Structural basis for thioredoxin-mediated suppression of NLRP1 inflammasome.

Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis1. NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer2-6. However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated4,7,8. Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system.

Live-cell epigenome manipulation by synthetic histone acetylation catalyst system.

Chemical modifications of histones, such as lysine acetylation and ubiquitination, play pivotal roles in epigenetic regulation of gene expression. Methods to alter the epigenome thus hold promise as tools for elucidating epigenetic mechanisms and as therapeutics. However, an entirely chemical method to introduce histone modifications in living cells without genetic manipulation is unprecedented. Here, we developed a chemical catalyst, PEG-LANA-DSSMe 11, that binds with nucleosome's acidic patch and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells. The size of polyethylene glycol in the catalyst was a critical determinant for its in-cell metabolic stability, binding affinity to histones, and high activity. The synthetic acetylation promoted by 11 without genetic manipulation competed with and suppressed physiological H2B ubiquitination, a mark regulating chromatin functions, such as transcription and DNA damage response. Thus, the chemical catalyst will be a useful tool to manipulate epigenome for unraveling epigenetic mechanisms in living cells.

Epigenetic control of rDNA loci in response to intracellular energy status.

Intracellular energy balance is important for cell survival. In eukaryotic cells, the most energy-consuming process is ribosome biosynthesis, which adapts to changes in intracellular energy status. However, the mechanism that links energy status and ribosome biosynthesis is largely unknown. Here, we describe eNoSC, a protein complex that senses energy status and controls rRNA transcription. eNoSC contains Nucleomethylin, which binds histone H3 dimethylated Lys9 in the rDNA locus, in a complex with SIRT1 and SUV39H1. Both SIRT1 and SUV39H1 are required for energy-dependent transcriptional repression, suggesting that a change in the NAD(+)/NADH ratio induced by reduction of energy status could activate SIRT1, leading to deacetylation of histone H3 and dimethylation at Lys9 by SUV39H1, thus establishing silent chromatin in the rDNA locus. Furthermore, eNoSC promotes restoration of energy balance by limiting rRNA transcription, thus protecting cells from energy deprivation-dependent apoptosis. These findings provide key insight into the mechanisms of energy homeostasis in cells.

Contribution of pre-existing neoantigen-specific T cells to a durable complete response after tumor-pulsed dendritic cell vaccine plus nivolumab therapy in a patient with metastatic salivary duct carcinoma.

Although immune checkpoint inhibitors (ICIs) have emerged as new therapeutic options for refractory cancer, they are only effective in select patients. Tumor antigen-pulsed dendritic cell (DC) vaccine therapy activates tumor-specific cytotoxic T lymphocytes, making it an important immunotherapeutic strategy. Salivary ductal carcinoma (SDC) carries a poor prognosis, including poor long-term survival after metastasis or recurrence. In this study, we reported a case of refractory metastatic SDC that was treated with a tumor lysate-pulsed DC vaccine followed by a single injection of low-dose nivolumab, and a durable complete response was achieved. We retrospectively analyzed the immunological factors that contributed to these long-lasting clinical effects. First, we performed neoantigen analysis using resected metastatic tumor specimens obtained before treatment. We found that the tumor had 256 non-synonymous mutations and 669 class I high-affinity binding neoantigen peptides. Using synthetic neoantigen peptides and ELISpot analysis, we found that peripheral blood mononuclear leukocytes cryopreserved before treatment contained pre-existing neoantigen-specific T cells, and the cells obtained after treatment exhibited greater reactivity to neoantigens than those obtained before treatment. Our results collectively suggest that the rapid and long-lasting effect of this combination therapy in our patient may have resulted from the presence of pre-existing neoantigen-specific T cells and stimulation and expansion of those cells following tumor lysate-pulsed DC vaccine and ICI therapy.

Identification of a novel nucleolar protein complex required for mitotic chromosome segregation through centromeric accumulation of Aurora B.

The nucleolus is a membrane-less nuclear structure that disassembles when cells undergo mitosis. During mitosis, nucleolar factors are thus released from the nucleolus and dynamically change their subcellular localization; however, their functions remain largely uncharacterised. Here, we found that a nucleolar factor called nucleolar protein 11 (NOL11) forms a protein complex with two tryptophan-aspartic acid (WD) repeat proteins named WD-repeat protein 43 (WDR43) and Cirhin in mitotic cells. This complex, referred to here as the NWC (NOL11-WDR43-Cirhin) complex, exists in nucleoli during interphase and translocates to the periphery of mitotic chromosomes, i.e., perichromosomal regions. During mitotic progression, both the congression of chromosomes to the metaphase plate and sister chromatid cohesion are impaired in the absence of the NWC complex, as it is required for the centromeric enrichment of Aurora B and the associating phosphorylation of histone H3 at threonine 3. These results reveal the characteristics of a novel protein complex consisting of nucleolar proteins, which is required for regulating kinetochores and centromeres to ensure faithful chromosome segregation.

