Second-Generation AUTACs for Targeted Autophagic Degradation.

Targeted protein degradation via the ubiquitin-proteasome system has emerged as one of the most promising drug discovery modalities. Autophagy, another intracellular degradation system, can target a wide range of nonproteinous substrates as well as proteins, but its application to targeted degradation is still in its infancy. Our previous work revealed a relationship between guanine modification of cysteine residues on intracellular proteins and selective autophagy, resulting in the first autophagy-based degraders, autophagy-targeted chimeras (AUTACs). Based on the research background, all the reported AUTACs compounds contain cysteine as a substructure. Here, we examine the importance of this substructure by conducting SAR studies and report significant improvements in the degrader's activity by replacing cysteine with other moieties. Several derivatives showed sub-µM range degrading activity, demonstrating the increased practical value of AUTACs.

Effects of Streamer Discharge on PM2.5 Containing Endotoxins and Polyaromatic Hydrocarbons and Their Biological Responses In Vitro.

Experimental and epidemiological studies have demonstrated that fine particulate matter with a diameter of <2.5 µm (PM2.5) affects both the respiratory and immune systems. However, effective approaches to reduce PM2.5-induced hazardous effects have not been discovered yet. Streamer discharge is a category of plasma discharge in which high-speed electrons collide with oxygen and nitrogen molecules. Although streamer discharge can reportedly eliminate bacteria, molds, chemical substances, and allergens, its ability to decontaminate PM2.5 has not been previously demonstrated. The present study explored whether streamer discharge treatment could reduce PM2.5-induced inflammatory responses by employing an in vitro system. PM2.5 was collected under four conditions (Bangkok (Sep.−Dec.), Bangkok (Dec.−Mar.), Singapore, and Taipei). Airway epithelial cells and antigen-presenting cells exposed to non-treated PM2.5 in several conditions resulted in inflammatory responses. Streamer-discharged PM2.5 (Bangkok (Sep.−Dec.)) decreased the expression of interleukin (IL)-6 and IL-8 compared to non-treated PM2.5. Moreover, composition analysis demonstrated that streamer discharge reduced some compounds, such as endotoxins and polycyclic aromatic hydrocarbons, included in PM2.5 that can elicit inflammatory responses. Streamer discharge treatment can reduce endotoxins, polycyclic aromatic hydrocarbons, and the subsequent inflammatory responses induced by PM2.5 in vitro.

[Hemobilia due to ruptured superior pancreaticoduodenal pseudoaneurysm after chemoradiotherapy for pancreatic head cancer: an autopsy case report].

A 78-year-old man came to our department because of obstructive jaundice, and was diagnosed as pancreatic head cancer. He underwent chemoradiation therapy. A metal stent was inserted into the common bile duct and the patient was followed up on an outpatient basis. The patient visited our emergency department 46 days after stent insertion due to abdominal pain. The patient was diagnosed with ruptured pseudoaneurysm of the superior pancreaticoduodenal artery by angiography and treated with coil embolization. He died due to sudden deterioration the next day. Pathological autopsy revealed that the cause of the ruptured pseudoaneurysm appeared to be vasculopathy due to radiation therapy.

Anti-nucleocapsid antibodies enhance the production of IL-6 induced by SARS-CoV-2 N protein.

A cytokine storm induces acute respiratory distress syndrome, the main cause of death in coronavirus disease 2019 (COVID-19) patients. However, the detailed mechanisms of cytokine induction due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unclear. To examine the cytokine production in COVID-19, we mimicked the disease in SARS-CoV-2-infected alveoli by adding the lysate of SARS-CoV-2-infected cells to cultured macrophages or induced pluripotent stem cell-derived myeloid cells. The cells secreted interleukin (IL)-6 after the addition of SARS-CoV-2-infected cell lysate. Screening of 25 SARS-CoV-2 protein-expressing plasmids revealed that the N protein-coding plasmid alone induced IL-6 production. The addition of anti-N antibody further enhanced IL-6 production, but the F(ab')2 fragment did not. Sera from COVID-19 patients also enhanced IL-6 production, and sera from patients with severer disease induced higher levels of IL-6. These results suggest that anti-N antibody promotes IL-6 production in SARS-CoV-2-infected alveoli, leading to the cytokine storm of COVID-19.

