulated PBMCs were subjected to flow cytometry analysis. RESULTS: A total of 24 patients (7 asymptomatic, 9 with mild disease, and 8 with severe disease) and 6 healthy volunteers were analyzed. SARS-CoV-2-specific OX40+CD137+CD4+ T cells and CD69+CD137+CD8+ T cells persisted at 8 months PSO. Also, antigen-specific cytokine-producing or polyfunctional CD4+ T cells were maintained for up to 8 months PSO. Memory CD4+ T-cell responses tended to be greater in patients who had severe illness than in those with mild or asymptomatic disease. CONCLUSIONS: Memory response to SARS-CoV-2, based on the frequency and functionality, persists for 8 months PSO. Further investigations involving its longevity and protective effect from reinfection are warranted.

Roles of Mis18α in epigenetic regulation of centromeric chromatin and CENP-A loading.

The Mis18 complex has been identified as a critical factor for the centromeric localization of a histone H3 variant, centromeric protein A (CENP-A), which is responsible for the specification of centromere identity in the chromosome. However, the functional role of Mis18 complex is largely unknown. Here, we generated Mis18α conditional knockout mice and found that Mis18α deficiency resulted in lethality at early embryonic stage with severe defects in chromosome segregation caused by mislocalization of CENP-A. Further, we demonstrate Mis18α's crucial role for epigenetic regulation of centromeric chromatin by reinforcing centromeric localization of DNMT3A/3B. Mis18α interacts with DNMT3A/3B, and this interaction is critical for maintaining DNA methylation and hence regulating epigenetic states of centromeric chromatin. Mis18α deficiency led to reduced DNA methylation, altered histone modifications, and uncontrolled noncoding transcripts in centromere region by decreased DNMT3A/3B enrichment. Together, our findings uncover the functional mechanism of Mis18α and its pivotal role in mammalian cell cycle.

EZH2 generates a methyl degron that is recognized by the DCAF1/DDB1/CUL4 E3 ubiquitin ligase complex.

Ubiquitination plays a major role in protein degradation. Although phosphorylation-dependent ubiquitination is well known for the regulation of protein stability, methylation-dependent ubiquitination machinery has not been characterized. Here, we provide evidence that methylation-dependent ubiquitination is carried out by damage-specific DNA binding protein 1 (DDB1)/cullin4 (CUL4) E3 ubiquitin ligase complex and a DDB1-CUL4-associated factor 1 (DCAF1) adaptor, which recognizes monomethylated substrates. Molecular modeling and binding affinity studies reveal that the putative chromo domain of DCAF1 directly recognizes monomethylated substrates, whereas critical binding pocket mutations of the DCAF1 chromo domain ablated the binding from the monomethylated substrates. Further, we discovered that enhancer of zeste homolog 2 (EZH2) methyltransferase has distinct substrate specificities for histone H3K27 and nonhistones exemplified by an orphan nuclear receptor, RORα. We propose that EZH2-DCAF1/DDB1/CUL4 represents a previously unrecognized methylation-dependent ubiquitination machinery specifically recognizing "methyl degron"; through this, nonhistone protein stability can be dynamically regulated in a methylation-dependent manner.

RORalpha attenuates Wnt/beta-catenin signaling by PKCalpha-dependent phosphorylation in colon cancer.

Wnt family members play diverse roles in development and disease. Noncanonical Wnt ligands can inhibit canonical Wnt signaling depending on the cellular context; however, the underlying mechanism of this antagonism remains poorly understood. Here we identify a specific mechanism of orphan nuclear receptor RORalpha-mediated inhibition of canonical Wnt signaling in colon cancer. Wnt5a/PKCalpha-dependent phosphorylation on serine residue 35 of RORalpha is crucial to link RORalpha to Wnt/beta-catenin signaling, which exerts inhibitory function of the expression of Wnt/beta-catenin target genes. Intriguingly, there is a significant correlation of reduction of RORalpha phosphorylation in colorectal tumor cases compared to their normal counterpart, providing the clinical relevance of the findings. Our data provide evidence for a role of RORalpha, functioning at the crossroads between the canonical and the noncanonical Wnt signaling pathways, in mediating transrepression of the Wnt/beta-catenin target genes, thereby providing new approaches for the development of therapeutic agents for human cancers.

Negative regulation of hypoxic responses via induced Reptin methylation.

Lysine methylation within histones is crucial for transcriptional regulation and thus links chromatin states to biological outcomes. Although recent studies have extended lysine methylation to nonhistone proteins, underlying molecular mechanisms such as the upstream signaling cascade that induces lysine methylation and downstream target genes modulated by this modification have not been elucidated. Here, we show that Reptin, a chromatin-remodeling factor, is methylated at lysine 67 in hypoxic conditions by the methyltransferase G9a. Methylated Reptin binds to the promoters of a subset of hypoxia-responsive genes and negatively regulates transcription of these genes to modulate cellular responses to hypoxia.

Inhibitory effect of ephedrannins A and B from roots of Ephedra sinica STAPF on melanogenesis.

