Electron holography observation of electron spin polarization around charged insulating wire.

We report direct observation by electron holography of the spin polarization of electrons in a vacuum region around a charged SiO2 wire coated with Pt-Pd. Irradiating the SiO2 wire with 300-keV electrons caused the wire to become positively charged due to the emission of secondary electrons. The spin polarization of these electrons interacting with the charged wire was observed in situ using a phase reconstruction process under an external magnetic field. The magnetic field of the spin-polarized electrons was simulated taking into account the distribution of secondary electrons and the effect of the external magnetic field.

Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis.

Primary tumor cells metastasize to a distant preferred organ. However, the most decisive host factors that determine the precise locations of metastases in cancer patients remain unknown. We have demonstrated that post-translational citrullination of fibrinogen creates a metastatic niche in the vulnerable spots. Pulmonary endothelial cells mediate the citrullination of fibrinogen, changing its conformation, surface charge, and binding properties with serum amyloid A proteins (SAAs), to make it a host tissue-derived metastatic pathogen. The human-specific SAAs-citrullinated fibrinogen (CitFbg) complex recruits cancer cells to form a protein-metastatic cell aggregation in humanized SAA cluster mice. Furthermore, a CitFbg peptide works as a competitive inhibitor to block the homing of metastatic cells into the SAAs-CitFbg sites. The potential metastatic sites in the lungs of patients are clearly visualized by our specific antibody for CitFbg. Thus, CitFbg deposition displays metastatic risks for cancer patients, and the citrullinated peptide is a new type of metastasis inhibitor.

Suppression of CEBPδ recovers exhaustion in anti-metastatic immune cells.

The pre-metastatic microenvironment consists of pro-metastatic and anti-metastatic immune cells in the early stages of cancer, when the primary tumor begins to proliferate. Redundantly, pro-inflammatory immune cells predominated during tumor growth. Although it is well known that pre-metastatic innate immune cells and immune cells fighting primary tumor cells become exhausted, the mechanism by which this occurs is unknown. We discovered that anti-metastatic NK cells were mobilized from the liver to the lung during primary tumor progression and that the transcription factor CEBPδ, which was upregulated in a tumor-stimulated liver environment, inhibited NK cell attachment to the fibrinogen-rich bed in pulmonary vessels and sensitization to the environmental mRNA activator. CEBPδ-siRNA treated anti-metastatic NK cells regenerated the binding proteins that support sitting in fibrinogen-rich soil, such as vitronectin and thrombospondin, increasing fibrinogen attachment. Furthermore, CEBPδ knockdown restored an RNA-binding protein, ZC3H12D, which captured extracellular mRNA to increase tumoricidal activity. Refreshed NK cells using CEBPδ-siRNA with anti-metastatic abilities would work at metastatic risk areas in the pre-metastatic phase, resulting in a reduction in lung metastasis. Furthermore, tissue-specific siRNA-based therapy in lymphocyte exhaustion may be beneficial in the treatment of early metastases.

Extracellular mRNA transported to the nucleus exerts translation-independent function.

RNA in extracellular vesicles (EVs) are uptaken by cells, where they regulate fundamental cellular functions. EV-derived mRNA in recipient cells can be translated. However, it is still elusive whether "naked nonvesicular extracellular mRNA" (nex-mRNA) that are not packed in EVs can be uptaken by cells and, if so, whether they have any functions in recipient cells. Here, we show the entrance of nex-mRNA in the nucleus, where they exert a translation-independent function. Human nex-interleukin-1ß (IL1ß)-mRNA outside cells proved to be captured by RNA-binding zinc finger CCCH domain containing protein 12D (ZC3H12D)-expressing human natural killer (NK) cells. ZC3H12D recruited to the cell membrane binds to the 3'-untranslated region of nex-IL1ß-mRNA and transports it to the nucleus. The nex-IL1ß-mRNA in the NK cell nucleus upregulates antiapoptotic gene expression, migration activity, and interferon-γ production, leading to the killing of cancer cells and antimetastasis in mice. These results implicate the diverse actions of mRNA.

Regulation of vascular permeability in cancer metastasis.

