Search

Knowledge of the histologic type and primary origin of pulmonary tumors is essential when preparing a surgical strategy. Intraoperative diagnosis of hematoxylin and eosin (HE)-stained frozen sections is the gold standard, but reliable pathology requires time-consuming immunohistochemistry (IHC) to distinguish among histological types/organ origins and to analyze molecular status. The aim of this study was to evaluate the clinical reliability of a new rapid-IHC technique for intraoperative diagnosis of pulmonary tumors. In total, 169 patients with undiagnosed pulmonary tumors were enrolled in a multicenter prospective observational study. At three institutes, pulmonary tumor samples were collected through core needle biopsy and/or surgery to determine surgical strategies. Using a new device for rapid IHC, we applied a high-voltage, low-frequency alternating current (AC) field, which mixes the available antibody as the voltage is switched on/off. Rapid IHC can provide tumor histologic type/origin diagnoses within 20 min, as opposed to the 3-6 h required for conventional IHC. No false diagnoses of malignancy were rendered in any of the cases when using simple HE staining. With HE staining alone, the overall definitive diagnosis rate, the rate of defined tumor origin, and the rate of determined histological type were 76.92%, 85.80%, and 90.53%, respectively. When rapid IHC was added, those rates were significantly improved to 88.76%, 94.67%, and 91.72%, respectively. By providing prompt and accurate intraoperative histological/molecular analysis, rapid IHC driven by AC mixing could serve as an effective clinical tool guiding the surgical strategy for undiagnosed pulmonary tumors.

The human epidermis is formed by the proliferation and differentiation of keratinocytes adjacent to the basement membrane. The outermost layer, the stratum corneum, is equipped with a barrier function that prevents water evaporation, and intercellular lipids play an important role in this barrier function. When the barrier is functioning normally, evaporation is prevented; however, when barrier function is impaired, moisture evaporates, resulting in dry and rough skin. Therefore, maintenance of normal barrier function is critical for maintaining normal skin function. Peroxisome proliferator-activated receptor α (PPARα) is mainly not only involved in lipid metabolism in the liver but is also expressed in the epidermis and is involved in inducing keratinocyte differentiation, promoting lipid production, maintaining barrier function and suppressing skin inflammation. Hence, compounds that activate PPARα are expected to control skin function. Therefore, we identified PPARα activators from among extracts of natural resources that have been approved for use in humans and analysed the effects of these extracts on skin function.First, extracts of 474 natural resources were screened using a PPARα activator screening cell line independently constructed in our laboratory. Next, reporter assays were performed using the Gal4-chimera system to evaluate whether these extracts act as ligands for PPARα. We then analysed their effect on primary normal human epidermal keratinocyte cells by using real-time RT-PCR. Finally, we evaluated PPARα activation effect by the combination of these extracts.We identified 36 extracts having the effect of activating PPARα. In particular, #419, a Typha angustifolia spike extract, showed concentration-dependent transcriptional activation through PPARα-LBD and was considered to be likely to contain a compound that is a ligand of PPARα. #419 increased the expression of PPARα target genes and genes related to skin function in primary cultured human epidermal keratinocytes. Finally, the use of #419 in combination with nine extracts increased PPAR activity more than twice as much as #419 alone treatment.These results showed that the reporter cell line could be useful for discovering extracts of natural resources and that the identified Typha angustifolia spike extract could be used in cosmetics that activate PPARα, which expected to improve skin function.L’épiderme humain se forme grâce à la prolifération et à la différenciation des kératinocytes adjacents à la membrane basale. La couche externe, dite « couche cornée », possède une fonction barrière qui empêche l’évaporation de l’eau, dans laquelle les lipides intercellulaires jouent un rôle important. Lorsque la barrière fonctionne normalement, l’évaporation est évitée ; mais lorsqu’elle est altérée, l’évaporation a lieu et la peau, privée d’hydratation, devient sèche et rêche. Par conséquent, il est capital de maintenir cette fonction barrière normale pour que la peau conserve son fonctionnement normal. Le récepteur alpha activé par proliférateurs de peroxysomes (PPARα) intervient surtout non seulement dans le métabolisme lipidique du foie, mais également dans l’épiderme ; il joue en effet un rôle dans l’induction de la différenciation des kératinocytes, la promotion de la production lipidique, le maintien de la fonction barrière et la suppression de l’inflammation de l’épiderme. Par conséquent, les activateurs du PPAR-α devraient être déterminants pour une bonne fonction cutanée. Nous avons donc identifié des activateurs du PPAR-α parmi des extraits de ressources naturelles dont l’utilisation chez l’homme est approuvée, et nous avons analysé les effets de ces extraits sur la fonction cutanée. MÉTHODES: Tout d’abord, des extraits de 474 ressources naturelles ont été sélectionnés à l’aide d’une lignée cellulaire de détection des activateurs du PPAR-α, construite indépendamment dans notre laboratoire. Ensuite, des tests de gènes rapporteurs ont été effectués à l’aide du système Gal4-chimera pour voir si ces extraits jouaient le rôle de ligands pour le PPAR-α. Nous avons ensuite analysé leur effet sur les cellules kératinocytaires épidermiques humaines normales primaires par RT-PCR en temps réel. Enfin, nous avons évalué l’effet d’activation du PPAR-α par l’association de ces extraits. RÉSULTATS: Nous avons identifié 36 extraits ayant pour effet d’activer le PPAR-α. En particulier, le n° 419, un extrait d’épi de Typha angustifolia, a montré une activation transcriptionnelle dépendante de la concentration par le PPAR-α-LBD et a été considéré comme susceptible de contenir un composé qui est un ligand du PPAR-α. Le n° 419 a augmenté l’expression des gènes cibles du PPAR-α et des gènes liés au fonctionnement de la peau dans les kératinocytes épidermiques humains primaires mis en culture. Enfin, l’utilisation du n° 419 en association avec neuf extraits a augmenté de plus du double l’activité du PPAR par rapport au traitement par le n° 419 seul.Ces résultats ont montré que la lignée cellulaire rapporteuse pourrait être utile pour découvrir des extraits de ressources naturelles et que l’extrait d’épi de Typha angustifolia identifié pourrait être utilisé dans des cosmétiques qui activent le PPAR-α, ce qui devrait améliorer la fonction cutanée.