Clinical trials of Cry j 1 and Cry j 2 T-cell epitope peptide-expressing rice in patients with Japanese cedar pollinosis.

BACKGROUND: In allergic models, administration of rice that expresses a hybrid peptide consisting of 7 major T cell epitopes of Cry j 1 and Cry j 2 (7Crp), suppressed allergic symptoms, IgE elevation and specific T cell response to Japanese cedar pollen. OBJECTIVE: To evaluate the efficacy and safety of 7Crp-expressing rice in patients with Japanese cedar pollinosis. METHODS: A 24-week randomized, double-blind, placebo-controlled study was performed to see the efficacy of 7Crp on allergic symptoms using scoring systems, in which 45 patients were assigned to take either 5 g, 20 g test rice, or placebo daily. A 96-week open study was also conducted to determine its inhibitory effect on serum IgE and T cell proliferative response for Japanese cedar pollen, in which 10 patients consumed 5 g test rice daily. RESULTS: No adverse events associated with the test rice occurred, and the intake rate was more than 96%. The test rice did not show suppression of symptoms related to Japanese cedar pollinosis within 24 weeks. However, intake of 5 g test rice led to a significant decrease in T cell response to Japanese cedar pollen during and after the second disperse season in a 96-week open trial, whereas the specific IgE titer remained unchanged. CONCLUSIONS: Tolerability and safety of 7Crp-expressing rice was accepted. Daily intake of up to 20 g transgenic rice did not provide beneficial effects on Japanese cedar pollinosis within 24 weeks, however, continuous intake of 5 g rice might reduce allergen specific T cell response.

[A Case of Laparoscopic Surgery for Sigmoid Lymph Node Metastases after Surgery for Rectal Cancer].

Herein, we report a case of laparoscopic surgery for sigmoid lymph node metastases after surgery for rectal cancer. A 58- year-old man underwent laparoscopic surgery for rectal cancer. He underwent D2 lymph node dissection, and he was undergoing dialysis for renal disease as a complication of diabetes. CT imaging performed 15 months after surgery revealed recurrence of tumors in the sigmoid lymph nodes. Subsequently, laparoscopic removal of the sigmoid lymph nodes was planned, as the patient had no tumor recurrence at any other location, and because his condition was not suitable for chemotherapy. The postoperative course was uneventful, and the patient was discharged a few days after surgery.

Adipocytokine Profile Reveals Resistin Forming a Prognostic-Related Cytokine Network in the Acute Phase of Sepsis.

INTRODUCTION: Cytokines compose a network and play crucial roles in the pathogenesis and prognosis of sepsis. Adipose tissue is an important immune endocrine organ that releases adipocytokines. This study aimed to evaluate adipocytokines in sepsis from a network perspective. MATERIALS AND METHODS: This retrospective study of 37 patients with sepsis and 12 healthy controls was conducted from February 2014 to July 2015. Blood samples were collected from patients on days 1 (within 24 h of diagnosis), 2, 4, 6, 8, 11, and 15 and from healthy controls. Adipocytokines (adiponectin, leptin, resistin, chemerin, visfatin, vaspin, CXCL-12/SDF-1, angiotensinogen), inflammatory cytokines (IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-12/IL-23p40, TNF-α, monocyte chemotactic protein [MCP-1]), and plasminogen activator inhibitor-1 were measured. Acute Physiology and Chronic Health Evaluation II score was evaluated on day 1, and Sequential Organ Failure Assessment (SOFA) score and Japanese Association for Acute Medicine (JAAM) and International Society of Thrombosis and Hemostasis overt disseminated intravascular coagulation (DIC) scores were assessed at the times of blood sampling. RESULTS: Hierarchical clustering analysis showed the cluster formed by resistin, IL-6, IL-8, MCP-1, and IL-10 on days 1, 2, and 4 represented the cytokine network throughout the acute phase of sepsis. Each cytokine in this network was significantly associated with SOFA and JAAM DIC scores over the acute phase. A Cox proportional hazards model focusing on the acute phase showed a significant relation of these five cytokines with patient prognosis. CONCLUSIONS: Adipocytokines and an inflammatory cytokine profile assessed over time in sepsis patients showed that resistin was involved in an inflammatory cytokine network including IL-6, IL-8, IL-10, and MCP-1 in the acute phase of sepsis, and this network was associated with severity and prognosis of sepsis.

