TRIM40 is a pathogenic driver of inflammatory bowel disease subverting intestinal barrier integrity.

The cortical actin cytoskeleton plays a critical role in maintaining intestinal epithelial integrity, and the loss of this architecture leads to chronic inflammation, as seen in inflammatory bowel disease (IBD). However, the exact mechanisms underlying aberrant actin remodeling in pathological states remain largely unknown. Here, we show that a subset of patients with IBD exhibits substantially higher levels of tripartite motif-containing protein 40 (TRIM40), a gene that is hardly detectable in healthy individuals. TRIM40 is an E3 ligase that directly targets Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), an essential kinase involved in promoting cell-cell junctions, markedly decreasing the phosphorylation of key signaling factors critical for cortical actin formation and stabilization. This causes failure of the epithelial barrier function, thereby promoting a long-lived inflammatory response. A mutant TRIM40 lacking the RING, B-box, or C-terminal domains has impaired ability to accelerate ROCK1 degradation-driven cortical actin disruption. Accordingly, Trim40-deficient male mice are highly resistant to dextran sulfate sodium (DSS)-induced colitis. Our findings highlight that aberrant upregulation of TRIM40, which is epigenetically silenced under healthy conditions, drives IBD by subverting cortical actin formation and exacerbating epithelial barrier dysfunction.

Prediction of Growth Characteristics and Migration Period of Spodoptera frugiperda (Lepidoptera: Noctuidae) According to Temperature.

In 2019, an outbreak of Spodoptera frugiperda (Smith) was first reported in Korea. This study aimed to determine the growth rate and feeding amount of S. frugiperda by temperature to establish the right time window for its control and management. Linear regression analysis was used to determine the growth period and thermal requirements of S. frugiperda. The longest growth period of 97.2 ± 1.2 days was observed at 16 °C, and the shortest growth period of 15.5 ± 0.7 days was observed at 36 °C. In terms of each growth stage, the pupal period was the longest at all temperatures, followed by the egg period. The maximum corn leaf feeding amount (6.61 g) was observed for the larvae grown at 16 °C, and the minimum (2.9 g) was observed at 36 °C. However, the daily feeding amount of S. frugiperda larvae was the highest at 28 °C and 32 °C. The hatching rate according to temperature exceeded 70% at 24 °C, 28 °C, and 32 °C, and the survival rate of larvae and pupae was 100% at 24 °C to 32 °C. Based on these results, a temperature range of 28 °C to 32 °C is proposed as the optimum temperature for the growth of S. frugiperda.

The nucleolus is the site for inflammatory RNA decay during infection.

Inflammatory cytokines are key signaling molecules that can promote an immune response, thus their RNA turnover must be tightly controlled during infection. Most studies investigate the RNA decay pathways in the cytosol or nucleoplasm but never focused on the nucleolus. Although this organelle has well-studied roles in ribosome biogenesis and cellular stress sensing, the mechanism of RNA decay within the nucleolus is not completely understood. Here, we report that the nucleolus is an essential site of inflammatory pre-mRNA instability during infection. RNA-sequencing analysis reveals that not only do inflammatory genes have higher intronic read densities compared with non-inflammatory genes, but their pre-mRNAs are highly enriched in nucleoli during infection. Notably, nucleolin (NCL) acts as a guide factor for recruiting cytosine or uracil (C/U)-rich sequence-containing inflammatory pre-mRNAs and the Rrp6-exosome complex to the nucleolus through a physical interaction, thereby enabling targeted RNA delivery to Rrp6-exosomes and subsequent degradation. Consequently, Ncl depletion causes aberrant hyperinflammation, resulting in a severe lethality in response to LPS. Importantly, the dynamics of NCL post-translational modifications determine its functional activity in phases of LPS. This process represents a nucleolus-dependent pathway for maintaining inflammatory gene expression integrity and immunological homeostasis during infection.

KS10076, a chelator for redox-active metal ions, induces ROS-mediated STAT3 degradation in autophagic cell death and eliminates ALDH1+ stem cells.