Adhesiolysis is often necessary in intrathoracic adhesion during ipsilateral repeat lung resection. This procedure has a risk of surgical complications, including unintentional intraoperative damage of the pulmonary vessels or lung parenchyma. We used an oxidized regenerated cellulose (ORC) sheet to prevent intrathoracic adhesion after lung resection in 55 patients. The sheet was placed on the surface of the resected region and on the lung surface under the wound. No major postoperative complications were observed. Three cases underwent ipsilateral thoracic surgery for the treatment of lung malignancies, and there were no intrathoracic adhesions around the ORC sheet-covered area.

Purpose Many effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed, but a weaker response in individuals undergoing anticancer treatment has been reported. This study evaluates the immunogenic status and safety of SARS-CoV-2 vaccines for patients with non-small-cell lung cancer (NSCLC), receiving tegafur–uracil (UFT) as postoperative adjuvant chemotherapy. Methods The subjects of this prospective study were 40 patients who underwent surgery for NSCLC and received SARS-CoV-2 vaccines postoperatively. We compared the antibody titers of SARS-CoV-2 vaccines and the adverse events between patients who received adjuvant UFT and patients who did not. Results The mean anti-S1 IgG titers were not significantly different between the UFT and without-UFT groups (mean optimal density, 0.194 vs. 0.205; P = 0.76). Multivariate analysis identified the period after the second vaccination as an independent predictor of anti-S1 IgG titer (P = 0.049), but not the UFT status (with or without-UFT treatment; P = 0.47). The prevalence of adverse events did not differ significantly between the groups, and no severe adverse events occurred. Conclusions The efficacy and safety of the SARS-CoV-2 vaccines for NSCLC patients who received postoperative adjuvant UFT chemotherapy were comparable to those for NSCLC patients who did not receive postoperative adjuvant UFT chemotherapy. Clinical trial registration This study was registered with the University Hospital Medical Information Network (UMIN) in Japan (UMIN000047380).

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

Background Myasthenia gravis (MG) is the most common paraneoplastic syndrome in thymoma. However, the association between MG and postoperative outcomes is controversial. Therefore, we examined the effect of MG on the surgical outcomes of patients with thymoma. Methods This study enrolled 145 consecutive patients with thymoma who underwent surgical resection at our institution between January 2000 and December 2020. The patients were classified into thymoma with MG (MG group) and without MG (non-MG group). Data about characteristics of patients, surgical outcomes, and prognostic factors were compared between the two groups. Results Of 145 patients, 47 (32%) presented with MG and 98 (68%) did not. There was no significant difference in terms of the incidence of postoperative complications, overall survival (OS), and recurrence-free survival (RFS) between the two groups. The deaths were not caused by thymoma. Among the patients aged >60 years, the MG group had a lower survival rate than the non-MG group. In the univariate analysis, age ≥60 years was a poor prognostic factor for OS, whereas in the multivariate analysis, Masaoka stage III and IV classifications were poor prognostic factors for RFS. Conclusion The incidence of postoperative complications did not differ between patients with thymoma and without MG. In the MG group, age ≥60 years was a poor prognostic factor for OS. The postoperative follow-up of patients aged ≥60 years with thymoma with MG should focus on not only recurrence but also progression of diseases other than thymoma.

