Engineering Epigenetic Regulation Using Synthetic Read-Write Modules.

Chemical modifications to DNA and histone proteins are involved in epigenetic programs underlying cellular differentiation and development. Regulatory networks involving molecular writers and readers of chromatin marks are thought to control these programs. Guided by this common principle, we established an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system utilizes synthetic factors that write and read m6A and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we used our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits were able to program epigenetic functions de novo, conceptually validating "read-write" architectures. This work provides a toolkit for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells.

Structural elements promote architectural stripe formation and facilitate ultra-long-range gene regulation at a human disease locus.

Enhancer clusters overlapping disease-associated mutations in Pierre Robin sequence (PRS) patients regulate SOX9 expression at genomic distances over 1.25 Mb. We applied optical reconstruction of chromatin architecture (ORCA) imaging to trace 3D locus topology during PRS-enhancer activation. We observed pronounced changes in locus topology between cell types. Subsequent analysis of single-chromatin fiber traces revealed that these ensemble-average differences arise through changes in the frequency of commonly sampled topologies. We further identified two CTCF-bound elements, internal to the SOX9 topologically associating domain, which promote stripe formation, are positioned near the domain's 3D geometric center, and bridge enhancer-promoter contacts in a series of chromatin loops. Ablation of these elements results in diminished SOX9 expression and altered domain-wide contacts. Polymer models with uniform loading across the domain and frequent cohesin collisions recapitulate this multi-loop, centrally clustered geometry. Together, we provide mechanistic insights into architectural stripe formation and gene regulation over ultra-long genomic ranges.

Loop stacking organizes genome folding from TADs to chromosomes.

Although population-level analyses revealed significant roles for CTCF and cohesin in mammalian genome organization, their contributions at the single-cell level remain incompletely understood. Here, we used a super-resolution microscopy approach to measure the effects of removal of CTCF or cohesin in mouse embryonic stem cells. Single-chromosome traces revealed cohesin-dependent loops, frequently stacked at their loop anchors forming multi-way contacts (hubs), bridging across TAD boundaries. Despite these bridging interactions, chromatin in intervening TADs was not intermixed, remaining separated in distinct loops around the hub. At the multi-TAD scale, steric effects from loop stacking insulated local chromatin from ultra-long range (>4 Mb) contacts. Upon cohesin removal, the chromosomes were more disordered and increased cell-cell variability in gene expression. Our data revise the TAD-centric understanding of CTCF and cohesin and provide a multi-scale, structural picture of how they organize the genome on the single-cell level through distinct contributions to loop stacking.

Enhancement of High-Density Lipoprotein (HDL) Quantity and Quality by Regular and Habitual Exercise in Middle-Aged Women with Improvements in Lipid and Apolipoprotein Profiles: Larger Particle Size and Higher Antioxidant Ability of HDL.

Regular exercise, especially aerobic exercise, is beneficial for increasing serum high-density lipoprotein-cholesterol (HDL-C) levels in the general population. In addition to the HDL-C quantity, exercise enhances HDL functionality, antioxidants, and cholesterol efflux. On the other hand, the optimal intensity and frequency of exercise to increase HDL quantity and enhance HDL quality in middle-aged women need to be determined. The current study was designed to compare the changes in HDL quantity and quality among middle-aged women depending on exercise intensity, frequency, and duration; participants were divided into a sedentary group (group 1), a middle-intensity group (group 2), and a high-intensity group (group 3). There were no differences in anthropometric parameters among the groups, including blood pressure, muscle mass, and handgrip strength. Although there was no difference in serum total cholesterol (TC) among the groups, the serum HDL-C and apolipoprotein (apo)A-I levels remarkably increased to 17% and 12%, respectively, in group 3. Serum low-density lipoprotein-cholesterol (LDL-C), glucose, triglyceride, and the apo-B/apoA-I ratio were remarkably decreased in the exercise groups depending on the exercise intensity; group 3 showed 13%, 10%, and 45% lower LDL-C, glucose, and triglyceride (TG), respectively, than group 1. The hepatic and muscle damage parameter, aspartate aminotransferase (AST), was significantly decreased in the exercise groups, but high-sensitivity C-reactive protein (CRP), alanine aminotransferase (ALT), and γ-glutamyl transferase (γ-GTP) were similar in the three groups. In LDL, the particle size was increased 1.5-fold (p < 0.001), and the oxidation extent was decreased by 40% with a 23% lower TG content in group 3 than in group 1. In the exercise groups (groups 2 and 3), LDL showed the slowest electromobility with a distinct band intensity compared to the sedentary group (group 1). In HDL2, the particle size was 2.1-fold increased (p < 0.001) in the exercise group (group 3) with a 1.5-fold increase in TC content compared to that in group 1, as well as significantly enhanced antioxidant abilities, paraoxonase (PON) activity, and ferric ion reduction ability (FRA). In HDL3, the particle size was increased 1.2-fold with a 45% reduction in TG in group 3 compared to group 1. With increasing exercise intensity, apoA-I expression was increased in HDL2 and HDL3, and PON activity and FRA were enhanced (p < 0.001). In conclusion, regular exercise in middle-aged women is associated with the elevation of serum HDL-C and apoA-I with the enhancement of HDL quality and functionality and an increase in the TC content, particle size, and antioxidant abilities. With the reduction in TG and oxidized products in LDL and HDL, lipoproteins could have more anti-atherogenic properties through regular exercise in an intensity-dependent manner.