Redox-active metal ions are pivotal for rapid metabolism, proliferation, and aggression across cancer types, and this presents metal chelation as an attractive cancer cell-targeting strategy. Here, we identify a metal chelator, KS10076, as a potent anti-cancer drug candidate. A metal-bound KS10076 complex with redox potential for generating hydrogen peroxide and superoxide anions induces intracellular reactive oxygen species (ROS). The elevation of ROS by KS10076 promotes the destabilization of signal transducer and activator of transcription 3, removes aldehyde dehydrogenase isoform 1-positive cancer stem cells, and subsequently induces autophagic cell death. Bioinformatic analysis of KS10076 susceptibility in pan-cancer cells shows that KS10076 potentially targets cancer cells with increased mitochondrial function. Furthermore, patient-derived organoid models demonstrate that KS10076 efficiently represses cancer cells with active KRAS, and fluorouracil resistance, which suggests clinical advantages.

Suppression of DYRK1A/B Drives Endoplasmic Reticulum Stress-mediated Autophagic Cell Death Through Metabolic Reprogramming in Colorectal Cancer Cells.

BACKGROUND/AIM: We previously identified KS40008 (4-(3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzene-1,2-diol), a novel inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase family (DYRK) 1A/B, which exhibited high enzymatic activity and cell proliferation-inhibitory effects in colorectal cancer (CRC) cell lines. In the present study, we aimed to elucidate the antitumor mechanisms of KS40008. MATERIALS AND METHODS: To assess the cytotoxicity of KS40008, we utilized a human cell line and organoid model and performed a CCK-8 assay and real-time cell analysis. Mitochondrial function was determined through mitochondrial staining, mito-stress test, and glycolysis test. In addition, we investigated the mechanisms of cancer cell death induced by KS40008 through immunoblotting, real-time quantitative polymerase chain reaction, reactive oxygen species staining, and immunofluorescence staining. RESULTS: KS40008 exhibited significant cytotoxicity in CRC and non-CRC cell lines, and organoid models compared to 5-fluorouracil, a conventional chemotherapeutic drug. Moreover, KS40008-induced inhibition of DYRK1A/B led to mitochondrial dysfunction and endoplasmic reticulum stress, promoting autophagic cancer cell death. CONCLUSION: KS40008 exerts antitumor activity through the inhibition of DYRK1A/B. Here, we demonstrated a mechanism by which KS40008 affects endoplasmic reticulum stress-mediated autophagy through the induction of mitochondrial stress, leading to cytotoxicity in CRC.

Synthesis of novel 1H-Pyrazolo[3,4-b]pyridine derivatives as DYRK 1A/1B inhibitors.

As DYRK1A and 1B inhibitors, 1H-pyrazolo[3,4-b]pyridine derivatives were synthesized. Mostly, 3-aryl-5-arylamino compounds (6) and 3,5-diaryl compounds (8 and 9) were prepared and especially, 3,5-diaryl compound 8 and 9 showed excellent DYRK1B inhibitory enzymatic activities with IC50 Values of 3-287 nM. Among them, 3-(4-hydroxyphenyl), 5-(3,4-dihydroxyphenyl)-1H-pyrazolo[3,4-b]pyridine (8h) exhibited the highest inhibitory enzymatic activity (IC50 = 3 nM) and cell proliferation inhibitory activity (IC50 = 1.6 µM) towards HCT116 colon cancer cells. Also compound 8h has excellent inhibitory activities in patient-derived colon cancer organoids model as well as in 3D spheroid assay model of SW480 and SW620. The docking study supported that we confirmed that compound 8h binds to DYRK1B through various hydrogen bonding interactions and hydrophobic interactions.

Synthesis of 1H-Indazoles via Silver(I)-Mediated Intramolecular Oxidative C-H Bond Amination.

We described a silver(I)-mediated intramolecular oxidative C-H amination that enables the construction of assorted 1H-indazoles that are widely applicable in medicinal chemistry. The developed amination was found to be efficient for the synthesis of a variety of 3-substituted indazoles that are otherwise difficult to be synthesized by other means of C-H aminations. Preliminary mechanistic studies suggested that the current amination proceeds via single electron transfer (SET) mediated by Ag(I) oxidant.

Solution-Based Indium-Zinc Oxide/Indium-Gallium-Zinc Oxide Double-Channel Thin-Film Transistors with Incorporated Hydrogen Peroxide.