Background Fibrous bands (FBs) are one of the histological features in tumors which can be confirmed by hematoxylin and eosin (H&E)‐stained slides. FBs have been reported to correlate with malignancy in various tumors. This study aimed to investigate whether the presence of FBs is associated with malignancy in thymoma. Methods A total of 123 consecutive patients with thymoma who underwent microscopically complete resections from January 2000 to December 2018 were enrolled into this study. H&E‐stained slides of all thymoma patients were re‐examined. Study patients were classified into two groups: with FBs (n = 36) and without FBs (n = 87). Clinicopathological characteristics, overall survival (OS), and recurrence‐free survival (RFS) were compared between the two groups. Furthermore, multivariate analyses were performed to identify whether the presence of FBs was associated with higher Masaoka stage and poor prognosis in patients with thymoma. Results The Masaoka stage was found to be higher and recurrence more likely in thymoma patients with FBs than in those without. RFS was significantly poorer in thymoma patients with FBs than in those without, although no significant difference was observed in OS between them. The presence of FBs was significantly associated with higher Masaoka stage in the multivariate analysis using logistic regression. Additionally, the presence of FBs was an independent prognostic factor for poor RFS in multivariate analysis using Cox's proportional hazards model. Conclusions The presence of FBs in patients with thymoma was associated with higher Masaoka stage, higher recurrence rate, and poorer RFS. Key points Significant findings of the study Fibrous bands (FBs) are bands of fibrosis dividing tumors into different‐sized irregular islands. The presence of FBs is associated with higher Masaoka stage and poor recurrence‐free survival in patients with thymoma. What this study adds The presence of fibrous bands might be associated with the malignant behavior of thymoma. Confirming the presence or absence of FBs may result in personalized medication for patients with thymoma., • Fibrous bands (FBs) are bands of fibrosis dividing tumors into different‐sized irregular islands. • The presence of FBs is associated with the malignant behavior of thymomas. • Confirmation of FBs might lead to individualized medications in patients with thymoma.

Roundabout4 (Robo4) is an endothelial cell-specific protein that stabilizes the vasculature in pathological angiogenesis and inflammation. We previously determined a 3-kb Robo4 promoter and demonstrated the importance of the upstream region for nuclear factor-kappaB (NF-κB)-mediated promoter activation induced by tumor necrosis factor α (TNFα). This region contains unique genomic features, including promoter region-specific DNA hypermethylation and chromatin condensation; however, the function of the region remains poorly understood. In this study, we analyzed the DNA sequences of the region and identified a motif for polycomb repressive complex 2 (PRC2). Chromatin immunoprecipitation assay indicates the binding of the PRC2 component, SUZ12, to the motif. A mutation in the motif decreased DNA methylation in embryonic stem cells and increased Robo4 promoter activity in endothelial cells. An inhibitor for the PRC2 component, EZH2, induced the promoter activity and expression of Robo4 in endothelial cells treated with or without TNFα. Taken together, these results indicate that the PRC2 components maintain DNA hypermethylation and suppress Robo4 expression via the PRC2 binding motif in the upstream promoter.

Background: The efficacy of diaphragmatic plication (DP) has been proven in many studies. However, there are few reports on DP for patients with severe respiratory conditions requiring mechanical ventilation. The study aim was to demonstrate the efficacy of DP for patients with severe respiratory insufficiency after cardiothoracic surgeries. Methods: We retrospectively reviewed 10 patients who underwent DP for severe respiratory insufficiency due to postoperative diaphragmatic paralysis; eight of them required mechanical ventilation, and two needed high-flow oxygen therapy prior to DP. The symptoms, lung function, and elevation of the diaphragm were assessed before and after DP. Results: All patients were successfully withdrawn from mechanical ventilation after DP and discharged without the need for oxygen therapy. The mean perioperative Medical Research Council (MRC) dyspnea scale (ATS/ERS 2004) score improved in 30 days (from 4 to 1.8) and in 90 days (from 4 to 0.6) after DP. Lung dynamic compliance was also ameliorated (mean improvement: 41.9 to 60.7 mL/cmH 2 O). Radiography revealed improved elevation of the diaphragm (mean improvement of 1.8 intercostal spaces, range, 1–2). Mean hospital stay after DP was 65.5 days (range, 25–187 days). One patient who underwent DP with endostapler-only suturing required re-operation because of staple line ruptures. Conclusions: DP was found to be an effective form of treatment for patients with severe respiratory insufficiency after cardiothoracic surgery.