NFκB and TGFß contribute to the expression of PTPN3 in activated human lymphocytes.

We previously reported that protein tyrosine phosphatase non-receptor type 3 (PTPN3), which is upregulated in activated lymphocytes, acts as an immune checkpoint. However, the mechanism by which PTPN3 expression is enhanced in activated lymphocytes is unknown. In this study, we analyzed the mechanism of PTPN3 expression in activated lymphocytes with a view for developing a novel immune checkpoint inhibitor that suppresses PTPN3. Through the activation process, lymphocytes showed enhanced NFκB activation as well as increased PTPN3 expression. NFκB enhanced proliferation, migration, and cytotoxicity of lymphocytes. Furthermore, NFκB enhanced PTPN3 expression and tyrosine kinase activation. TGFß reduced PTPN3 expression and NFκB activation in the cancer microenvironment, and suppressed the biological activity of lymphocytes. The results of this study are expected to provide significant implications for improving existing immunotherapy and developing novel immunotherapy.

PTPN3 expressed in activated T lymphocytes is a candidate for a non-antibody-type immune checkpoint inhibitor.

It has been shown that protein tyrosine phosphatase non-receptor type (PTPN) 3 inhibits T-cell activation. However, there is no definitive conclusion about how the inhibition of PTPN3 in lymphocytes affects immune functions in human lymphocytes. In the present study, we showed that PTPN3 inhibition significantly contributes to the enhanced activation of activated human lymphocytes. The PTPN3 expression of lymphocytes was significantly increased through the activation process using IL-2 and anti-CD3 mAb. Interestingly, inhibiting the PTPN3 expression in activated lymphocytes significantly augmented the proliferation, migration, and cytotoxicity through the phosphorylation of zeta-chain-associated protein kinase 70 (ZAP-70), lymphocyte-specific protein tyrosine kinase (LCK), and extracellular signal-regulated kinases (ERK). Lymphocyte activation by PTPN3 inhibition was observed only in activated CD3+ T cells and not in NK cells or resting T cells. In therapy experiments using autologous tumors and lymphocytes, PTPN3 inhibition significantly augmented the number of tumor-infiltrated lymphocytes and the cytotoxicity of activated lymphocytes. Our results strongly imply that PTPN3 acts as an immune checkpoint in activated lymphocytes and that PTPN3 inhibitor may be a new non-antibody-type immune checkpoint inhibitor for cancer therapy.

Hedgehog signaling regulates PDL-1 expression in cancer cells to induce anti-tumor activity by activated lymphocytes.

We investigated whether hypoxia-induced activation of Hh signaling contributes to PDL-1 expression in cancer and whether it affects the anti-tumor function of activated lymphocytes. Hypoxia augmented PDL-1 expression and inhibition of Hh signaling reduced PDL-1 expression under hypoxia. When activated lymphocytes were cocultured with cancers treated with a Hh inhibitor, activated lymphocyte cell numbers increased under hypoxia. In contrast, this increase was abrogated when cancer cells were treated with a PDL-1 neutralizing antibody. These results suggest that Hh signaling is one of regulatory pathways of PDL-1 expression under hypoxia and that inhibiting Hh signaling may induce lymphocyte anti-tumor activity.

Inhibition of PTPN3 Expressed in Activated Lymphocytes Enhances the Antitumor Effects of Anti-PD-1 Therapy in Head and Neck Cancer, Especially in Hypoxic Environments.