BACKGROUND: Melanogenesis, a process producing the pigment melanin in human skin, eyes and hair, is a major physiological response against various environmental stresses, in particular exposure to ultraviolet radiation, and its pathway is regulated by a key enzyme, tyrosinase. In this study, we evaluated the effects of ephedrannins A and B, which are polyphenols from the roots of Ephedra sinica, commonly used in herbalism in oriental countries, on mushroom tyrosinase and melanogenesis in B16F10 melanoma cells. METHODS: Their effects on mushroom tyrosinase were determined via kinetic studies using a spectrophotometric analysis and those on melanin and tyrosinase production in melanoma cells treated with α-MSH (melanin stimulating hormone) were examined using PCR and ELISA. RESULTS: Both ephedrannins A and B exhibited concentration-dependent inhibitory effects on L-tyrosine oxidation by mushroom tyrosinase, and the inhibition mechanism was competitive and reversible with L-tyrosine as the substrate. In addition, melanin production in melanoma cells was also suppressed in a concentration-dependent manner by ephedrannins A and B without significant effects on cell proliferation at the concentrations tested. Both compounds showed inhibitory effects on melanin production by suppressing the transcription of tyrosinase in the cells. CONCLUSION: Both compounds exhibited significant inhibitory effects, but the inhibition by ephedrannin B was much more effective than that by ephedrannin A. Both ephedrannins A and B may be good candidates for a whitening agent for skin. GENERAL SIGNIFICANCE: This is the first report that describes effective inhibition of melanin production by ephedrannins A and B isolated from Ephedra roots.

ßTrCP-mediated ubiquitylation regulates protein stability of Mis18ß in a cell cycle-dependent manner.

Ubiquitin E3 ligases including SCF complex are key regulators of cell cycle. Here, we show that Mis18ß, a component of Mis18 complex governing CENP-A localization, is a new substrate of ßTrCP-containing SCF complex. ßTrCP interacted with Mis18ß exclusively during interphase but not during mitosis and mediated proteasomal degradation of Mis18ß leading to the inactivation of Mis18 complex during interphase. In addition, uncontrolled stabilization of Mis18ß caused cell death. Together, we propose that ßTrCP-mediated regulation of Mis18ß stability is a mechanism to restrict centromere function of Mis18 complex from late mitosis to early G1 phase.

Roles of sumoylation of a reptin chromatin-remodelling complex in cancer metastasis.

Defining the functional modules within transcriptional regulatory factors that govern switching between repression and activation events is a central issue in biology. Recently, we have reported the dynamic role of a beta-catenin-reptin chromatin remodelling complex in regulating a metastasis suppressor gene KAI1 (ref.1), which is capable of inhibiting the progression of tumour metastasis. Here, we identify signalling factors that confer repressive function on reptin and hence repress the expression of KAI1. Biochemical purification of a reptin-containing complex has revealed the presence of specific desumoylating enzymes that reverse the sumoylation of reptin that underlies its function as a repressor. Desumoylation of reptin alters the repressive function of reptin and its association with HDAC1. Furthermore, the sumoylation status of reptin modulates the invasive activity of cancer cells with metastatic potential. These data clearly define a functional model and provide a novel link for SUMO modification in cancer metastasis.

Single-Cell Molecular Barcoding to Decode Multimodal Information Defining Cell States.

Single-cell research has provided a breakthrough in biology to understand heterogeneous cell groups, such as tissues and organs, in development and disease. Molecular barcoding and subsequent sequencing technology insert a singlecell barcode into isolated single cells, allowing separation cell by cell. Given that multimodal information from a cell defines precise cellular states, recent technical advances in methods focus on simultaneously extracting multimodal data recorded in different biological materials (DNA, RNA, protein, etc.). This review summarizes recently developed singlecell multiomics approaches regarding genome, epigenome, and protein profiles with the transcriptome. In particular, we focus on how to anchor or tag molecules from a cell, improve throughputs with sample multiplexing, and record lineages, and we further discuss the future developments of the technology.

DNA Barcoding Technology for Lineage Recording and Tracing to Resolve Cell Fate Determination.

In various biological contexts, cells receive signals and stimuli that prompt them to change their current state, leading to transitions into a future state. This change underlies the processes of development, tissue maintenance, immune response, and the pathogenesis of various diseases. Following the path of cells from their initial identity to their current state reveals how cells adapt to their surroundings and undergo transformations to attain adjusted cellular states. DNA-based molecular barcoding technology enables the documentation of a phylogenetic tree and the deterministic events of cell lineages, providing the mechanisms and timing of cell lineage commitment that can either promote homeostasis or lead to cellular dysregulation. This review comprehensively presents recently emerging molecular recording technologies that utilize CRISPR/Cas systems, base editing, recombination, and innate variable sequences in the genome. Detailing their underlying principles, applications, and constraints paves the way for the lineage tracing of every cell within complex biological systems, encompassing the hidden steps and intermediate states of organism development and disease progression.

Amorphous Carbon Films for Electronic Applications.