Background: Large-cell neuroendocrine carcinoma (LCNEC) and small cell lung cancer (SCLC) are categorized as high-grade neuroendocrine carcinoma (HGNEC). We analyzed the efficacy of perioperative chemotherapy for HGNEC and the prognostic factors. Methods: We retrospectively reviewed the medical records of patients who underwent tumor resection and were diagnosed with HGNEC between January 2001 and December 2014. The overall survival (OS) was estimated by the Kaplan-Meier method. Propensity score matching was performed to compare the OS between the treatment groups. Multivariate analyses using a Cox proportional hazards model were performed to search for prognostic factors for HGNEC. Results: We analyzed 146 HGNEC patients (LCNEC n=92, SCLC n=54) without synchronous multiple cancers, who underwent complete resection. Seventy patients (LCNEC n=31, SCLC n=32) received perioperative chemotherapy and all of them received a platinum-based anticancer drug. Perioperative chemotherapy significantly improved the 5-year OS rates of HGNEC patients (all stages: 74.5% vs. 34.7%, P

Roundabout guidance receptor 4 (Robo4) is an endothelial-specific membrane protein that suppresses pathological angiogenesis and vascular hyperpermeability by stabilizing endothelial cells. Robo4 suppresses severe systemic inflammation induced by pathogens and endotoxins and inhibits tumor growth and metastasis, therefore serving as a potential therapeutic target. Although the regulation of Robo4 expression through transcription factors and epigenetic mechanisms has been studied, the role of histone deacetylases (HDACs) has not been explored. In the present study, we investigated the involvement of HDACs in the regulation of Robo4 expression. An HDAC inhibitor, MS-275, which inhibits HDAC1, HDAC2, and HDAC3, was found to suppress Robo4 expression in endothelial cells. Small interfering RNA (siRNA)-mediated knockdown of HDAC3, but not of HDAC1 and 2, also decreased its expression level. MS-275 downregulated the expression of the transcription factor complex GABP, in addition to suppressing Robo4 promoter activity. GABP expression was also downregulated by the siRNA against HDAC3. MS-275 decreased the transendothelial electrical resistance of a monolayer of mouse endothelial cells and increased the rate of leakage of Evans blue dye in the mouse lungs. In addition, MS-275 accelerated cell migration through the endothelial cell monolayer and augmented cell extravasation in the mouse lungs. Taken together, we demonstrated that MS-275 suppresses Robo4 expression by inhibiting HDAC3 in endothelial cells and enhances endothelial and vascular permeability. Thus, we demonstrated a novel mechanism regulating Robo4 expression and vascular permeability, which is anticipated to contribute to future therapies for infectious and inflammatory diseases.

The lysine methyltransferase SETDB1, an enzyme responsible for methylation of histone H3 at lysine 9, plays a key role in H3K9 tri-methylation-dependent silencing of endogenous retroviruses and developmental genes. Recent studies have shown that ubiquitination of human SETDB1 complements its catalytic activity and the silencing of endogenous retroviruses in human embryonic stem cells. However, it is not known whether SETDB1 ubiquitination is essential for its other major role in epigenetic silencing of developmental gene programs. We previously showed that SETDB1 contributes to the formation of H3K4/H3K9me3 bivalent chromatin domains that keep adipogenic Cebpa and Pparg genes in a poised state for activation and restricts the differentiation potential of pre-adipocytes. Here, we show that ubiquitin-resistant K885A mutant of SETDB1 represses adipogenic genes and inhibits pre-adipocyte differentiation similar to wild-type SETDB1. We show this was due to a compensation mechanism for H3K9me3 chromatin modifications on the Cebpa locus by other H3K9 methyltransferases Suv39H1 and Suv39H2. In contrast, the K885A mutant did not repress other SETDB1 target genes such as Tril and Gas6 suggesting SETDB1 represses its target genes by two mechanisms; one that requires its ubiquitination and another that still requires SETDB1 but not its enzyme activity.