In the tumor microenvironment, wherein cytotoxic lymphocytes interact with cancer cells, lymphocyte exhaustion, an immune checkpoint inhibitor target, is promoted. However, the efficacy of these inhibitors is limited, and improving response rates remains challenging. We previously reported that protein tyrosine phosphatase nonreceptor type (PTPN) 3 is a potential immune checkpoint molecule for activated lymphocytes and that PTPN3 inhibition should be a focus area for cancer immunotherapy development. Therefore, in this study, we focused on PTPN3-suppressive therapy in terms of lymphocyte exhaustion under hypoxic conditions, which are a cancer microenvironment, and investigated measures for improving the response to anti-programmed death receptor (PD)-1 antibody drugs. We found that PTPN3 expression was upregulated in activated lymphocytes under hypoxic conditions, similar to the findings for other immune checkpoint molecules, such as PD-1, T cell immunoglobulin mucin-3, and lymphocyte-activation gene-3; furthermore, it functioned as a lymphocyte exhaustion marker. In addition, PTPN3-suppressed activated lymphocytes promoted the mammalian target of rapamycin (mTOR)-Akt signaling pathway activation and enhanced proliferation, migration, and cytotoxic activities under hypoxic conditions. Furthermore, PTPN3 suppression in activated lymphocytes increased PD-1 expression and enhanced the antitumor effects of anti-PD-1 antibody drugs against head and neck cancer in vitro and in vivo. These results suggest that the suppression of PTPN3 expression in activated lymphocytes enhances the therapeutic effect of anti-PD-1 antibody drugs in head and neck cancer, especially under hypoxic conditions that cause lymphocyte exhaustion.

Difficulties and innovations in teaching anatomy and physiology in nursing.

AIM: This study aimed to clarify the difficulties and innovations in anatomy and physiology education from the perspective of faculty members in charge of anatomy and physiology education in Japanese nursing universities. BACKGROUND: Many nursing students have difficulty acquiring anatomy and physiology knowledge and applying it in clinical situations; therefore, effective teaching and learning methods for anatomy and physiology must be provided in nursing education. DESIGN: This study followed a cross-sectional observational design using a web-based self-administered questionnaire. METHODS: A web-based self-administered open-ended survey regarding the difficulties and innovations in teaching anatomy and physiology in nursing was conducted involving nursing faculty members at universities in Japan. After inviting 280 nursing universities to participate in the survey, 37 completed questionnaires were collected and descriptively analyzed. RESULTS: In approximately 30% of the universities, multiple professions were involved in collaboratively teaching anatomy and physiology. Furthermore, 59.5% of the universities offered a combination of didactic lectures and laboratory learning. Difficulties in teaching anatomy and physiology included the limitations of the physical teaching environment, the difficulty of teaching the importance of anatomy and physiology in nursing practice, the difficulty of adjusting educational content to learner readiness, and the difficulty of terminology and concepts specific to anatomy and physiology. Innovations used in teaching anatomy and physiology from a nursing perspective included integrating anatomy and physiology with related disciplines, encouraging students to visualize human anatomy in three dimensions, and linking anatomy and physiology to clinical nursing practice. CONCLUSION: Despite some difficulties in teaching the subjects, faculty members have implemented effective educational methods on their own. Due to the various constraints of the learning environment, it is difficult for students to learn anatomy and physiology on campus in depth and acquire a deep understanding of its significance in clinical practice. Therefore, educational involvement that fosters students' self-directed learning skills and encourages flexible learning is expected. In addition, by collaborating with multiple disciplines to provide anatomy and physiology education, the participants of this study innovated pedagogies for teaching nursing students about the usefulness of anatomy and physiology and motivated their students. Moving forward, teachers need to relate their knowledge of anatomy and physiology to actual nursing care and clinical cases and convey the importance of deeply understanding anatomy and physiology in nursing practice to support their students' active learning.

TLR3 forms a laterally aligned multimeric complex along double-stranded RNA for efficient signal transduction.

Toll-like receptor 3 (TLR3) is a member of the TLR family, which plays an important role in the innate immune system and is responsible for recognizing viral double-stranded RNA (dsRNA). Previous biochemical and structural studies have revealed that a minimum length of approximately 40-50 base pairs of dsRNA is necessary for TLR3 binding and dimerization. However, efficient TLR3 activation requires longer dsRNA and the molecular mechanism underlying its dsRNA length-dependent activation remains unknown. Here, we report cryo-electron microscopy analyses of TLR3 complexed with longer dsRNA. TLR3 dimers laterally form a higher multimeric complex along dsRNA, providing the basis for cooperative binding and efficient signal transduction.

Lacrimal Sac Tumors: A Single-institution Experience, Including New Insights.