Long-Term Alcohol Consumption Caused a Significant Decrease in Serum High-Density Lipoprotein (HDL)-Cholesterol and Apolipoprotein A-I with the Atherogenic Changes of HDL in Middle-Aged Korean Women.

Light-to-moderate alcohol drinking is associated with a low incidence of cardiovascular disease (CVD) via an elevation of high-density lipoproteins-cholesterol (HDL-C), particularly with the short-term supplementation of alcohol. However, there is no information on the change in the HDL qualities and functionalities between non-drinkers and mild drinkers in the long-term consumption of alcohol. This study analyzed the lipid and lipoprotein profiles of middle-aged Korean female non-drinkers, mild-drinkers, and binge-drinkers, who consumed alcohol for at least 10 years. Unexpectedly, the serum levels of HDL-C and apolipoprotein A-I (apoA-I) were decreased significantly depending on the alcohol amount; the binge-drinker group showed 18% and 13% lower HDL-C (p = 0.011) and apoA-I levels (p = 0.024), respectively, than the non-drinker group. Triglyceride (TG) and oxidized species, malondialdehyde (MDA), and low-density lipoproteins (LDL) levels were significantly elevated in the drinker groups. Interestingly, the binge-drinker group showed 1.4-fold higher (p = 0.020) cholesterol contents in HDL2 and 1.7-fold higher (p < 0.001) TG contents in HDL3 than those of the non-drinker group. The mild-drinker group also showed higher TG contents in HDL3 (p = 0.032) than the non-drinker group, while cholesterol contents were similar in the HDL3 of all groups. Transmission electron microscopy (TEM) showed that the non-drinker group showed a more distinct and clear particle shape of the LDL and HDL image with a larger particle size than the drinker group. Electrophoresis of LDL showed that the drinker group had faster electromobility with a higher smear band intensity and aggregation in the loading position than the non-drinker group. The HDL level of binge drinkers showed the lowest paraoxonase activity, the highest glycated extent, and the most smear band intensity of HDL and apoA-I, indicating that HDL quality and functionality were impaired by alcohol consumption. In conclusion, long-term alcohol consumption in middle-aged women, even in small amounts, caused a significant decrease in the serum HDL-C and apoA-I with atherogenic changes in LDL and HDL, such as an increase in TG and MDA content with a loss of paraoxonase activity.

A classical swine fever virus E2 fusion protein produced in plants elicits a neutralizing humoral immune response in mice and pigs.

Classical swine fever (CSF) is one of the most important viral diseases of swine worldwide. Although live or attenuated virus vaccines have been used to control CSFV, it is difficult to distinguish vaccinated pigs from infected pigs; this leads to restrictions on import and export. Subunit vaccines based on the CSFV E2 glycoprotein have been developed using baculovirus or insect cell systems, but some weaknesses remain. Here, we describe production of an E2 recombinant protein using a Nicotiana benthamiana plant expression system. To do this, we took advantage of the ability of the swine Fc domain to increase solubility and stability of the fusion protein and to strengthen immune responses in target animals. N. benthamiana expressed high amounts of pFc2-fused E2 proteins, which were isolated and purified by affinity chromatography to yield a high pure recombinant protein in a cost-effective manner. Native-polyacrylamide gel electrophoresis and size exclusion chromatography confirmed that the pmE2:pFc2 fusion exists as a multimer rather than as a dimer. Injection of recombinant pmE2 protein into mice or piglets generated anti-pmE2 antibodies with efficient neutralizing activity against CSFV. These results suggest that a purified recombinant E2 protein produced in N. benthamiana generates high titers of neutralizing antibodies in vivo; as such, the protein could be developed as a subunit vaccine against CSFV.