Small-molecule agonism of peroxisome proliferator-activated receptor α (PPARα), a ligand-activated transcriptional factor involved in regulating fatty acid metabolism, is an important approach for treating dyslipidemia. Here, we determined the structures of the ligand-binding domain (LBD) of PPARα in complex with 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives, which were recently identified as PPARα-selective activators with markedly different structures from those of the well-known PPARα agonists fibrates. The crystal structures of the complexes showed that they form a canonical hydrogen-bond network involving helix 12 in the LBD, which is thought to be essential for PPARα activation, as also observed for fibrates. However, the phenyl side chain of the compounds occupies a small cavity between Ile272 and Ile354, which is rarely accessed by fibrates. This unique feature may be essential for subtype selectivity and combine with the well-characterized binding mode of fibrates to improve activity. These findings demonstrate the advantage of using 1H-pyrazolo-[3,4-b]pyridine as a skeleton of PPARα agonists and provide insight into the design of molecules for treating dyslipidemia.

In advanced cancer patients, malignant cells invade and disseminate within normal cells and develop resistance to therapy with additional genetic mutations, which makes radical cure very difficult. Precision medicine against advanced cancer is hampered by the lack of systems aimed at multiple target molecules within multiple loci. Here, we report the development of a versatile diagnostic and therapeutic system for advanced cancer, named the Cupid and Psyche system. Based on the strong non-covalent interaction of streptavidin and biotin, a low immunogenic mutated streptavidin, Cupid, and a modified artificial biotin, Psyche, have been designed. Cupid can be fused with various single-chain variable fragment antibodies and forms tetramer to recognize cancer cells precisely. Psyche can be conjugated to a wide range of diagnostic and therapeutic agents against malignant cells. The Cupid and Psyche system can be used in pre-targeting therapy as well as photo-immunotherapy effectively in animal models supporting the concept of a system for precision medicine for multiple targets within multiple loci.

Claudin (CLDN) proteins, a tetra-transmembrane family containing over 20 members, have been identified as key structural and functional components of intercellular seals, tight junctions (TJs). CLDNs are involved in the barrier and fence functions of TJs. Loosening the TJ barrier is one strategy for increasing drug absorption and delivery to the brain. Due to aberrant CLDN expression, the TJ fence function is frequently dysregulated in carcinogenesis. In addition, CLDN-1 is a co-receptor for the hepatitis C virus. Together these characteristics indicate CLDNs as promising targets for drug development, and CLDN binders are potential candidates for delivering drugs, treating cancer, and preventing viral infection. Before 2008, a receptor-binding fragment of Clostridium perfringens enterotoxin was the only CLDN binder available. Since then, several challenges regarding the generation of monoclonal antibodies against CLDNs have been surmounted, leading to breakthroughs in CLDN-targeted drug development. Here, we provide an overview of the recent progress in technology using created CLDN binders-anti-CLDN monoclonal antibodies.

Background: Advances in the development of staplers for pulmonary vessels have contributed to safe and convenient procedures in thoracic surgery. However, adverse events (AEs) can occur during vascular stapling and cause fatal hemorrhage. We aimed to assess the risk level of using the vessel stapling procedure to reduce such AEs. Methods: First, an animal experiment using pig cardiopulmonary blocks was conducted. Pulmonary arteries were closed with staplers under stressful conditions such as lifting or twisting; vessel stump endurance was analyzed through different methods. Second, AEs associated with clinical stapler use for pulmonary vessels were retrospectively reviewed by studying clinical videos of 263 patients. Results: In the animal experiment, the pressure resistance was significantly lower in the twisted group than in the no strain group (no strain vs. lifting: P=0.2008, no strain vs. twisting: P=0.002, no strain vs. twisting and lifting: P