While various crystalline carbon allotropes, including graphene, have been actively investigated, amorphous carbon (a-C) thin films have received relatively little attention. The a-C is a disordered form of carbon bonding with a broad range of the CC bond length and bond angle. Although accurate structural analysis and theoretical approaches are still insufficient, reproducible structure-property relationships have been accumulated. As the a-C thin film is now adapted as a hardmask in the semiconductor industry and new properties are reported continuously, expectations are growing that it can be practically used as active materials beyond as a simple sacrificial layer. In this perspective review article, after a brief introduction to the synthesis and properties of the a-C thin films, their potential practical applications are proposed, including hardmasks, extreme ultraviolet (EUV) pellicles, diffusion barriers, deformable electrodes and interconnects, sensors, active layers, electrodes for energy, micro-supercapacitors, batteries, nanogenerators, electromagnetic interference (EMI) shielding, and nanomembranes. The article ends with a discussion on the technological challenges in a-C thin films.

Nanocomplexes of doxorubicin and DNA fragments for efficient and safe cancer chemotherapy.

Cancer-targeted therapy by a chemotherapeutic agent formulated in a nanoscale platform has been challenged by complex and inefficient manufacturing, low drug loading, difficult characterization, and marginally improved therapeutic efficacy. This study investigated facile-to-produce nanocomplexes of doxorubicin (DOX), a widely used cancer drug, and clinically approved DNA fragments that are extracted from a natural source. DOX was found to self-assemble DNA fragments into relatively monodispersed nanocomplexes with a diameter of ∼70 nm at 14.3% (w/w) drug loading by simple and scalable mixing. The resulting DOX/DNA nanocomplexes showed sustained DOX release, unlike overly stable Doxil®, cellular uptake via multiple endocytosis pathways, and high hematological and immunological compatibility. DOX/DNA nanocomplexes eradicated EL4 T lymphoma cells in a time-dependent manner, eventually surpassing free DOX. Extended circulation of DOX/DNA nanocomplexes, while avoiding off-target accumulation in the lung and being cleared from the liver, resulted in rapid accumulation in tumor and lowered cardio toxicity. Finally, tumor growth of EL4-challenged C57BL/6 mice (syngeneic model) and OPM2-challenged NSG mice (human xenograft model) were efficiently inhibited by DOX/DNA nanocomplexes with enhanced overall survival, in comparison with free DOX and Doxil®, especially upon repeated administrations. DOX/DNA nanocomplexes are a promising chemotherapeutics delivery platform for their ease of manufacturing, high biocompatibility, desired drug release and accumulation, efficient tumor eradication with improved safety, and further engineering versatility for extended therapeutic applications.

Corrigendum: Distinct Immune Response at 1 Year Post-COVID-19 According to Disease Severity.

[This corrects the article DOI: 10.3389/fimmu.2022.830433.].

Distinct Immune Response at 1 Year Post-COVID-19 According to Disease Severity.

Background: Despite the fact of ongoing worldwide vaccination programs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), understanding longevity, breadth, and type of immune response to coronavirus disease-19 (COVID-19) is still important to optimize the vaccination strategy and estimate the risk of reinfection. Therefore, we performed thorough immunological assessments 1 year post-COVID-19 with different severity. Methods: We analyzed peripheral blood mononuclear cells and plasma samples at 1 year post-COVID-19 in patients who experienced asymptomatic, mild, and severe illness to assess titers of various isotypes of antibodies (Abs) against SARS-CoV-2 antigens, phagocytic capability, and memory B- and T-cell responses. Findings: A total of 24 patients (7, 9, and 8 asymptomatic, mild, and severe patients, respectively) and eight healthy volunteers were included in this study. We firstly showed that disease severity is correlated with parameters of immune responses at 1 year post-COVID-19 that play an important role in protecting against reinfection with SARS-CoV-2, namely, the phagocytic capacity of Abs and memory B-cell responses. Interpretation: Various immune responses at 1 year post-COVID-19, particularly the phagocytic capacity and memory B-cell responses, were dependent on the severity of the prior COVID-19. Our data could provide a clue for a tailored vaccination strategy after natural infection according to the severity of COVID-19.

Breast cancer metastasis suppressor 1 (BRMS1) is destabilized by the Cul3-SPOP E3 ubiquitin ligase complex.

Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis without affecting primary tumorigenesis. The regulatory mechanism of BRMS1 at the protein level has not been revealed until recently. Here, we found that cullin 3 (Cul3), a component of E3 ubiquitin ligase, is a new binding partner of BRMS1 and the interaction between BRMS1 and Cul3 is mediated by the SPOP adaptor protein. Intriguingly, BRMS1 turns out to be a potent substrate that is ubiquitinated by the Cul3-SPOP complex. Knockdown of SPOP increases the level of BRMS1 protein and represses the expression of BRMS1 repressive target genes such as OPN and uPA in breast cancer cells. These results suggest that the novel regulatory mechanism of BRMS1 by Cul3-SPOP complex is important for breast cancer progression.