Enhancement of vascular permeability is indispensable for cancer metastasis. Weakened endothelial barrier function enhances vascular permeability. Circulating tumor cells moving in the microvasculature tend to invade into stromal tissue at the location where vascular permeability is enhanced. Many basic studies have identified permeability factors by using gene-modified animals and cells. These factors directly/indirectly interact with endothelial cells. Here, we review vascular permeability factors and their molecular mechanisms. Interactions between tumor cells and endothelial cells are also discussed in the process of extravasation, one of the most critical steps in tumor metastasis. In some cases, primary tumors can manipulate permeability in a remote organ by secreting permeability factors. In addition, the importance of glycocalyx, which covers the endothelial cell surface, in controlling vascular permeability and tumor metastasis is also described. Furthermore, analysis of the hyperpermeable region found in a mouse model study is introduced. It clearly showed that tumor-bearing mouse lungs had a hyperpermeable region due to the influence of a remote primary tumor, and fibrinogen deposition was observed in that region. Given that fibrinogen was reported to be a permeability factor and a key regulator of inflammation, eliminating fibrinogen deposition may prevent future metastasis.

Analysis of ADAM12-Mediated Ephrin-A1 Cleavage and Its Biological Functions.

Accumulating evidence indicates that an elevated ephrin-A1 expression is positively correlated with a worse prognosis in some cancers such as colon and liver cancer. The detailed mechanism of an elevated ephrin-A1 expression in a worse prognosis still remains to be fully elucidated. We previously reported that ADAM12-cleaved ephrin-A1 enhanced lung vascular permeability and thereby induced lung metastasis. However, it is still unclear whether or not cleaved forms of ephrin-A1 are derived from primary tumors and have biological activities. We identified the ADAM12-mediated cleavage site of ephrin-A1 by a Matrix-assisted laser desorption ionization mass spectrometry and checked levels of ephrin-A1 in the serum and the urine derived from the primary tumors by using a mouse model. We found elevated levels of tumor-derived ephrin-A1 in the serum and the urine in the tumor-bearing mice. Moreover, inhibition of ADAM-mediated cleavage of ephrin-A1 or antagonization of the EphA receptors resulted in a significant reduction of lung metastasis. The results suggest that tumor-derived ephrin-A1 is not only a potential biomarker to predict lung metastasis from the primary tumor highly expressing ephrin-A1 but also a therapeutic target of lung metastasis.

Lung Tumor Cell Recruitment Assay.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

Influence of Hemofiltration on Intradialytic Plasma Volume Decrease.

BACKGROUND/AIMS: Compared with hemodialysis (HD), hemodiafiltration (HDF) reduces the frequency of episodes of intradialytic hypotension. Intradialytic plasma volume decrease (IPVD) induced by ultrafiltration is a leading cause of the episodes, and hemofiltration might have a preventive effect on IPVD. This study examined whether online HDF (ol-HDF) prevented IPVD compared with HD. METHODS: Online HDF of pre-dilution mode (pre-ol-HDF) and post-dilution mode (post-ol-HDF) and HD were performed in 22 patients on maintenance dialysis. In each session, IPVD was calculated by using an intradialytic change in hematocrit, and IPVD in pre-ol-HDF and post-ol-HDF was compared with that in HD in a crossover manner. RESULTS: While the ratios of intradialytic body weight loss to post-dialysis BW (IBWL/BW) in ol-HDF were generally smaller than those in HD, the levels of IPVD and IPVD/IBWL/BW were generally larger than those in HD; the IPVD levels were 0.108 ± 0.058, 0.113 ± 0.053, and 0.101 ± 0.057 (P = 0.67), and those of IPVD/IWL/BW were 2.21 ± 0.97, 2.32 ± 0.91, and 1.98 ± 1.14 (P = 0.21) in pre-ol-HDF, post-ol-HDF, and HD, respectively. CONCLUSION: Online mode hemofiltration, in either pre-dilution mode or post-dilution mode, performed in combination with hemodialysis has no preventive effect on IPVD.

A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death.