We fabricated and evaluated solution-based double-channel thin-film transistors (TFTs) that consisted of an indium-zinc oxide (IZO) front layer and an indium-gallium-zinc oxide (IGZO) back channel with the addition of hydrogen peroxide (H2O2). The devices showed superior electrical properties with regard to saturation mobility (12.9 cm2/V·s), the on-off ratio (5 × 107), and the subthreshold swing (0.21 V/decade). All the devices were subjected under bias and illumination stress for reliability assessment. The threshold voltage shift stability of positive and negative bias illumination stress under different wavelengths was also enhanced. Thus, we achieved improved performance using IZO/IGZO TFTs with back channels that incorporated H2O2.

Hafnium Incorporation in InZnO Thin Film Transistors as a Carrier Suppressor.

In this study, we fabricated Hf-doped indium zinc oxide thin-film transistors (HIZO TFTs) using a solution process. Channel layers of the TFTs were optimized by varying the molar ratio of Hf in the channel layers. The electrical properties of the fabricated devices were compared to gallium indium zinc oxide (GIZO). HIZO TFTs showed 0.12 V threshold voltage, 0.45 V/decade subthreshold swing and 1.24 × 106 on-off current ratio, which were excellent compared to that of GIZO. In particular, when a positive gate bias stress of 10 V was applied for 10³ s, the HIZO TFT exhibited a lower threshold voltage shift of 1.11 V than the GIZO TFT (1.88 V). These results originate from the higher oxygen bonding with Hf in IZO compared to Ga atoms. We confirmed that Hf acts as an excellent carrier suppressor whose properties exceed those of Ga.

Defect Analysis and Reliability Characteristics of (HfZrO4)1-x(SiO2)x High-κ Dielectrics.

Hafnium zirconium silicon oxide ((HfZrO4)1-x(SiO2)x) materials were investigated through the defect analysis and reliability characterization for next generation high-κ dielectric. Silicate doped hafnium zirconium oxide (HfZrO4) films showed a reduction of negative flat-band voltage (Vfb) shift compared to pure HfZrO4. This result was caused by a decrease in donor-like interface traps (Dit) and positive border traps (Nbt). As the silicon oxide (SiO2) content increased, the Vfb was shifted in the positive direction from -1.23 to -1.10 to -0.91 V and the slope of the capacitance-voltage (C-V) curve increased. The nonparallel shift of the C-V characteristics was affected by the Dit, while the Nbt was responsible for the parallel C-V curve shift. The values of Dit reduced from 4.3 × 1011, 3.5 × 1011, and 3.0 × 1011 cm-2eV-1, as well as the values of Nbt were decreased from 5.24, 3.90 to 2.26 × 1012 cm-2. Finally, reduction of defects in the HfZrO4-base film with an addition of SiO2 affected the gate oxide reliability characteristics, such as gate leakage current (JG), bias temperature stress instability (BTSI), and time dependent gate dielectric breakdown (TDDB).

Flagellin-Stimulated Production of Interferon-ß Promotes Anti-Flagellin IgG2c and IgA Responses.

Flagellin, a major structural protein of the flagellum found in all motile bacteria, activates the TLR5- or NLRC4 inflammasomedependent signaling pathway to induce innate immune responses. Flagellin can also serve as a specific antigen for the adaptive immune system and stimulate anti-flagellin antibody responses. Failure to recognize commensal-derived flagellin in TLR5-deficient mice leads to the reduction in antiflagellin IgA antibodies at steady state and causes microbial dysbiosis and mucosal barrier breach by flagellated bacteria to promote chronic intestinal inflammation. Despite the important role of anti-flagellin antibodies in maintaining the intestinal homeostasis, regulatory mechanisms underlying the flagellin-specific antibody responses are not well understood. In this study, we show that flagellin induces interferon-ß (IFN-ß) production and subsequently activates type I IFN receptor signaling in a TLR5- and MyD88-dependent manner in vitro and in vivo . Internalization of TLR5 from the plasma membrane to the acidic environment of endolysosomes was required for the production of IFN-ß, but not for other proinflammatory cytokines. In addition, we found that antiflagellin IgG2c and IgA responses were severely impaired in interferon-alpha receptor 1 (IFNAR1)-deficient mice, suggesting that IFN-ß produced by the flagellin stimulation regulates anti-flagellin antibody class switching. Our findings shed a new light on the regulation of flagellin-mediated immune activation and may help find new strategies to promote the intestinal health and develop mucosal vaccines.

HCMV-encoded US7 and US8 act as antagonists of innate immunity by distinctively targeting TLR-signaling pathways.