Combination Antitumor Effect of Sorafenib via Calcium-Dependent Deactivation of Focal Adhesion Kinase Targeting Colorectal Cancer Cells.

Sorafenib has been recently used for the treatment of patients with advanced colorectal cancer (CRC) and is recognized for its therapeutic value. However, the continuous use of sorafenib may cause resistance in the treatment of cancer patients. In this study, we investigated whether sorafenib exerts an enhanced anticancer effect on CRC cells via the calcium-mediated deactivation of the focal adhesion kinase (FAK) signaling pathways. The appropriate dose of sorafenib and lactate calcium salt (CaLa) for a combination treatment were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Then, cell cycle analysis was performed following treatment with 2.5 µM sorafenib and/or 2.5 mM CaLa. CRC cells were found to be in the G1 phase by sorafenib treatment, and they accumulated in the sub-G1 phase with CaLa treatment. Western blots and enzyme-linked immunosorbent assays were performed to analyze the elements of the recombinant activated factor (RAF) and focal adhesion kinase (FAK) signaling cascades. Sorafenib-inhibited RAF-dependent signaling in CRC cells, however, either did not affect the expression of Akt or increased it. As the upstream signaling of FAK was suppressed by CaLa, we observed that the expression of the sub-signaling phospho (p) AKT and p-mammalian target of rapamycin was also suppressed. Treatment with a combination of sorafenib and CaLa enhanced the antitumor activity of CRC cells. The % viability of CRC cells was significantly decreased compared to the single treatment with sorafenib or CaLa, and the accumulation of Sub G1 of CRC cells was clearly confirmed. The migration ability of CRC cells was significantly reduced. The findings of this study indicate that sorafenib will show further improved antitumor efficacy against CRC due to overcoming resistance through the use of CaLa.

Antitumor Effect of Calcium-Mediated Destabilization of Epithelial Growth Factor Receptor on Non-Small Cell Lung Carcinoma.

Despite the development of numerous therapeutics targeting the epithelial growth factor receptor (EGFR) for non-small cell lung carcinoma (NSCLC), the application of these drugs is limited because of drug resistance. Here, we investigated the antitumor effect of calcium-mediated degradation of EGFR pathway-associated proteins on NSCLC. First, lactate calcium salt (LCS) was utilized for calcium supplementation. Src, α-tubulin and EGFR levels were measured after LSC treatment, and the proteins were visualized by immunocytochemistry. Calpeptin was used to confirm the calcium-mediated effect of LCS on NSCLC. Nuclear expression of c-Myc and cyclin D1 was determined to understand the underlying mechanism of signal inhibition following EGFR and Src destabilization. The colony formation assay and a xenograft animal model were used to confirm the in vitro and in vivo antitumor effects, respectively. LCS supplementation reduced Src and α-tubulin expression in NSCLC cells. EGFR was destabilized because of proteolysis of Src and α-tubulin. c-Myc and cyclin D1 expression levels were also reduced following the decrease in the transcriptional co-activation of EGFR and Src. Clonogenic ability and tumor growth were significantly inhibited by LSC treatment-induced EGFR destabilization. These results suggest that other than specifically targeting EGFR, proteolysis of associated molecules such as Src or α-tubulin may effectively exert an antitumor effect on NSCLC via EGFR destabilization. Therefore, LCS is expected to be a good candidate for developing novel anti-NSCLC therapeutics overcoming chemoresistance.

The effects of task-oriented training with altered sensory input on balance in patients with chronic stroke.