Intracellular lipopolysaccharide (LPS) triggers the non-canonical inflammasome pathway, resulting in pyroptosis of innate immune cells. In addition to its well-known proinflammatory role, LPS can directly cause regression of some tumors, although the underlying mechanism has remained unknown. Here we show that secretoglobin(SCGB)3A2, a small protein predominantly secreted in airways, chaperones LPS to the cytosol through the cell surface receptor syndecan-1; this leads to pyroptotic cell death driven by caspase-11. SCGB3A2 and LPS co-treatment significantly induced pyroptosis of macrophage RAW264.7 cells and decreased cancer cell proliferation in vitro, while SCGB3A2 treatment resulted in reduced progression of xenograft tumors in mice. These data suggest a conserved function for SCGB3A2 in the innate immune system and cancer cells. These findings demonstrate a critical role for SCGB3A2 as an LPS delivery vehicle; they reveal one mechanism whereby LPS enters innate immune cells leading to pyroptosis, and they clarify the direct effect of LPS on cancer cells.

C1D is not directly involved in the repair of UV-damaged DNA but protects cells from oxidative stress by regulating gene expressions in human cell lines.

A small nuclear protein, C1D, has roles in various cellular processes, transcription regulation, genome stability surveillance, DNA repair and RNA processing, all of which are required to maintain the host life cycles. In the previous report, C1D directly interacts with XPB, a component of the nucleotide excision repair complex, and C1D knockdown reduced cell survival of 27-1 cells, CHO derivative cells, after UV irradiation. To find out the role of C1D in UV-damaged cells, we used human cell lines with siRNA or shRNA to knockdown C1D. C1D knockdown reduced cell survival rates of LU99 and 786-O after UV irradiation, although C1D knockdown did not affect the efficiency of the nucleotide excision repair. Immunostaining data support that C1D is not directly involved in the DNA repair process in UV-damaged cells. However, H2O2 treatment reduced cell viability in LU99 and 786-O cells. We also found that C1D knockdown upregulated DDIT3 expression in LU99 cells and downregulated APEX1 in 786-O cells, suggesting that C1D functions as a co-repressor/activator. The data accounts for the reduction of cell survival rates upon UV irradiation.

Hepato-entrained B220+CD11c+NK1.1+ cells regulate pre-metastatic niche formation in the lung.

Primary tumours establish metastases by interfering with distinct organs. In pre-metastatic organs, a tumour-friendly microenvironment supports metastatic cells and is prepared by many factors including tissue resident cells, bone marrow-derived cells and abundant fibrinogen depositions. However, other components are unclear. Here, we show that a third organ, originally regarded as a bystander, plays an important role in metastasis by directly affecting the pre-metastatic soil. In our model system, the liver participated in lung metastasis as a leucocyte supplier. These liver-derived leucocytes displayed liver-like characteristics and, thus, were designated hepato-entrained leucocytes (HepELs). HepELs had high expression levels of coagulation factor X (FX) and vitronectin (Vtn) and relocated to fibrinogen-rich hyperpermeable regions in pre-metastatic lungs; the cells then switched their expression from Vtn to thrombospondin, both of which were fibrinogen-binding proteins. Cell surface marker analysis revealed that HepELs contained B220+CD11c+NK1.1+ cells. In addition, an injection of B220+CD11c+NK1.1+ cells successfully eliminated fibrinogen depositions in pre-metastatic lungs via FX Moreover, B220+CD11c+NK1.1+ cells demonstrated anti-metastatic tumour ability with IFNγ induction. These findings indicate that liver-primed B220+CD11c+NK1.1+ cells suppress lung metastasis.

Development of a secondary electron energy analyzer for a transmission electron microscope.

A secondary electron (SE) energy analyzer was developed for a transmission electron microscope. The analyzer comprises a microchannel plate (MCP) for detecting electrons, a coil for collecting SEs emitted from the specimen, a tube for reducing the number of backscattered electrons incident on the MCP, and a retarding mesh for selecting the energy of SEs incident on the MCP. The detection of the SEs associated with charging phenomena around a charged specimen was attempted by performing electron holography and SE spectroscopy using the energy analyzer. The results suggest that it is possible to obtain the energy spectra of SEs using the analyzer and the charging states of a specimen by electron holography simultaneously.

Cardiac hyaluronan may be associated with the persistence of atrial fibrillation.

