IGSF3 is a homophilic cell adhesion molecule that drives lung metastasis of melanoma by promoting adhesion to vascular endothelium.

The immunoglobulin superfamily (IgSF) is one of the largest families of cell-surface molecules involved in various cell-cell interactions, including cancer-stromal interactions. In this study, we undertook a comprehensive RT-PCR-based screening for IgSF molecules that promote experimental lung metastasis in mice. By comparing the expression of 325 genes encoding cell-surface IgSF molecules between mouse melanoma B16 cells and its highly metastatic subline, B16F10 cells, we found that expression of the immunoglobulin superfamily member 3 gene (Igsf3) was significantly enhanced in B16F10 cells than in B16 cells. Knockdown of Igsf3 in B16F10 cells significantly reduced lung metastasis following intravenous injection into C57BL/6 mice. IGSF3 promoted adhesion of B16F10 cells to vascular endothelial cells and functioned as a homophilic cell adhesion molecule between B16F10 cells and vascular endothelial cells. Notably, the knockdown of IGSF3 in either B16F10 cells or vascular endothelial cells suppressed the transendothelial migration of B16F10 cells. Moreover, IGSF3 knockdown suppressed the extravasation of B16F10 cells into the lungs after intravenous injection. These results suggest that IGSF3 promotes the metastatic potential of B16F10 cells in the lungs by facilitating their adhesion to vascular endothelial cells.

Trans-homophilic interaction of CADM1 promotes organ infiltration of T-cell lymphoma by adhesion to vascular endothelium.

The initial step of organ infiltration of malignant cells is the interaction with host vascular endothelial cells, which is often mediated by specific combinations of cell adhesion molecules. Cell adhesion molecule 1 (CADM1) is overexpressed in adult T-cell leukemia/lymphoma (ATL) and provides a cell-surface diagnostic marker. CADM1 promotes the adhesion of ATL cells to vascular endothelial cells and multiple organ infiltration in mice. However, its binding partner on host cells has not yet been identified. In this study, we show that CADM1 promotes transendothelial migration of ATL cells in addition to the adhesion to vascular endothelial cells. Moreover, CADM1 enhances liver infiltration of mouse T-cell lymphoma cells, EL4, after tail vein injection, whereas a CADM1 mutant lacking adhesive activity did not. Among the known CADM1-binding proteins expressed in primary endothelial cells, only CADM1 and CADM4 could induce morphological extension of ATL cells when plated onto glass coated with these proteins. Furthermore, CADM1-mediated liver infiltration of EL4 cells was canceled in conventional and vascular endothelium-specific Cadm1 knockout mice, whereas it was not canceled in Cadm4 knockout mice. These results suggest that CADM1 on host vascular endothelial cells is required for organ infiltration of ATL and other T-cell lymphomas expressing CADM1.

Mathematical analysis of gefitinib resistance of lung adenocarcinoma caused by MET amplification.

Gefitinib, one of the tyrosine kinase inhibitors of epidermal growth factor receptor (EGFR), is effective for treating lung adenocarcinoma harboring EGFR mutation; but later, most cases acquire a resistance to gefitinib. One of the mechanisms conferring gefitinib resistance to lung adenocarcinoma is the amplification of the MET gene, which is observed in 5-22% of gefitinib-resistant tumors. A previous study suggested that MET amplification could cause gefitinib resistance by driving ErbB3-dependent activation of the PI3K pathway. In this study, we built a mathematical model of gefitinib resistance caused by MET amplification using lung adenocarcinoma HCC827-GR (gefitinib resistant) cells. The molecular reactions involved in gefitinib resistance consisted of dimerization and phosphorylation of three molecules, EGFR, ErbB3, and MET were described by a series of ordinary differential equations. To perform a computer simulation, we quantified each molecule on the cell surface using flow cytometry and estimated unknown parameters by dimensional analysis. Our simulation showed that the number of active ErbB3 molecules is around a hundred-fold smaller than that of active MET molecules. Limited contribution of ErbB3 in gefitinib resistance by MET amplification is also demonstrated using HCC827-GR cells in culture experiments. Our mathematical model provides a quantitative understanding of the molecular reactions underlying drug resistance.