The mechanisms by which many human cytomegalovirus (HCMV)-encoded proteins help the virus to evade immune surveillance remain poorly understood. In particular, it is unknown whether HCMV proteins arrest Toll-like receptor (TLR) signaling pathways required for antiviral defense. Here, we report that US7 and US8 as key suppressors that bind both TLR3 and TLR4, facilitating their destabilization by distinct mechanisms. US7 exploits the ER-associated degradation components Derlin-1 and Sec61, promoting ubiquitination of TLR3 and TLR4. US8 not only disrupts the TLR3-UNC93B1 association but also targets TLR4 to the lysosome, resulting in rapid degradation of the TLR. Accordingly, a mutant HCMV lacking the US7-US16 region has an impaired ability to hinder TLR3 and TLR4 activation, and the impairment is reversed by the introduction of US7 or US8. Our findings reveal an inhibitory effect of HCMV on TLR signaling, which contributes to persistent avoidance of the host antiviral response to achieve viral latency.

Hypoxia-Triggered Transforming Immunomodulator for Cancer Immunotherapy via Photodynamically Enhanced Antigen Presentation of Dendritic Cell.

A key factor for successful cancer immunotherapy (CIT) is the extent of antigen presentation by dendritic cells (DCs) that phagocytize tumor-associated antigens (TAA) in the tumor site and migrate to tumor draining lymph nodes (TDLN) for the activation of T cells. Although various types of adjuvant delivery have been studied to enhance the activity of the DCs, poor delivery efficiency and depleted population of tumor infiltrating DCs have limited the efficacy of CIT. Herein, we report a hypoxia-responsive mesoporous silica nanocarrier (denoted as CAGE) for an enhanced CIT assisted by photodynamic therapy (PDT). In this study, CAGE was designed as a hypoxia-responsive transforming carrier to improve the intracellular uptake of nanocarriers and the delivery of adjuvants to DCs. Furthermore, PDT was exploited for the generation of immunogenic debris and recruitment of DCs in a tumor site, followed by enhanced antigen presentation. Finally, a significant inhibition of tumor growth was observed in vivo, signifying that the PDT would be a promising solution for DC-based immunotherapy.

Two-photon microscopy of Paneth cells in the small intestine of live mice.

Paneth cells are one of the principal epithelial cell types in the small intestine, located at the base of intestinal crypts. Paneth cells play key roles in intestinal host-microbe homeostasis via granule secretion, and their dysfunction is implicated in pathogenesis of several diseases including Crohn's disease. Despite their physiological importance, study of Paneth cells has been hampered by the limited accessibility and lack of labeling methods. In this study, we developed a simple in vivo imaging method of Paneth cells in the intact mouse small intestine by using moxifloxacin and two-photon microscopy (TPM). Moxifloxacin, an FDA-approved antibiotic, was used for labeling cells and its fluorescence was strongly observed in Paneth cell granules by TPM. Moxifloxacin labeling of Paneth cell granules was confirmed by molecular counterstaining. Comparison of Paneth cells in wild type, genetically obese (ob/ob), and germ-free (GF) mice showed different granule distribution. Furthermore, Paneth cell degranulation was observed in vivo. Our study suggests that TPM with moxifloxacin labeling can serve as a useful tool for studying Paneth cell biology and related diseases.

Human cytomegalovirus-encoded US9 targets MAVS and STING signaling to evade type I interferon immune responses.

Human cytomegalovirus (HCMV) has evolved sophisticated immune evasion mechanisms that target both the innate and adaptive immune responses. However, how HCMV encoded proteins are involved in this immune escape is not clear. Here, we show that HCMV glycoprotein US9 inhibits the IFN-ß response by targeting the mitochondrial antiviral-signaling protein (MAVS) and stimulator of interferon genes (STING)-mediated signaling pathways. US9 accumulation in mitochondria attenuates the mitochondrial membrane potential, leading to promotion of MAVS leakage from the mitochondria. Furthermore, US9 disrupts STING oligomerization and STING-TBK1 association through competitive interaction. Intriguingly, US9 blocks interferon regulatory factor 3 (IRF3) nuclear translocation and its cytoplasmic domain is essential for inhibiting IRF3 activation. Mutant HCMV lacking US7-16 is impaired in antagonism of MAVS/STING-mediated IFN-ß expression, an effect that is reversible by the introduction of US9. Our findings indicate that HCMV US9 is an antagonist of IFN signaling to persistently evade host innate antiviral responses.