Gap junction protein beta 4 plays an important role in cardiac function in humans, rodents, and zebrafish.

AIMS: GJB4 encodes a transmembrane connexin protein (Cx30.3) that is a component of gap junctions. This study investigated whether GJB4 plays an important role in human heart disease and function. METHODS AND RESULTS: We examined a patient and her older brother who both presented with complicated severe hypertrophic cardiomyopathy (HCM) and whose parents are healthy married cousins. The gene exome analysis showed 340 single nucleotide polymorphisms (SNPs) that caused amino acid changes for which the patient was homozygous and both parents were heterozygous. After excluding all known common (>10%) SNP gene mutations, the gene for GJB4 was the only identified gene that is possibly associated with cardiac muscle. The resultant E204A substitution exists in the 4th transmembrane domain. GJB4-E204A impaired the binding with gap junction protein A1 (GJA1) compared with GJB4-WT. The expression of GJB4 was induced in rat disease models of left and right ventricle hypertrophy and mouse disease models of adriamycin-induced cardiomyopathy and myocardial infarction, while it was not detected at all in control. An immunohistochemical study was performed for autopsied human hearts and the explanted heart of the patient. GJB4 was expressed and colocalized with GJA1 in intercalated discs in human diseased hearts, which was extensively enhanced in the explanted heart of the patient. The abnormal expression and localization of GJB4 were observed in beating spheres of patient's induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). We generated knockout zebrafish of GJB4 by CRISPR/Cas9 and the endodiastolic volume and the ventricular ejection fraction were significantly lower in GJB4-deficient than in wild-type zebrafish at five days post-fertilization. CONCLUSIONS: These results indicate both that GJB4 is defined as a new connexin in diseased hearts, of which mutation can cause a familial form of HCM, and that GJB4 may be a new target for the treatment of cardiac hypertrophy and dysfunction.

IL-6 trans-signaling induces plasminogen activator inhibitor-1 from vascular endothelial cells in cytokine release syndrome.

Cytokine release syndrome (CRS) is a life-threatening complication induced by systemic inflammatory responses to infections, including bacteria and chimeric antigen receptor T cell therapy. There are currently no immunotherapies with proven clinical efficacy and understanding of the molecular mechanisms of CRS pathogenesis is limited. Here, we found that patients diagnosed with CRS from sepsis, acute respiratory distress syndrome (ARDS), or burns showed common manifestations: strikingly elevated levels of the four proinflammatory cytokines interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and IL-10 and the coagulation cascade activator plasminogen activator inhibitor-1 (PAI-1). Our in vitro data indicate that endothelial IL-6 trans-signaling formed an inflammation circuit for robust IL-6, IL-8, and MCP-1 production and promoted PAI-1 production; additionally, an IL-6 signaling blockade by the human monoclonal antibody tocilizumab blunted endothelial cell activation. Plasma from severe COVID-19 patients similarly exhibited increased IL-6, IL-10, and MCP-1 levels, but these levels were not as high as those in patients with CRS from other causes. In contrast, the PAI-1 levels in COVID-19 patients were as highly elevated as those in patients with bacterial sepsis or ARDS. Tocilizumab treatment decreased the PAI-1 levels and alleviated critical illness in severe COVID-19 patients. Our findings suggest that distinct levels of cytokine production are associated with CRS induced by bacterial infection and COVID-19, but both CRS types are accompanied by endotheliopathy through IL-6 trans-signaling. Thus, the present study highlights the crucial role of IL-6 signaling in endothelial dysfunction during bacterial infection and COVID-19.

Significance of Quantitative Interferon-gamma Levels in Non-small-cell Lung Cancer Patients' Response to Immune Checkpoint Inhibitors.