Hyaluronan (HA), a primary component of the extracellular matrix, is associated with several cardiovascular diseases. However, its precise cardiac origin and role in atrial fibrillation (AF) remain unclear. We investigated chamber-specific HA levels in patients with paroxysmal AF (PAF) or persistent AF (PSAF). The levels of HA, a diacron-reactive oxygen metabolite (dROM) as a marker for oxidative stress, at different cardiac sites, and peripheral brain natriuretic peptide (BNP) levels were measured in patients with PAF (n = 50) or PSAF (n = 35). HA levels in the coronary sinus (CS-HA) were significantly higher than those other sites, in both PAF and PSAF patients, and CS-HA levels were significantly higher in PSAF patients than in PAF patients [37.1 (interquartile range, 31.2-48.3) vs. 30.6 (23.7-40.2) pg/mL, P < 0.01]. CS-HA levels were correlated with CS-dROM levels and peripheral BNP levels in PSAF patients (r = 0.417, P = 0.03 and r = 0.579, P < 0.001, respectively), but not in PAF patients (r = -0.115, P = 0.421 and r = 0.048, P = 0.740, respectively). CS-HA levels were elevated in both PAF and PSAF patients and were correlated with cardiac oxidative stress and BNP levels in PSAF patients. Cardiac HA may be associated with the persistence of AF.

Characterization of cardiac oxidative stress levels in patients with atrial fibrillation.

Atrial fibrillation (AF) is associated with oxidative stress and elevated brain natriuretic peptide (BNP) levels. However, the exact cardiac origin of oxidative stress and its association with BNP levels in AF patients remain unclear. Therefore, we investigated the chamber-specific plasma oxidative stress levels in patients with paroxysmal AF (PAF) and persistent AF (PSAF). Diacron-reactive oxygen metabolite (dROM) levels were measured in patients with PAF (n = 50) and PSAF (n = 35) at different cardiac sites before ablation and in peripheral vein 3 months after ablation. For all sites, dROM levels were higher in PSAF patients than in PAF patients; the levels were the highest in the coronary sinus at 429.0 (interquartile range: 392.0-449.0) vs. 374.0 (357.0-397.8) Carratelli units (P < 0.05). dROM levels in the coronary sinus were related to the BNP levels (r = 0.436, P < 0.001). Furthermore, the reduction in the peripheral dROM levels was related to that in the peripheral BNP levels in patients with symptomatic improvement (r = 0.473, P < 0.001). Cardiac oxidative stress may either be a cause or consequence of prolonged AF, and cardiac oxidative stress levels correlated with BNP levels, though a possible source of oxidative stress in AF patients may be systemic circulation.

Impact of oral beta-blocker therapy on mortality after primary percutaneous coronary intervention for Killip class 1 myocardial infarction.

The use of beta-blockers therapy has been recommended to reduce mortality in patients with left ventricular dysfunction after acute myocardial infarction (AMI). Primary percutaneous coronary intervention (PCI), which has become the mainstay of treatment for AMI, is associated with a lower mortality than fibrinolysis. The benefits of beta-blockers after primary PCI in AMI patients without pump failure are unclear. We hypothesized that oral beta-blocker therapy after primary PCI might reduce the mortality in AMI patients without pump failure. The assessment of lipophilic vs. hydrophilic statin therapy in acute myocardial infarction (ALPS-AMI) study was a multi-center study that enrolled 508 AMI patients to compare the efficacy of hydrophilic and lipophilic statins in secondary prevention after myocardial infarction. We prospectively tracked cardiovascular events for 3 years in 444 ALPS-AMI patients (median age 66 years; 18.2 % women) who had Killip class 1 on admission and were discharged alive. The primary endpoint was all-cause mortality. The 3-year follow-up was completed in 413 patients (93.0 %). During this follow-up, 21 patients (4.7 %) died. In Kaplan-Meier analysis, patients on beta-blockers had a significantly lower incidence of all-cause mortality (2.7 vs. 7.3 %, log-rank p = 0.025). After adjusting for the calculated propensity score for using beta-blockers, their use remained an independent predictor of all-cause mortality (hazard ratio 0.309; 95 % confidence interval 0.116-0.824; p = 0.019). In the statin era, the use of beta-blocker therapy after primary PCI is associated with lower mortality in AMI patients with Killip class 1 on admission.