BACKGROUND/AIM: Lacrimal sac tumors are rare tumor types, with a long time interval from disease onset to diagnosis. We aimed to investigate the characteristics and outcomes of patients with lacrimal sac tumors. PATIENTS AND METHODS: The medical records of 25 patients with lacrimal sac tumors initially treated at the Kyushu university hospital from January 1996 to July 2020 were reviewed. RESULTS: Our analysis included 3 epithelial benign tumors (12.0%) and 22 malignant (88.0%) tumors (squamous cell carcinoma, n=6; adenoid cystic carcinoma, n=2; sebaceous adenocarcinoma, n=2; mucoepidermoid carcinoma, n=1; malignant lymphoma, n=10). The average time from symptom onset to diagnosis was 14.7 months (median=8 months; range=1-96 months). The analysis of patients revealed that lacrimal sac mass (22/25, 88.0%) was the most frequent symptom and a possible tumor marker. Most epithelial benign (n=3) and malignant epithelial (n=12) tumors were treated surgically (14/15, 93.3%). One malignant case was treated with heavy ion beam therapy. Eight patients were treated with postoperative (chemo)radiation therapy because of positive surgical margins (including one unanalyzed case). Local control was ultimately achieved in all but one case. The patient survived for 24 months with immune checkpoint inhibitors and subsequent chemotherapy for local and metastatic recurrence. CONCLUSION: We report our experience in the diagnosis and treatment of lacrimal sac tumors and analyze the clinical trends in cases involving these tumors. Postoperative radiotherapy and pharmacotherapy, including immune checkpoint inhibitors, may be useful for recurrent cases.

Designer Adaptor Proteins for Functional Conversion of Peptides to Small-Molecule Ligands toward In-Cell Catalytic Protein Modification.

Peptides are privileged ligands for diverse biomacromolecules, including proteins; however, their utility is often limited due to low membrane permeability and in-cell instability. Here, we report peptide ligand-inserted eDHFR (PLIED) fusion protein as a universal adaptor for targeting proteins of interest (POI) with cell-permeable and stable synthetic functional small molecules (SFSM). PLIED binds to POI through the peptide moiety, properly orienting its eDHFR moiety, which then recruits trimethoprim (TMP)-conjugated SFSM to POI. Using a lysine-acylating BAHA catalyst as SFSM, we demonstrate that POI (MDM2 and chromatin histone) are post-translationally and synthetically acetylated at specific lysine residues. The residue-selectivity is predictable in an atomic resolution from molecular dynamics simulations of the POI/PLIED/TMP-BAHA (MTX was used as a TMP model) ternary complex. This designer adaptor approach universally enables functional conversion of impermeable peptide ligands to permeable small-molecule ligands, thus expanding the in-cell toolbox of chemical biology.

Enhancer of rudimentary homolog (ERH) plays an essential role in the progression of mitosis by promoting mitotic chromosome alignment.

The enhancer of rudimentary homolog (ERH) is a small eukaryotic protein that is highly conserved in animals, plants, and protists but not in fungi. ERH has several binding proteins and has been associated with various cellular processes, such as pyrimidine metabolism, cell cycle progression, and transcription control; however, little is known about the exact role of this protein and the underlying molecular mechanisms. We found that ERH has a critical role in the mitotic phase of the cell cycle. ERH depleted-cells showed severe chromosome misalignment and weakened kinetochore-microtubule attachment. ERH depletion also caused dissociation of centromere-associated protein E (CENP-E), a mitotic kinesin that is involved in stabilizing the kinetochore-microtubule attachment, from kinetochores of mitotic chromosomes. We propose that ERH contributes to chromosome alignment at the metaphase plate by localizing CENP-E at kinetochore regions.

Relationship between personality traits and health behavior among working women in Japan.

Considering personality traits is effective for cultivating health promotion habits. Therefore, individualized interventions that account for personality traits would be more beneficial for modifying health behaviors. The present study describes the relationship between personality traits and health behaviors among working women compared with non-working women in Japan. Secondary analysis was conducted using nationally representative data from the Japanese Household Panel Survey (JHPS/KHPS). In the 2019 wave, data were gathered from 1,141 women (939 who were working and 202 who were not) between the ages of 28 and 65. Associations among health behaviors, personality traits, and employment status were confirmed by phi coefficient and coefficient ratio. Logistic regression analysis was conducted to examine the association between health behaviors and personality traits. As for working women, daily fruit consumption was associated with extraversion (odds ratio [OR], 1.197; p = 0.003) and conscientiousness (OR, 1.238; p = 0.032). Conscientiousness has been found to significantly contribute to low-risk alcohol consumption (OR, 1.213; p = 0.035). Desirable physical activity habit was associated with extraversion (OR, 1.312; p = 0.000). In contrast, among non-working women, desirable physical activity habits was associated with extraversion (OR, 1.573; p = 0.007) and neuroticism (OR, 0.390; p = 0.001). Further research is needed to clarify the mechanisms underlying differences between working women and non-working women in the association between health behaviors and personality.