[Purpose] The purpose of this study was to identify the effects of task-oriented training with altered somatosensory input on the balance ability of chronic stroke patients. [Subjects and Methods] Twenty-six subjects with chronic stroke were divided into an experimental group (n=14) and a control group (n=12). Both groups attended physical therapy sessions five times a week for four weeks. The experimental group performed additional, task-oriented training with altered sensory input three times a week for four weeks. Limit-of-stability tests were conducted before and after the intervention. In addition, all subjects were evaluated using the Berg Balance Scale (BBS) and Korean Activities-Specific Balance Confidence Scale before and after the intervention. [Results] There was a significant interaction between time and group on BBS scores, on the total surface area of the limit of stability, and on the surface area of the limit of stability of the affected side. However, an analysis of covariance in which the baseline values of each variable served as the covariates showed that only the post-intervention BBS score of the experimental group was significantly higher than that of the control group. [Conclusion] Task-oriented training with altered somatosensory input can improve functional balance in patients with chronic stroke.

Distinct Protein Expression Profiles of Solid-Pseudopapillary Neoplasms of the Pancreas.

Solid-pseudopapillary neoplasm (SPN) is an uncommon pancreatic tumor with mutation in CTNNB1 and distinct clinical and pathological features. We compared the proteomic profiles of SPN to mRNA expression. Pooled SPNs and pooled non-neoplastic pancreatic tissues were examined with high-resolution mass spectrometry. We identified 329 (150 up-regulated and 179 down-regulated) differentially expressed proteins in SPN. We identified 191 proteins (58.1% of the 329 dysregulated proteins) with the same expression tendencies in SPN based on mRNA data. Many overexpressed proteins were related to signaling pathways known to be activated in SPNs. We found that several proteins involved in Wnt signaling, including DKK4 and ß-catenin, and proteins that bind ß-catenin, such as FUS and NONO, were up-regulated in SPNs. Molecules involved in glycolysis, including PKM2, ENO2, and HK1, were overexpressed in accordance to their mRNA levels. In summary, SPN showed (1) distinct protein expression changes that correlated with mRNA expression, (2) overexpression of Wnt signaling proteins and proteins that bind directly to ß-catenin, and (3) overexpression of proteins involved in metabolism. These findings may help develop early diagnostic biomarkers and molecular targets.

Characterization of gene expression and activated signaling pathways in solid-pseudopapillary neoplasm of pancreas.

Solid-pseudopapillary neoplasm is an uncommon pancreatic tumor with distinct clinicopathologic features. Solid-pseudopapillary neoplasms are characterized by mutations in exon 3 of CTNNB1. However, little is known about the gene and microRNA expression profiles of solid-pseudopapillary neoplasms. Thus, we sought to characterize solid-pseudopapillary neoplasm-specific gene expression and identify the signaling pathways activated in these tumors. Comparisons of gene expression in solid-pseudopapillary neoplasm to pancreatic ductal carcinomas, neuroendocrine tumors, and non-neoplastic pancreatic tissues identified solid-pseudopapillary neoplasm-specific mRNA and microRNA profiles. By analyzing 1686 (1119 upregulated and 567 downregulated) genes differentially expressed in solid-pseudopapillary neoplasm, we found that the Wnt/ß-catenin, Hedgehog, and androgen receptor signaling pathways, as well as genes involved in epithelial mesenchymal transition, are activated in solid-pseudopapillary neoplasms. We validated these results experimentally by assessing the expression of ß-catenin, WIF-1, GLI2, androgen receptor, and epithelial-mesenchymal transition-related markers with western blotting and immunohistochemistry. Our analysis also revealed 17 microRNAs, especially the miR-200 family and miR-192/215, closely associated with the upregulated genes associated with the three pathways activated in solid-pseudopapillary neoplasm and epithelial mesenchymal transition. Our results provide insight into the molecular mechanisms underlying solid-pseudopapillary neoplasm tumorigenesis and its characteristic less epithelial cell differentiation than the other common pancreatic tumors.

Comparison of the Predictors of Smoking Cessation Plans between Adolescent Conventional Cigarette Smokers and E-Cigarette Smokers Using the Transtheoretical Model.