A young patient with atypical type-B Wolff-Parkinson-White syndrome accompanied by left ventricular dysfunction.

A 15-year-old asymptomatic male patient presented with an electrocardiographic abnormality and left ventricular (LV) dysfunction (left ventricle ejection fraction of 40%) in a physical examination performed 2 years previously. LV dysfunction did not improve despite optimal medical therapy for dilated cardiomyopathy. Twelve-lead electrocardiography revealed a normal PR interval (138 ms) with a small delta-like wave in V2, but not a typical diagnostic wave that could be diagnosed as Wolff-Parkinson-White (WPW) syndrome by an electrocardiogram auto-analysis. Transthoracic echocardiography showed a remarkable asynchronous septal motion. An electrophysiological study was performed to exclude WPW syndrome. An accessory pathway (AP) was revealed on the lateral wall of the right ventricle, and radiofrequency catheter ablation was successfully performed to disconnect the AP. Thereafter, the dyssynchrony disappeared, and LV function improved. The intrinsic atrioventricular nodal conduction was very slow (A-H, 237 ms). The results of electrocardiogram auto-analysis could not be used to confirm the diagnosis of WPW syndrome because of the atypical delta wave. Conduction via the right lateral AP caused electrical dyssynchrony in the LV. This case suggests that atypical delta waves should be evaluated without depending on electrocardiographic auto-analyses in patients with LV dysfunction accompanied by dyssynchrony.

Successful transjugular extraction of a lead in front of the anterior scalene muscle by using snare technique.

The incidence of cardiovascular implantable electronic device infection is increasing. We report a case of and successful device removal in a 79-year-old man with implantable cardioverter-defibrillator infection. Right phrenic nerve paralysis was evident on chest radiography. The lead was in front of the anterior scalene muscle, close to the left phrenic nerve. Therefore, extraction carried a risk of bilateral phrenic nerve paralysis. The lead was successfully extracted from the right internal jugular vein by using the snare technique. No complications occurred, and the extraction was successful.

Transgenically-expressed secretoglobin 3A2 accelerates resolution of bleomycin-induced pulmonary fibrosis in mice.

BACKGROUND: Secretoglobin (SCGB) 3A2, a cytokine-like secretory protein of small molecular weight, is predominantly expressed in airway epithelial cells. While SCGB3A2 is known to have anti-inflammatory, growth factor, and anti-fibrotic activities, whether SCGB3A2 has any other roles, particularly in lung homeostasis and disease has not been demonstrated in vivo. The aim of this study was to address these questions in mice. METHODS: A transgenic mouse line that expresses SCGB3A2 in the lung using the human surfactant protein-C promoter was established. Detailed histological, immunohistochemical, physiological, and molecular characterization of the Scgb3a2-transgenic mouse lungs were carried out. Scgb3a2-transgenic and wild-type mice were subjected to bleomycin-induced pulmonary fibrosis model, and their lungs and bronchoalveolar lavage fluids were collected at various time points during 9 weeks post-bleomycin treatment for further analysis. RESULTS: Adult Scgb3a2-transgenic mouse lungs expressed approximately five-fold higher levels of SCGB3A2 protein in comparison to wild-type mice as determined by western blotting of lung tissues. Immunohistochemistry showed that expression was localized to alveolar type II cells in addition to airway epithelial cells, thus accurately reflecting the site of surfactant protein-C expression. Scgb3a2-transgenic mice showed normal lung development and histology, and no overt gross phenotypes. However, when subjected to a bleomycin-induced pulmonary fibrosis model, they initially exhibited exacerbated fibrosis at 3 weeks post-bleomycin administration that was more rapidly resolved by 6 weeks as compared with wild-type mice, as determined by lung histology, Masson Trichrome staining and hydroxyproline content, inflammatory cell numbers, expression of collagen genes, and proinflammatory cytokine levels. The decrease of fibrosis coincided with the increased expression of SCGB3A2 in Scgb3a2-transgenic lungs. CONCLUSIONS: These results demonstrate that SCGB3A2 is an anti-fibrotic agent, and suggest a possible therapeutic use of recombinant SCGB3A2 in the treatment of pulmonary fibrosis.