Loss of YAP1 defines neuroendocrine differentiation of lung tumors.

YAP1, the main Hippo pathway effector, is a potent oncogene and is overexpressed in non-small-cell lung cancer (NSCLC); however, the YAP1 expression pattern in small-cell lung cancer (SCLC) has not yet been elucidated in detail. We report that the loss of YAP1 is a special feature of high-grade neuroendocrine lung tumors. A hierarchical cluster analysis of 15 high-grade neuroendocrine tumor cell lines containing 14 SCLC cell lines that depended on the genes of Hippo pathway molecules and neuroendocrine markers clearly classified these lines into two groups: the YAP1-negative and neuroendocrine marker-positive group (n = 11), and the YAP1-positive and neuroendocrine marker-negative group (n = 4). Among the 41 NSCLC cell lines examined, the loss of YAP1 was only observed in one cell line showing the strong expression of neuroendocrine markers. Immunostaining for YAP1, using the sections of 189 NSCLC, 41 SCLC, and 30 large cell neuroendocrine carcinoma (LCNEC) cases, revealed that the loss of YAP1 was common in SCLC (40/41, 98%) and LCNEC (18/30, 60%), but was rare in NSCLC (6/189, 3%). Among the SCLC and LCNEC cases tested, the loss of YAP1 correlated with the expression of neuroendocrine markers, and a survival analysis revealed that YAP1-negative cases were more chemosensitive than YAP1-positive cases. Chemosensitivity test for cisplatin using YAP1-positive/YAP1-negative SCLC cell lines also showed compatible results. YAP1-sh-mediated knockdown induced the neuroendocrine marker RAB3a, which suggested the possible involvement of YAP1 in the regulation of neuroendocrine differentiation. Thus, we showed that the loss of YAP1 has potential as a clinical marker for predicting neuroendocrine features and chemosensitivity.

Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.

Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro.

Factors Contributing to Well-Being in Japanese Community-Dwelling Older Adults Who Experienced Spousal Bereavement.

PURPOSE: Providing support for older adults after spousal bereavement is crucial for psychological recovery through the grieving process and for promoting a healthy and happy remainder of life. The current study aimed to understand factors influencing well-being among Japanese community-dwelling older adults who experienced spousal bereavement by evaluating how their roles and activities affected their psychological well-being following the loss. METHOD: We conducted a cross-sectional, anonymous, self-administered questionnaire survey of 332 older adults who had experienced death of a spouse in later adulthood. RESULTS: Identified factors that affected well-being after spousal loss were sex, ability to go out without assistance from others, long duration since spousal loss, having a hobby, and adopting a life-oriented approach to cope with bereavement. Well-being following spousal loss was influenced by participants' activities and roles. CONCLUSION: Findings of the current study provide potentially valuable suggestions for surviving spouses and their supporters when coping with grief following spousal loss. [Research in Gerontological Nursing, 17(3), 121-130.].

Paroxysmal Sympathetic Hyperactivity in Stroke.

OBJECTIVE: Paroxysmal sympathetic hyperactivity (PSH) is a life-threatening neurological emergency associated with severe brain injury. Stroke-related PSH, particularly post-aneurysmal subarachnoid hemorrhage (aSAH) PSH, has been relatively understudied and is often misdiagnosed as an aSAH-related hyperadrenergic crisis. This study aims to clarify the feature of stroke-related PSH. METHODS: This study discusses the case of a patient with post-aSAH PSH and identifies 19 articles (25 cases) on stroke-related PSH by searching the PubMed database from 1980 to 2021. RESULTS: In the total cohort, 15 (60.0%) patients were male and the average age was 40.1 ± 16.6 years. The primary diagnoses included intracranial hemorrhage (13 cases, 52.0%), cerebral infarction (7 cases, 28.0%), subarachnoid hemorrhage (4 cases, 16.0%), and intraventricular hemorrhage (1 case, 4.0%). The sites of stroke damage were predominantly the cerebral lobe (10 cases, 40.0%), basal ganglia (8 cases, 32.0%), and the pons (4 cases, 16.0%). The median time of PSH onset after admission was 5 (1-180) days. Most cases employed combination therapy with sedation drugs, beta-blockers, gabapentin, and clonidine. On the Glasgow Outcome Scale, outcomes included death (4 cases, 21.1%), vegetative state (2 cases, 10.5%), severe disability (7 cases, 36.8%), and in only one case (5.3%) was a good recovery noted. CONCLUSIONS: The clinical features and treatment of post-aSAH PSH differed from those of aSAH-related hyperadrenergic crises. Early diagnosis and treatment can prevent severe complications. PSH should be acknowledged as a potential complication of aSAH. Differential diagnosis can aid in developing individualized treatment plans and improving patient prognosis.