Correction of a pathogenic gene mutation in human embryos.

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.

Axl acts as a tumor suppressor by regulating LIGHT expression in T lymphoma.

Axl is an oncogenic receptor tyrosine kinase that plays a role in many cancers. LIGHT (Lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells) is a ligand that induces robust anti-tumor immunity by enhancing the recruitment and activation of effector immune cells at tumor sites. We observed that mouse EL4 and human Jurkat T lymphoma cells that stably overexpressed Axl also showed high expression of LIGHT. When Jurkat-Axl cells were treated with Gas6, a ligand for Axl, LIGHT expression was upregulated through activation of the PI3K/AKT signaling pathway and transcriptional induction by Sp1. The lytic activity of cytotoxic T lymphocytes and natural killer cells was enhanced by EL4-Axl cells. In addition, tumor volume and growth were markedly reduced due to enhanced apoptotic cell death in EL4-Axl tumor-bearing mice as compared to control mice. We also observed upregulated expression of CCL5 and its receptor, CCR5, and enhanced intratumoral infiltration of cytotoxic T lymphocytes and natural killer cells in EL4-Axl-bearing mice as compared to mock controls. These data strongly suggested that Axl exerts novel tumor suppressor effects by inducing upregulation of LIGHT in the tumor microenvironment of T lymphoma.

Gut-Specific Delivery of T-Helper 17 Cells Reduces Obesity and Insulin Resistance in Mice.

BACKGROUND & AIMS: Obesity and metabolic syndrome have been associated with alterations to the intestinal microbiota. However, few studies examined the effects of obesity on the intestinal immune system. We investigated changes in subsets of intestinal CD4+ T-helper (TH) cells with obesity and the effects of gut-tropic TH17 cells in mice on a high-fat diet (HFD). METHODS: We isolated immune cells from small intestine and adipose tissue of C57BL/6 mice fed a normal chow diet or a HFD for 10 weeks and analyzed the cells by flow cytometry. Mice fed a vitamin A-deficient HFD were compared with mice fed a vitamin A-sufficient HFD. Obese RAG1-deficient mice were given injections of only regulatory T cells or a combination of regulatory T cells and TH17 cells (wild type or deficient in integrin ß7 subunit or interleukin 17 [IL17]). Mice were examined for weight gain, fat mass, fatty liver, glucose tolerance, and insulin resistance. Fecal samples were collected before and after T cell transfer and analyzed for microbiota composition by metagenomic DNA sequencing and quantitative polymerase chain reaction. RESULTS: Mice placed on a HFD became obese, which affected the distribution of small intestinal CD4+ TH cells. Intestinal tissues from obese mice had significant reductions in the proportion of TH17 cells but increased proportion of TH1 cells, compared with intestinal tissues from nonobese mice. Depletion of vitamin A in obese mice further reduced the proportion of TH17 cells in small intestine; this reduction correlated with more weight gain and worsening of glucose intolerance and insulin resistance. Adoptive transfer of in vitro-differentiated gut-tropic TH17 cells to obese mice reduced these metabolic defects, which required the integrin ß7 subunit and IL17. Delivery of TH17 cells to intestines of mice led to expansion of commensal microbes associated with leanness. CONCLUSIONS: In mice, intestinal TH17 cells contribute to development of a microbiota that maintains metabolic homeostasis, via IL17. Gut-homing TH17 cells might be used to reduce metabolic disorders in obese individuals.

CNBP acts as a key transcriptional regulator of sustained expression of interleukin-6.

The transcription of inflammatory genes is an essential step in host defense activation. Here, we show that cellular nucleic acid-binding protein (CNBP) acts as a transcription regulator that is required for activating the innate immune response. We identified specific CNBP-binding motifs present in the promoter region of sustained inflammatory cytokines, thus, directly inducing the expression of target genes. In particular, lipopolysaccharide (LPS) induced cnbp expression through an NF-κB-dependent manner and a positive autoregulatory mechanism, which enables prolonged il-6 gene expression. This event depends strictly on LPS-induced CNBP nuclear translocation through phosphorylation-mediated dimerization. Consequently, cnbp-depleted zebrafish are highly susceptible to Shigella flexneri infection in vivo. Collectively, these observations identify CNBP as a key transcriptional regulator required for activating and maintaining the immune response.