Recently, there has been a shift in smoking patterns among adolescents, with a decrease in the prevalence of conventional cigarette smoking and an increase in the use of electronic cigarettes (e-cigarettes). The harmful effects of e-cigarettes are remarkable, highlighting the need for proactive interventions for adolescent users and smoking cessation that consider the characteristics of both conventional cigarette smokers and e-cigarette users. This study aims to investigate the smoking status of adolescent conventional cigarette and e-cigarette smokers and to analyze the predictors of their smoking cessation plans (SCPs) based on the transtheoretical model. Self-rated health, prior smoking cessation education, consciousness-raising, and dramatic relief as types of experiential processes of change, and formation of helping relationships as a type of behavioral process of change significantly differed according to the type of cigarette behavior among adolescents. The predictors of SCP among adolescents were perceived pros of smoking and academic performance among conventional cigarette smokers and behavioral process of change, perceived pros of smoking, and economic status among e-cigarette users. This study identified differences in the characteristics and predictors of SCP. Strategies tailored to each specific adolescent smoking population are further required to promote smoking cessation.

Advances in the multimodal analysis of the 3D chromatin structure and gene regulation.

Recent studies have demonstrated that the three-dimensional conformation of the chromatin plays a crucial role in gene regulation, with aberrations potentially leading to various diseases. Advanced methodologies have revealed a link between the chromatin conformation and biological function. This review divides these methodologies into sequencing-based and imaging-based methodologies, tracing their development over time. We particularly highlight innovative techniques that facilitate the simultaneous mapping of RNAs, histone modifications, and proteins within the context of the 3D architecture of chromatin. This multimodal integration substantially improves our ability to establish a robust connection between the spatial arrangement of molecular components in the nucleus and their functional roles. Achieving a comprehensive understanding of gene regulation requires capturing diverse data modalities within individual cells, enabling the direct inference of functional relationships between these components. In this context, imaging-based technologies have emerged as an especially promising approach for gathering spatial information across multiple components in the same cell.

Additive-Driven Nanoscale Architecture of Solid Electrolyte Interphase Revealed by Cryogenic Transmission Electron Microscopy.

In Li metal batteries (LMBs), which boast the highest theoretical capacity, the chemical structure of the solid electrolyte interphase (SEI) serves as the key component that governs the growth of reactive Li. Various types of additives have been developed for electrolyte optimization, representing one of the most effective strategies to enhance the SEI properties for stable Li plating. However, as advanced electrolyte systems become more chemically complicated, the use of additives is empirically optimized. Indeed, their role in SEI formation and the resulting cycle life of LMBs are not well-understood. In this study, we employed cryogenic transmission electron microscopy combined with Raman spectroscopy, theoretical studies including molecular dynamics (MD) simulations and density functional theory (DFT) calculations, and electrochemical measurements to explore the nanoscale architecture of SEI modified by the most representative additives, lithium nitrate (LiNO3) and vinylene carbonate (VC), applied in a localized high-concentration electrolyte. We found that LiNO3 and VC play distinct roles in forming the SEI, governing the solvation structure, and influencing the kinetics of electrochemical reduction. Their collaboration leads to the desired SEI, ensuring prolonged cycle performance for LMBs. Moreover, we propose mechanisms for different Li growth and cycling behaviors that are determined by the physicochemical properties of SEI, such as uniformity, elasticity, and ionic conductivity. Our findings provide critical insights into the appropriate use of additives, particularly regarding their chemical compatibility.

Examining Factors Associated with E-Cigarette Use among Current Smokers.

We present a secondary data analysis of the raw data from the eighth Korea National Health and Nutrition Examination Survey (KNHANES). A total of 827 current smokers who responded that they had smoked >5 packs (100 cigarettes) of cigarettes in their lifetime and were currently smoking traditional cigarettes were selected. This study was conducted to identify sociodemographic, smoking-related, and health-related characteristics that influence the use of e-cigarettes in adult smokers. To examine these factors, general characteristics such as age, marital status, education level, and occupation were included in Model 1, while health-related characteristics such as the level of smoking and depression were included in Model 2. In Model 1, age, a high level of education, and working in an office were found to be significantly correlated with e-cigarette use among smokers, while age and working in the office were found to be significantly correlated with e-cigarette use in Model 2. Therefore, e-cigarette use was high among adult smokers of young ages who were office workers. Although evidence is lacking regarding its safety and use as smoking cessation aids, many smokers have been reported to use e-cigarettes as smoking cessation aids, making it necessary to provide accurate information on e-cigarettes.

Interplay between epigenome and 3D chromatin structure.