Future stroke risk in the chronic phase of post-percutaneous coronary intervention.

A percutaneous coronary intervention (PCI) is widely performed for acute coronary syndromes or chronic coronary syndromes. Periprocedural stroke is a clinically significant complication during PCI. The incidence of cerebrovascular events (CVEs) after PCI in the chronic phase is obscure. This study aimed to investigate the prevalence of CVEs after PCI in the chronic phase and evaluate the usefulness of a simple coronary artery calcification (CAC) evaluation method. This prospective observational study included 179 patients who underwent PCI between January 2016 and December 2018. The incidence of cerebral infarction was examined from one month after PCI to December 2019. In total, 171 individuals (134 men; mean age, 69.8 ± 9.8 years) were recruited. During a median follow-up period of 33 months, the onset of cerebral infarction was observed in 20 individuals (11.7%). More CAC sites (p = 0.009) and post-PCI for the chronic coronary syndrome (p = 0.049) showed a significant association with future CVEs. There was no significant cervical internal carotid artery stenosis for patients who occurred CVEs. The cutoff value for the number of CAC sites for predicting future CVEs was 4.5. The new and easy method accurately reflected future CVEs risk and may be clinically applicable.

Post-traumatic growth of family members of deceased cancer patients and related factors in Japan: A cross-sectional study.

PURPOSE: This study examines the post-traumatic growth (PTG) of bereaved families who care for cancer patients and related factors in Japan. METHODS: Participants included 1298 members of bereaved families of cancer patients (aged 20 or older). An anonymous self-administered questionnaire on PTG, coping, and social support was mailed to 496 bereaved families who provided written informed consent. RESULTS: Responses were obtained from 476 bereaved families; however, since 28 families had missing data, 448 were included for the analyses. The mean age of participants was 61.4 years: 69% women and 45% spouses. The average age of the deceased was 72.8 years old for men (59%). The PTG score of the bereaved families was higher for women than for men (p < 0.0001). Moreover, the Post-Traumatic Growth Inventory Score for those above 65 years of age was higher than of those below 65 years of age (p < 0.0001). A regression analysis confirmed that emotion-focused coping, problem-focused coping, relationship with the deceased, advanced age of bereaved families, and emotional support impacted PTG. CONCLUSION: The significance of the deceased for the bereaved, bereaved family members being older in age, emotion-focused coping, problem-focused coping, and emotional support suggest that these aspects are associated with psychological growth in terms of accepting the death of a loved one and moving forward. It is necessary to evaluate the relationship between the bereaved family and the deceased, the age and gender of the bereaved, coping behaviors, and support status and establish a higher quality bereaved family care system.

Hepatocyte growth factor increases uptake of estradiol 17beta-D-glucuronide and Oatp1 protein level in rat hepatocytes.