Peroxisome proliferator–activated receptor α (PPARα) is a ligand-activated transcription factor that belongs to the superfamily of nuclear hormone receptors. PPARα is mainly expressed in the liver, where it activates fatty acid oxidation and lipoprotein metabolism and improves plasma lipid profiles. Therefore, PPARα activators are often used to treat patients with dyslipidemia. To discover additional PPARα activators as potential compounds for use in hypolipidemic drugs, here we established human hepatoblastoma cell lines with luciferase reporter expression from the promoters containing peroxisome proliferator–responsive elements (PPREs) and tetracycline-regulated expression of full-length human PPARα to quantify the effects of chemical ligands on PPARα activity. Using the established cell-based PPARα-activator screening system to screen a library of >12,000 chemical compounds, we identified several hit compounds with basic chemical skeletons different from those of known PPARα agonists. One of the hit compounds, a 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivative we termed compound 3, selectively up-regulated PPARα transcriptional activity, leading to PPARα target gene expression both in vitro and in vivo. Of note, the half-maximal effective concentrations of the hit compounds were lower than that of the known PPARα ligand fenofibrate. Finally, fenofibrate or compound 3 treatment of high fructose–fed rats having elevated plasma triglyceride levels for 14 days indicated that compound 3 reduces plasma triglyceride levels with similar efficiency as fenofibrate. These observations raise the possibility that 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives might be effective drug candidates for selective targeting of PPARα to manage dyslipidemia.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis and closely related to exposure to asbestos. MPM is a heterogeneous tumor with three main histological subtypes, epithelioid, sarcomatoid, and biphasic types, among which sarcomatoid type shows the poorest prognosis. The Ror-family of receptor tyrosine kinases, Ror1 and Ror2, is expressed in various types of tumor cells at higher levels and affects their aggressiveness. However, it is currently unknown whether they are expressed in and involved in aggressiveness of MPM. Here, we show that Ror1 and Ror2 are expressed in clinical specimens and cell lines of MPM with different histological features. Studies using MPM cell lines indicate that expression of Ror2 is associated tightly with high invasiveness of MPM cells, whereas Ror1 can contribute to their invasion in the absence of Ror2. However, both Ror1 and Ror2 promote proliferation of MPM cells. We also show that promoted invasion and proliferation of MPM cells by Ror signaling can be mediated by the Rho-family of small GTPases, Rac1, and Cdc42. These findings elucidate the critical role of Ror signaling in promoting invasion and proliferation of MPM cells.

Disruption of the gastrointestinal epithelial barrier is a hallmark of chronic inflammatory bowel diseases (IBDs). The transmembrane protein claudin 2 (CLDN2) is a component of epithelial tight junctions (TJs). In the intestines of patients with IBDs, the expression of the pore-forming TJ protein CLDN2 is upregulated. Although CLDN2 is involved in these leaky barriers, whether it can be a target to enhance TJ integrity is unknown because a CLDN2-specific inhibitor has not been developed. Here, we used DNA immunization to generate a monoclonal antibody (mAb) that recognized an extracellular loop of CLDN2. Treatment of epithelial cell monolayers with the mAb increased barrier integrity. In addition, the anti-CLDN2 mAb attenuated the decrease in TJ integrity induced by the proinflammatory cytokine tumor necrosis factor-α (TNF-α), and cotreatment of cells with anti-TNF-α mAb and anti-CLDN2 mAb showed additive attenuating effects. These findings indicate that CLDN2 may be a target for enhancing TJ integrity, and CLDN2 binder may be an enhancer of mucosal barrier integrity and a potential therapeutic option for IBDs.

A limiting barrier for mucosal absorption of drugs is the tight junction (TJ). TJs exist between two adjacent cells (bicellular TJ, bTJ) and at the sites where three cells meet (tricellular TJ, tTJ). We present a novel approach which employs a physiologically regulated pathway for the passage of large molecules through the tTJ. Main barrier-relevant tTJ proteins are tricellulin and angulin-1 to -3. We developed an angulin binder from Clostridium perfringens iota-toxin (Ib) whose receptor is angulin-1. An Ib fragment corresponding to amino acids 421-664 (Ib421-664) of iota-toxin proved to bind in cells expressing angulin-1 and -3, but not angulin-2. This binding led to removal of angulin-1 and tricellulin from the tTJ which enhanced the permeation of macromolecular solutes. Ib421-664 enhanced intestinal absorption in rats and mice. Our findings indicate that Ib421-664, which we designate angubindin-1, is a modulator of the tTJ barrier and that modulation of that barrier qualifies for a new strategy of developing a mucosal absorption enhancer.

A 39-year-old man was admitted to our hospital with back pain and numbness of the left leg. Computed tomography (CT) showed a giant bulla and tumor in the right lung, mediastinal shift to the left side and lesions suggestive of metastatic sacral tumor. Three days later, the patient visited the emergency room with dyspnea and tachycardia. Chest CT showed the progression of mediastinal shift due to the rapid expansion of the giant bulla, and an emergency surgery was performed. After induction of anesthesia, sudden respiratory and circulatory failure occurred. Considering further expansion of the giant bulla by positive pressure ventilation, veno-arterial (V-A) extracorporeal membrane oxygenation (ECMO) was applied. After establishing ECMO, the condition of the patient became stable and the giant bulla could be resected successfully.