Epigenetic mechanisms, primarily mediated through histone and DNA modifications, play a pivotal role in orchestrating the functional identity of a cell and its response to environmental cues. Similarly, the spatial arrangement of chromatin within the threedimensional (3D) nucleus has been recognized as a significant factor influencing genomic function. Investigating the relationship between epigenetic regulation and 3D chromatin structure has revealed correlation and causality between these processes, from the global alignment of average chromatin structure with chromatin marks to the nuanced correlations at smaller scales. This review aims to dissect the biological significance and the interplay between the epigenome and 3D chromatin structure, while also exploring the underlying molecular mechanisms. By synthesizing insights from both experimental and modeling perspectives, we seek to provide a comprehensive understanding of cellular functions. [BMB Reports 2023; 56(12): 633-644].

Transcriptional kinetic synergy: A complex landscape revealed by integrating modeling and synthetic biology.

Transcription factors (TFs) control gene expression, often acting synergistically. Classical thermodynamic models offer a biophysical explanation for synergy based on binding cooperativity and regulated recruitment of RNA polymerase. Because transcription requires polymerase to transition through multiple states, recent work suggests that "kinetic synergy" can arise through TFs acting on distinct steps of the transcription cycle. These types of synergy are not mutually exclusive and are difficult to disentangle conceptually and experimentally. Here, we model and build a synthetic circuit in which TFs bind to a single shared site on DNA, such that TFs cannot synergize by simultaneous binding. We model mRNA production as a function of both TF binding and regulation of the transcription cycle, revealing a complex landscape dependent on TF concentration, DNA binding affinity, and regulatory activity. We use synthetic TFs to confirm that the transcription cycle must be integrated with recruitment for a quantitative understanding of gene regulation.

Exploring Optimal Water Splitting Bifunctional Alloy Catalyst by Pareto Active Learning.

Design of bifunctional multimetallic alloy catalysts, which are one of the most promising candidates for water splitting, is a significant issue for the efficient production of renewable energy. Owing to large dimensions of the components and composition of multimetallic alloys, as well as the trade-off behavior in terms of the hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) overpotentials for bifunctional catalysts, it is difficult to search for high-performance bifunctional catalysts with multimetallic alloys using conventional trial-and-error experiments. Here, an optimal bifunctional catalyst for water splitting is obtained by combining Pareto active learning and experiments, where 110 experimental data points out of 77946 possible points lead to effective model development. The as-obtained bifunctional catalysts for HER and OER exhibit high performance, which is revealed by model development using Pareto active learning; among the catalysts, an optimal catalyst (Pt0.15 Pd0.30 Ru0.30 Cu0.25 ) exhibits a water splitting behavior of 1.56 V at a current density of 10 mA cm-2 . This study opens avenues for the efficient exploration of multimetallic alloys, which can be applied in multifunctional catalysts as well as in other applications.

A Plant-Derived Maternal Vaccine against Porcine Epidemic Diarrhea Protects Piglets through Maternally Derived Immunity.

Newborn piglets are susceptible to a highly contagious enteritis caused by the porcine epidemic diarrhea virus (PEDV), associated with high levels of mortality worldwide. There is pressing need for a rapid, safe, and cost-effective vaccine to safeguard pigs from getting infected by PEDV. PEDV belongs to the coronavirus family and is characterized by high levels of mutability. The primary goal of a PEDV vaccine is to provide immunity to newborn piglets through vaccination of sows. Plant-based vaccines are becoming more popular because they have low manufacturing costs, are easily scalable, have high thermostability, and a long shelf life. This is in contrast to conventional vaccines which include inactivated, live, and/or recombinant types that can be expensive and have limited ability to respond to rapidly mutating viruses. The binding of the virus to host cell receptors is primarily facilitated by the N-terminal subunit of the viral spike protein (S1), which also contains several epitopes that are recognized by virus-neutralizing antibodies. As a result, we generated a recombinant S1 protein using a plant-based vaccine platform. We found that the recombinant protein was highly glycosylated, comparable to the native viral antigen. Vaccination of pregnant sows at four and two weeks before farrowing led to the development of humoral immunity specific to S1 in the suckling piglets. In addition, we noted significant viral neutralization titers in both vaccinated sows and piglets. When challenged with PEDV, piglets born from vaccinated sows displayed less severe clinical symptoms and significantly lower mortality rates compared to piglets born from non-vaccinated sows.

High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells.

High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-ζ, λ, and ι) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-ζ by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-ζ in colon cancer tissues. Our findings suggest that PKC-ζ is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.