Hepatocyte growth factor (HGF) ameliorates liver injuries in hepatectomized cholestatic rats. On the other hand, the protein level of organic anion-transporting polypeptide (Oatp1), which is responsible for the uptake of bile salts into hepatocytes, decreases in cholestatic humans and rats. However, the relationship between the ameliorative effects of HGF and the decrease in Oatp1 levels in cholestasis remains to be understood. Therefore, in order to investigate this relationship, we evaluated the effects of HGF on the function and protein level of Oatp1. HGF treatment significantly increased the uptake of radiolabeled estradiol 17beta-d-glucuronide ([(3)H]E(2)17betaG), a predominant Oatp1 substrate, in primary cultured rat hepatocytes. Additionally, there was an increase in the Oatp1 protein levels. The increased [(3)H]E(2)17betaG uptake was significantly inhibited by simultaneous incubation with the HGF receptor antibody and treatment with non-radiolabeled E(2)17betaG. However, inhibition by taurocholic acid, a Na(+)-taurocholate co-transporting polypeptide (Ntcp) substrate, was weaker than that caused by non-radiolabeled E(2)17betaG. Further, the increase was not altered by replacing Na(+) in the medium with Li(+). In the inhibition study, the increased [(3)H]E(2)17betaG uptake was inhibited by Oatp1 substrates, including bromosulfophthalein, ochratoxin A, and ouabain, but not by digoxin, which is an Oatp2-specific substrate. Furthermore, HGF did not alter the Oatp1 mRNA expression. In contrast, HGF treatment suppressed the ubiquitination of Oatp1 protein. In conclusion, this is the first report suggesting that HGF regulates Oatp1 protein level and that the ameliorative effects of HGF in cholestasis was induced, at least in part, by correcting the down-regulation of the Oatp1 protein level.

Quantitative Analysis of Interaction Between CADM1 and Its Binding Cell-Surface Proteins Using Surface Plasmon Resonance Imaging.

The cell adhesion molecule (CADM) family of the immunoglobulin superfamily (IgSF) comprises four members, CADM1-CADM4, and participates in the formation of epithelial and synaptic adhesion through cell-cell homophilic and heterophilic interactions. To identify the partners that interact with each member of the CADM family proteins, we set up a platform for multiple detection of the extracellular protein-protein interactions using surface plasmon resonance imaging (SPRi) and analyzed the interactions between the CADM family proteins and 10 IgSF of their structurally related cell adhesion molecules. SPRi analysis identified a new interaction between CADM1 and CADM4, where this heterophilic interaction was shown to be involved in morphological spreading of adult T-cell leukemia (ATL) cells expressing CADM1 when incubated on CADM4-coated glass. Moreover, class-I MHC-restricted T-cell-associated molecule (CRTAM) was identified to show the highest affinity to CADM1 among its binding partners by comparing the dissociation constants calculated from the SPR sensorgrams. These results suggest that the SPRi platform would provide a novel screening tool to characterize extracellular protein-protein interactions among cell-surface and secreted proteins, including IgSF molecules.

A One-Pot Three-Component Double-Click Method for Synthesis of [67Cu]-Labeled Biomolecular Radiotherapeutics.

A one-pot three-component double-click process for preparing tumor-targeting agents for cancer radiotherapy is described here. By utilizing DOTA (or NOTA) containing tetrazines and the TCO-substituted aldehyde, the two click reactions, the tetrazine ligation (an inverse electron-demand Diels-Alder cycloaddition) and the RIKEN click (a rapid 6π-azaelectrocyclization), could simultaneously proceed under mild conditions to afford covalent attachment of the metal chelator DOTA or NOTA to biomolecules such as to albumin and anti-IGSF4 antibody without altering their activities. Subsequently, radiolabeling of DOTA- or NOTA-attached albumin and anti-IGSF4 antibody (an anti-tumor-targeting antibody) with [67Cu], a ß--emitting radionuclide, could be achieved in a highly efficient manner via a simple chelation with DOTA proving to be a more superior chelator than NOTA. Our work provides a new and operationally simple method for introducing the [67Cu] isotope even in large quantities to biomolecules, thereby representing an important process for preparations of clinically relevant tumor-targeting agents for radiotherapy.

Exogenous expression of homeoprotein EGAM1N prevents in vitro cardiomyogenesis by impairing expression of T and Nkx2.5, but not Mef2c, in mouse embryonic stem cells.