OBJECTIVES During video-assisted thoracoscopic surgery (VATS), blood oozing from the surface of the access port wound can hamper the surgical view. Although this oozing is difficult to prevent, it can be decreased by placing a wound edge protector with oxidized regenerated cellulose (ORC) on the surface of the access port wound, thereby improving the surgical outcomes and safety of VATS. METHODS We conducted a prospective, single-centre, open-label, randomized clinical trial to evaluate the operative outcomes of VATS when using the ORC (ORC group) compared with operative outcomes without using the ORC (non-ORC group). The primary end point was interruption of the operation as a result of blood oozing from the surface of the access port wound. The secondary end points were the other intraoperative and postoperative outcomes. RESULTS A total of 108 patients were divided into the ORC group (n = 54) and the non-ORC group (n = 54). Compared with the non-ORC group, the ORC group had fewer patients with an interruption in the operation (11.1% vs 51.8%; P CONCLUSION The use of an ORC sheet around a wound edge protector for haemostasis can ensure a clear view during VATS. Clinical trial registration number UMIN000031112.

We previously reported that 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid derivative 6 is an agonist of human peroxisome proliferator-activated receptor alpha (hPPARα). Here, we prepared a series of 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid derivatives in order to examine the structure-activity relationships (SAR). SAR studies clearly indicated that the steric bulkiness of the substituent on 1H-pyrazolo-[3,4-b]pyridine ring, the position of the distal hydrophobic tail part, and the distance between the distal hydrophobic tail part and the acidic head part are critical for hPPARα agonistic activity. These SAR results are somewhat different from those reported for fibrate-class hPPARα agonists. A representative compound (10f) was as effective as fenofibrate in reducing the elevated plasma triglyceride levels in a high-fructose-fed rat model.

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily and include three subtypes (PPARα, PPARδ, and PPARγ). They regulate gene expression in a ligand-dependent manner. PPARα plays an important role in lipid metabolism. PPARγ is involved in glucose metabolism and is a potential therapeutic target in Type 2 diabetes. PPARδ ligands are candidates for the treatment of metabolic disorders. Thus, the detection of PPAR ligands may facilitate the treatment of various diseases. In this study, to identify PPAR ligands, we engineered reporter cell lines that can be used to quantify PPARγ and PPARδ activity. We evaluated several known ligands using these reporter cell lines and confirmed that they are useful for PPAR ligand detection. Furthermore, we evaluated extracts of approximately 200 natural resources and found various extracts that enhance reporter gene activity. Finally, we identified a main alkaloid of the Evodia fruit, evodiamine, as a PPARγ activator using this screening tool. These results suggest that the established reporter cell lines may serve as a useful cell-based screening tool for finding PPAR ligands to ameliorate metabolic syndromes.

Roundabout4 (Robo4) is an endothelial cell-specific receptor that regulates vascular stability. Recently, Robo4 has been shown to regulate vascular permeability in inflammation. However, the mechanisms regulating the Robo4 gene in the context of inflammation are poorly understood. In this study, we found that intravenous injection of tumor necrosis factor (TNF) α increased Robo4 expression in mouse organs. In vitro analyses showed that TNFα increased Robo4 expression in human primary endothelial cells, but not in cells pretreated with a nuclear factor (NF)-κB inhibitor. Reporter assays using wild-type and mutant Robo4 promoters indicated that TNFα activated the Robo4 promoter and that both the -2753 and -2220 NF-κB motifs were essential for this activation. Electrophoretic mobility shift assays demonstrated that the NF-κB p65-p50 heterodimer bound to these motifs. These findings were further supported by chromatin immunoprecipitation assays in endothelial cells. Taken together, these results indicated that TNFα induced Robo4 expression by facilitating NF-κB p65-p50 heterodimer binding to the -2753 and -2220 motifs in the Robo4 promoter in endothelial cells in the context of inflammation.