BACKGROUND/AIM: We aimed to study the association between the quantitative interferon-gamma (IFN-γ) levels and clinical outcomes in non-small-cell lung cancer (NSCLC) patients receiving immune checkpoint inhibitors (ICIs). PATIENTS AND METHODS: Sample collection for IFN-γ release assay (IGRA) was performed within 14 days before treatment (T1), on day 22±7 (T3), and on day 43±7 (T4). The stored specimens over 10 IU/ml in IGRA were re-examined using the dilution method (with saline as the dilution medium). The patients were classified into Lower and Higher groups by 7.06 IU/ml as a cut-off of IFN-γ levels at T1. RESULTS: Median progression-free survival in the Higher group was significantly longer than that in the Lower group. IFN-γ levels in the non-progression disease group were significantly higher than those in the progression disease group. IFN-γ levels at T1 in patients with immune-related adverse events were significantly lower compared to those at T3. CONCLUSION: IFN-γ could be a biomarker for NSCLC patients receiving ICIs.

Noncanonical STAT1 phosphorylation expands its transcriptional activity into promoting LPS-induced IL-6 and IL-12p40 production.

The lipopolysaccharide (LPS)-induced endocytosis of Toll-like receptor 4 (TLR4) is an essential step in the production of interferon-ß (IFN-ß), which activates the transcription of antiviral response genes by STAT1 phosphorylated at Tyr701 Here, we showed that STAT1 regulated proinflammatory cytokine production downstream of TLR4 endocytosis independently of IFN-ß signaling and the key proinflammatory regulator NF-κB. In human macrophages, TLR4 endocytosis activated a noncanonical phosphorylation of STAT1 at Thr749, which subsequently promoted the production of interleukin-6 (IL-6) and IL-12p40 through distinct mechanisms. STAT1 phosphorylated at Thr749 activated the expression of the gene encoding ARID5A, which stabilizes IL6 mRNA. Moreover, STAT1 phosphorylated at Thr749 directly enhanced transcription of the gene encoding IL-12p40 (IL12B). Instead of affecting STAT1 nuclear translocation, phosphorylation of Thr749 facilitated the binding of STAT1 to a noncanonical DNA motif (5'-TTTGANNC-3') in the promoter regions of ARID5A and IL12B The endocytosis of TLR4 induced the formation of a complex between the kinases TBK1 and IKKß, which mediated the phosphorylation of STAT1 at Thr749 Our data suggest that noncanonical phosphorylation in response to LPS confers STAT1 with distinct DNA binding and gene-regulatory properties that promote both IL12B expression and IL6 mRNA stabilization. Thus, our study provides a potential mechanism for how TLR4 endocytosis might regulate proinflammatory cytokine production.

Identification of a selective DDX3X inhibitor with newly developed quantitative high-throughput RNA helicase assays.

The DEAD-box family of RNA helicases plays essential roles in both transcriptional and translational mRNA degradation; they unwind short double-stranded RNA by breaking the RNA-RNA interactions. Two DEAD-box RNA helicases, eukaryotic translation initiation factor 4A3 (eIF4A3) and DEAD-box helicase 3 (DDX3X), show high homology in the ATP-binding region and are considered key molecules for cancer progression. Several small molecules that target eIF4A3 and DDX3X have been reported to inhibit cancer cell growth; however, more potent compounds are required for cancer therapeutics, and there is a critical need for high-throughput assays to screen for RNA helicase inhibitors. In this study, we developed novel fluorescence resonance energy transfer-based high-throughput RNA helicase assays for eIF4A3 and DDX3X. Using these assays, we identified several eIF4A3 allosteric inhibitors whose inhibitory effect on eIF4A3 ATPase showed a strong correlation with inhibitory effect on helicase activity. From 102 compounds that exhibited eIF4A3 ATPase inhibition, we identified a selective DDX3X inhibitor, C1, which showed stronger inhibition of DDX3X than of eIF4A3. Small-molecule helicase inhibitors can be valuable for clarifying the molecular machinery of DEAD-box RNA helicases. The high-throughput quantitative assays established here should facilitate the evaluation of the helicase inhibitory activity of compounds.