Generation of multiple cell types from embryonic stem (ES) cells and induced pluripotent stem cells is crucial to provide materials for regenerative medicine. EGAM1N has been found in preimplantation mouse embryos and mouse ES cells as a functionally unclassified homeoprotein. Recently, we reported that expression of EGAM1N suppressed the in vitro differentiation of ES cells into progenitor cells that arise in early embryogenesis. To clarify the effect of EGAM1N on terminal differentiation, embryoid bodies (EBs) were prepared from ES cells expressing exogenous Egam1n. In EBs expressing Egam1n, cardiomyogenesis was inhibited by impairing the expression of crucial transcription factors Brachyury T and Nkx2.5 in the generation of mesoderm and cardiomyocytes, respectively. Expression levels of Mef2c, another crucial gene for cardiomyogenesis, were unaffected. Conversely, the expression levels of Gata6 and Plat, markers for the primitive endoderm lineage, and Cdx2, a marker for the trophectoderm lineage, were increased. These results suggested that certain cell populations in EBs expressing Egam1n preferentially differentiated to such cell lineages. Our results suggest that EGAM1N not only affects the generation of progenitor cells during early embryogenesis, but also the progression of terminal differentiation, such as cardiomyogenesis, in mouse ES cells.

Prognostic items for the last 10 and 3 days of life of cancer patients at home.

BACKGROUND: Prognostic indices are needed to optimize end-of-life care for cancer patients at home, but few prognostic indices predict the last 10 days. OBJECTIVE: The purpose of this study was to identify predictors for the last 10 and 3 days of life in patients with lung, gastric, or colorectal cancer at home. METHODS: Symptoms and signs were initially identified by literature review, and questionnaire was developed. Evaluation of these items and identification of additional items were then performed by 72 visiting nurses using the 3-round Delphi approach. RESULTS: The evaluation of 31 third-round responses is reported. The items for gastric and colorectal cancers were almost same; these cancers were treated as gastrointestinal cancer. To predict the last 10 and 3 days, there were 6 and 0 specific items for lung cancer, respectively, and 5 and 13 specific items for gastrointestinal cancer, respectively. There were 9 common items to predict the last 10 days and 29 common items to predict the last 3 days. CONCLUSION: The specific and common items that could be used to predict the last 10 and 3 days in patients with lung or gastrointestinal cancer were identified. The prognostic items for the last 3 days of life were more numerous among the gastrointestinal cancers than those for the last 10 days. IMPLICATIONS FOR PRACTICE: Specific prognostic items for each cancer are useful for visiting nurses to offer individualized care to patients and families. Using the specific and common prognostic items, end-of-life care may be improved.

Inhibition of carrier-mediated uptake of epirubicin reduces cytotoxicity in primary culture of rat hepatocytes.

Epirubicin, an antineoplastic drug, is considered to be taken up by tumor cells via a common carrier by facilitated diffusion and is then pumped out in an energy-dependent manner because epirubicin is a substrate for P-glycoprotein (P-gp). However, this study investigated the details of the influx mechanism of epirubicin and demonstrated that epirubicin uptake was mediated by active carrier systems in addition to facilitated diffusion in the primary culture of rat hepatocytes. The uptake of epirubicin gradually increased in a saturated manner when the concentrations were between 1 x 10(-7) M and 1 x 10(-6) M. In contrast, the uptake increased progressively in a linear manner when the concentration was high (greater than 1 x 10(-6) M). The uptake of epirubicin at a clinical concentration (7.5 x 10(-7) M) was significantly reduced at 4 degrees C and significantly inhibited when pretreated with metabolic inhibitors (carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), rotenone and sodium azide) by nearly 25%. Furthermore, an organic anion transporter inhibitor, namely, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS); organic anion transport substrates, namely, para-aminohippurate (PAH), taurocholic acid and estradiol 17-beta-D-glucuronide; and organic cation transporter inhibitors, namely, verapamil and tetraethylammonium significantly reduced the uptake of epirubicin. Furthermore, pretreatment with verapamil and PAH significantly prevented epirubicin-induced reduction of proliferative activity in rat hepatocytes. These results indicated that the uptake of epirubicin was induced, at least in part, by the active transport protein in rat hepatocytes; the inhibition of the probable transport protein protected the intact normal cells from the injury induced by the cytotoxicity of epirubicin.