Invasion has a significant role in cancer progression, including expansion to surrounding tissue and metastasis. Previously, we assessed the invasive ability of cancer cells using an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) method by which cancer cell invasion can be easily visualized. The present study examined multiple lung adenocarcinoma and malignant pleural mesothelioma (MPM) cell lines using the DL-CGH method and identified inherently invasive cell lines. Next, by comparing gene expression between invasive and non-invasive cells by cDNA microarray, the potential candidate gene brain-expressed x-linked protein 1 (Bex1) was identified to be involved in cancer invasion, as it was highly expressed in the invasive cell lines. Downregulation of Bex1 suppressed the invasion and proliferation of the invasive tumor cell lines. The findings of the present study suggested that Bex1 may promote metastasis in vivo and could be a potential oncogene and molecular therapeutic target in lung adenocarcinoma and MPM.

The undifferentiated state of muscle stem (satellite) cells (MuSCs) is maintained by the canonical Notch pathway. Although three bHLH transcriptional factors, Hey1, HeyL, and Hes1, are considered to be potential effectors of the Notch pathway exerting anti-myogenic effects, neither HeyL nor Hes1 inhibits myogenic differentiation of myogenic cell lines. Furthermore, whether these factors work redundantly or cooperatively is unknown. Here, we showed cell-autonomous functions of Hey1 and HeyL in MuSCs using conditional and genetic null mice. Analysis of cultured MuSCs revealed anti-myogenic activity of both HeyL and Hes1. We found that HeyL forms heterodimeric complexes with Hes1 in living cells. Moreover, our ChIP-Seq experiments demonstrated that, compared with HeyL alone, HeyL-Hes1 heterodimer bound with high affinity to specific sites in the chromatin including the cis-element of Hey1. Finally, the analyses of myogenin promoter activity showed that HeyL and Hes1 acted synergistically to suppress myogenic differentiation. Collectively, those results suggest that HeyL and Hey1 function redundantly in MuSCs, and that HeyL requires Hes1 for effective DNA binding and biological activity.

In the management of chest drain after thoracic surgery, it is important to egest fluid and air which accumulate in the pleural cavity and to gain information such as air leakage, bleeding or pus discharge. To achieve these purpose, continuous chest drainage system is necessary for thoracic surgery. In addition, we have to understand the particularity in the pleural cavity and the structure of continuous chest drainage system. Traditional drainage system is based on 3-bottle system. Recently, we can use new drainage system, such as Thopaz, which is called digital drainage system. There are several studies comparing digital drainage system with traditional drainage system, but the superiority of digital drainage system to traditional drainage system is not confirmed.

A 51-year-old woman underwent radical operation for a gastric cancer and adjuvant chemotherapy with tegafur, gimeracil, and oteracil potassium. Five and a half years later, screening chest computed tomography(CT)scan showed slight ground glass opacity(GGO)of the both lungs and they were regarded as inflammatory change. Several months later, she experienced a dry cough. CT scan showed aggravation of GGO, and she was refered to our hospital to investigate these lesions 6 years after operation. We performed a wedge resection of right middle lobe. A postoperative pathology revealed metastatic carcinomatous lymphangiosis of gastric cancer. Chemotherapy using capecitabine and oxaliplatin, and then docetaxel was performed and talc pleurodesis was done for the malignant pleural effusion. These treatments did not improve condition, and she died 7 months after lung operation. In conclusion, we need to consider GGO as the possibility of carcinomatous lymphangiosis in case of patient with gastric cancer.

Rationale Oligonucleotide therapeutics have recently gained much attention, but its pharmacokinetic evaluation methods are still not sufficient, and, in particular, more tools are needed to evaluate their tissue distribution and metabolites. We developed a matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS)-based method to evaluate the tissue distribution of oligonucleotide therapeutics. Methods We used an antisense oligonucleotide containing locked nucleic acids (LNA-A). Various washing protocols were examined using mouse kidney homogenate sections. Next, we applied a two-step matrix preparation strategy. As a first step, 3-hydroxypicolinic acid (3-HPA) matrix containing citrate and amines was sprayed using an airbrush and subsequently 3-HPA matrix containing citrate only was sprayed using the ImagePrep. Finally, kidney sections prepared from LNA-A-dosed mice were treated with our optimized method and analyzed with MALDI-IMS. Results The selected washing method made it possible to detect LNA-A with MALDI-IMS and, furthermore, our developed matrix pretreatment method enhanced signal intensity approximately two-fold. MALDI-IMS revealed that LNA-A localized in a portion presumed to be the renal cortex. We also obtained information on LNA-A metabolites, which showed the same distribution profile as LNA-A in kidneys. Conclusions This study shows that MALDI-IMS can be applied to evaluate the tissue distribution of oligonucleotide therapeutics. Our method can evaluate the tissue distribution along with metabolites and has the potential to help the development of novel oligonucleotide therapeutics.