Deletion of the Golgi Ca2+-ATPase PMR1 gene potentiates antifungal effects of dodecanol that depend on intracellular Ca2+ accumulation in budding yeast.

One strategy for overcoming infectious diseases caused by drug-resistant fungi involves combining drugs rendered inactive by resistance with agents targeting the drug resistance mechanism. The antifungal activity of n-dodecanol disappears as incubation time passes. In Saccharomyces cerevisiae, anethole, a principal component of anise oil, prolongs the transient antifungal effect of dodecanol by downregulating genes of multidrug efflux pumps, mainly PDR5. However, the detailed mechanisms of dodecanol's antifungal action and the anethole-induced prolonged antifungal action of dodecanol are unknown. Screening of S. cerevisiae strains lacking genes related to Ca2+ homeostasis and signaling identified a pmr1Δ strain lacking Golgi Ca2+-ATPase as more sensitive to dodecanol than the parental strain. Dodecanol and the dodecanol + anethole combination significantly increased intracellular Ca2+ levels in both strains, but the mutant failed to clear intracellular Ca2+ accumulation. Further, dodecanol and the drug combination reduced PMR1 expression and did not lead to specific localization of Pmr1p in the parental strain after 4-h treatment. By contrast with the parental strain, dodecanol did not stimulate PDR5 expression in pmr1Δ. Based on these observations, we propose that the antifungal activity of dodecanol is related to intracellular Ca2+ accumulation, possibly dependent on PMR1 function, with anethole enabling Ca2+ accumulation by restricting dodecanol efflux.

[A Case of Small Intestinal Cancer Resected by Laparoscopic Surgery].

We report a case of laparoscopic surgical resection of a small intestinal cancer. A woman in her 40s was referred to our department for prolonged abdominal problems(epigastralgia, nausea, diarrhea, and constipation). CT scan revealed a small intestinal tumor with dilatation of the oral side of the intestine. She was admitted to our hospital, and an ileus tube was introduced. One week after admission, she experienced laparoscopic partial resection of the small intestine. She was soon discharged without any problems and has had no recurrence of small intestinal cancer after 8 months of surgery without any adjuvant chemotherapy. Small intestinal cancer is frequently detected in an advanced stage, resulting in poor prognosis, but curative surgery can improve the prognosis. Optimal therapy for small intestinal cancer has not been established yet because it is rare. A multi-centered study of small intestinal cancer for the establishment of its diagnosis and therapy needs to be conducted.

The Levels of Interferon-gamma Release as a Biomarker for Non-small-cell Lung Cancer Patients Receiving Immune Checkpoint Inhibitors.

BACKGROUND/AIM: The present study aimed to prospectively examine the usefulness of interferon-gamma (IFN-γ) release (IGR) as a biomarker in non-small-cell lung cancer patients receiving immune checkpoint inhibitor treatment (ICI-Tx). PATIENTS AND METHODS: IGR was measured using enzyme-linked immunosorbent assay at four time points: within 14 days before ICI-Tx (T1), and 8±3 (T2), 22±7 (T3), and 43±7 (T4) days after ICI-Tx. RESULTS: Twenty-nine patients were divided into three groups based on IFN-γ levels in the IGR-positive control: Group-1 (n=8) with <10 IU/ml at T1, Group-2 (n=12) with a decrease in IFN-γ levels to <10 IU/ml at T3 and/or T4, and Group-3 (n=9) without changes in IFN-γ levels. Early progression and ICI-induced interstitial pneumonitis were frequently observed in Group-1 and Group-2, respectively. Group-3 exhibited more treatment cycles than the other groups. All three groups showed clear differences in clinical outcomes. CONCLUSION: IFN-γ levels could be a biomarker for ICI-Tx.

[Next generation zebrafish-based drug discovery and precision medicine].