Cancer therapy that targets the Hedgehog signaling pathway considering the cancer microenvironment (Review).

Recently, the cancer microenvironment (CME) has received significant attention. At the local site of the tumor, cancer progression is affected by secreted cytokines and conditions derived from the CME and stimulation by cancer­induced cytokines in an autocrine manner. The CME is characterized by various types of conditions, such as hypoxia, inflammation stimulation, and angiogenesis, and contains various components, such as reactive oxygen species, cancer­associated fibroblasts, infiltrated immune cells, exosomes, and cancer stem cells (CSCs). These conditions and components complicate the progression of cancer. The Hedgehog (HH) signaling pathway is a morphogenesis signaling pathway that is reactivated in some cancers. In these cancers, reactivated HH signaling is involved in the induction of the malignant phenotype. HH signaling is also activated under hypoxic conditions and is considered to be strongly correlated with the CME, including the induction of cancer fibrosis and maintenance of CSCs. The aim of the present review was to elucidate a cancer therapy that targets HH signaling by considering the CME, particularly focusing on hypoxia.

PTPN3 Could Βe a Therapeutic Target of Pancreatic Cancer.

BACKGROUND/AIM: Recently, protein tyrosine phosphatase non-receptor type 3 (PTPN3) has gained attention. However, the role of PTPN3 in cancer has not been fully elucidated. In the present study, we analyzed the role of PTPN3 in pancreatic cancer and investigated whether PTPN3 could be a new therapeutic target for pancreatic cancer. MATERIALS AND METHODS: Two pancreatic ductal adenocarcinoma (PDAC) cell lines were used as target cells. Cell proliferation was investigated using cell counting and a xenograft mouse model. Migration and invasion were analyzed using Transwell inserts. Activation-related signaling molecules were examined by western blotting. RESULTS: PTPN3 contributes to the proliferation, migration, and invasion of PDAC cells in vitro. PTPN3 promotes tumor growth in a mouse xenograft model, an action mediated partially through the MAPK pathway. CONCLUSION: PTPN3 could be a new therapeutic target for pancreatic cancer.

Estrogen inhibits transforming growth factor beta signaling by promoting Smad2/3 degradation.

Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERalpha and ERbeta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. On the other hand, TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. Although a number of studies have been made on the cross-talk between estrogen/ERalpha and TGF-beta/Smad signaling, whose molecular mechanisms remain to be determined. Here, we show that ERalpha inhibits TGF-beta signaling by decreasing Smad protein levels. ERalpha-mediated reductions in Smad levels did not require the DNA binding ability of ERalpha, implying that ERalpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ERalpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner. Our observations provide new insight into the molecular mechanisms governing the non-genomic functions of ERalpha.

GLI2 but not GLI1/GLI3 plays a central role in the induction of malignant phenotype of gallbladder cancer.

We previously reported that Hedgehog (Hh) signal was enhanced in gallbladder cancer (GBC) and was involved in the induction of malignant phenotype of GBC. In recent years, therapeutics that target Hh signaling have focused on molecules downstream of smoothened (SMO). The three transcription factors in the Hh signal pathway, glioma­associated oncogene homolog 1 (GLI1), GLI2, and GLI3, function downstream of SMO, but their biological role in GBC remains unclear. In the present study, the biological significance of GLI1, GLI2, and GLI3 were analyzed with the aim of developing novel treatments for GBC. It was revealed that GLI2, but not GLI1 or GLI3, was involved in the cell cycle­mediated proliferative capacity in GBC and that GLI2, but not GLI1 or GLI3, was involved in the enhanced invasive capacity through epithelial­mesenchymal transition. Further analyses revealed that GLI2 may function in mediating gemcitabine sensitivity and that GLI2 was involved in the promotion of fibrosis in a mouse xenograft model. Immunohistochemical staining of 66 surgically resected GBC tissues revealed that GLI2­high expression patients had fewer numbers of CD3+ and CD8+ tumor­infiltrating lymphocytes (TILs) and increased programmed cell death ligand 1 (PD­L1) expression in cancer cells. These results suggest that GLI2, but not GLI1 or GLI3, is involved in proliferation, invasion, fibrosis, PD­L1 expression, and TILs in GBC and could be a novel therapeutic target. The results of this study provide a significant contribution to the development of a new treatment for refractory GBC, which has few therapeutic options.