Even after entering the era of genomic drug discovery in the 21st century, development of a breakthrough therapeutic drug (first-in-class) for intractable diseases (unmet medical needs) has been extremely difficult, but to the US FDA 62% of the approved first-in-class drugs are found by phenotypic screening. The next-generation zebrafish drug discovery enables high-throughput quantitative live in vivo phenotypic screening, and has been impacting global drug discovery strategies now. Compared to severe immunodeficient mice, zebrafish is expected to become a true individualized medical tool as a clinical ex vivo diagnostic system because of the high efficiency and speed of engraftment of patient-derived cancer xenotransplantation. Phenomics-based personalized medicine with the patient-derived cancer xenograft zebrafish in addition to conventional omics platform of individualized medicine is a true next-generation precision medicine to utilize for selection of therapeutic drugs and decision of their doses for the patient, and emerging paradigm shift is realizing in this century.

Targeting Interleukin-6 Signaling in Clinic.

Interleukin-6 (IL-6) is a pleiotropic cytokine with roles in immunity, tissue regeneration, and metabolism. Rapid production of IL-6 contributes to host defense during infection and tissue injury, but excessive synthesis of IL-6 and dysregulation of IL-6 receptor signaling is involved in disease pathology. Therapeutic agents targeting the IL-6 axis are effective in rheumatoid arthritis, and applications are being extended to other settings of acute and chronic inflammation. Recent studies reveal that selective blockade of different modes of IL-6 receptor signaling has different outcomes on disease pathology, suggesting novel strategies for therapeutic intervention. However, some inflammatory diseases do not seem to respond to IL-6 blockade. Here, we review the current state of IL-6-targeting approaches in the clinic and discuss how to apply the growing understanding of the immunobiology of IL-6 to clinical decisions.

Toxicological Evaluation of SiO2 Nanoparticles by Zebrafish Embryo Toxicity Test.

As the use of nanoparticles (NPs) is increasing, the potential toxicity and behavior of NPs in living systems need to be better understood. Our goal was to evaluate the developmental toxicity and bio-distribution of two different sizes of fluorescently-labeled SiO2 NPs, 25 and 115 nm, with neutral surface charge or with different surface functionalization, rendering them positively or negatively charged, in order to predict the effect of NPs in humans. We performed a zebrafish embryo toxicity test (ZFET) by exposing the embryos to SiO2 NPs starting from six hours post fertilization (hpf). Survival rate, hatching time, and gross morphological changes were assessed at 12, 24, 36, 48, 60, and 72 hpf. We evaluated the effect of NPs on angiogenesis by counting the number of sub-intestinal vessels between the second and seventh intersegmental vessels and gene expression analysis of vascular endothelial growth factor (VEGF) and VEGF receptors at 72 hpf. SiO2 NPs did not show any adverse effects on survival rate, hatching time, gross morphology, or physiological angiogenesis. We found that SiO2 NPs were trapped by the chorion up until to the hatching stage. After chemical removal of the chorion (dechorionation), positively surface-charged SiO2 NPs (25 nm) significantly reduced the survival rate of the fish compared to the control group. These results indicate that zebrafish chorion acts as a physical barrier against SiO2 NPs, and removing the chorions in ZFET might be necessary for evaluation of toxicity of NPs.

Potential Beneficial Effects of Wine Flavonoids on Allergic Diseases.

Wine, a widely consumed beverage, comprises several biophenols that promote health. Flavonoids, majorly present in red wine, have been shown to have antioxidant, anti-inflammatory, anticancer, and immunomodulatory activities. Regular consumption of red wine (100 mL/day) is estimated to provide an average of 88 mg of flavonoids, whereas recent epidemiological studies indicate that wine is one of the major sources of flavonoid intake amongst wine lovers in European countries (providing an average intake of 291⁻374 mg/day of flavonoids). In addition to being antioxidants, in vitro studies suggest that flavonoids also have anti-allergic activities that inhibit IgE synthesis, activation of mast cells and basophils or other inflammatory cells, and production of inflammatory mediators, including cytokines. Furthermore, they affect the differentiation of naïve CD4+ T cells into effector T cell subsets. Moreover, several studies have reported the benefits of flavonoids in allergic models such as atopic dermatitis, asthma, anaphylaxis, and food allergy; however, evidence in humans is limited to allergic rhinitis and respiratory allergy. Although further evaluation is required, it is expected that an appropriate intake of flavonoids may be beneficial in preventing, and eventually managing, allergic diseases.