Distribution of Signal Peptides in Microvesicles from Activated Macrophage Cells.

Extracellular vesicles, such as microvesicles (LEV) and exosomes (SEV), play an important role in intercellular signaling by encapsulating functional molecules and delivering them to specific cells. Recent studies showed that signal peptides (SPs), which are derived from sequences at the N-terminal of newly synthesized proteins, exhibited biological activity in the extracellular fluid. We previously reported that SPs were secreted into the extracellular fluid via SEV; however, it remains unclear whether the release of SPs occurs via LEV. In the present study, we demonstrated that SP fragments from human placental secreted alkaline phosphatase (SEAP) were present in LEV as well as SEV released from RAW-Blue cells, which stably express an NF-κB-inducible SEAP reporter. When RAW-Blue cells were treated with LPS at 0-10,000 ng/mL, SEAP SP fragments per particle were more abundant in LEV than in SEV, with fragments in LEV and SEV reaching a maximum at 1000 and 100 ng/mL, respectively. The content of SEAP SP fragments in LEV from IFNγ-stimulated RAW-Blue cells was higher than those from TNFα-stimulated cells, whereas that in SEV from TNFα-stimulated RAW-Blue cells was higher than those from IFNγ-stimulated cells. Moreover, the content of SEAP SP fragments in LEV and SEV decreased in the presence of W13, a calmodulin inhibitor. Collectively, these results indicate that the transportation of SP fragments to extracellular vesicles was changed by cellular activation, and calmodulin was involved in their transportation to LEV and SEV.

Signal Sequence-Dependent Orientation of Signal Peptide Fragments to Exosomes.

Signal peptides (SPs) not only mediate targeting to the endoplasmic reticulum (ER) but also play important roles as biomarkers and substances with physiological activity in extracellular fluids including blood. SPs are thought to be degraded intracellularly, making it unclear how they are transported from the ER to the extracellular fluid. In a recent study, we showed that a C-terminal fragment of the SP of a type I membrane protein, amyloid precursor protein (APP), was secreted into the extracellular fluid via exosomes using transformed HEK293 cells expressing APP SP flanking a reporter protein. In the present study, we demonstrate that a N-terminal fragment of the SP from a type II membrane protein, human placental secreted alkaline phosphatase (SEAP), is contained in exosomes and secreted into the extracellular fluid using HEK-Blue hTLR3 cells, which express both a human toll-like receptor 3 gene and an inducible SEAP reporter gene. When HEK-Blue hTLR3 cells were stimulated with a TLR3 ligand, a N-terminal fragment of SEAP SP in exosomes was increased in parallel with SEAP secretion in a concentration-dependent manner. These results indicated that SP fragments are exosomal components. In addition, migrating SP fragments were determined by characteristics of the signal-anchor sequence of membrane proteins. Furthermore, we found that SP fragments could bind to calmodulin (CALM), which is a cytosolic protein and also a component of exosomes, suggesting its involvement in the transportation of SP fragments from the endoplasmic reticulum to exosomes.

Secretion of signal peptides via extracellular vesicles.

Signal peptides (SPs) consist of short peptide sequences present at the N-terminal of newly synthesizing proteins and act as a zip code for the translocation of the proteins to the endoplasmic reticulum (ER). It was thought that the SPs are intracellularly degraded after translocation to the ER; however, recent studies showed cleaved SPs have diverse roles for controlling cell functions in auto- and/or intercellular manners. In addition, it still remains obscure how SP fragments translocate away from the site where they are produced. Extracellular vesicles (EV) are important for intercellular communication and can transport functional molecules to specific cells. In this study, we show that SPs are involved in EV from T-REx AspALP cells that were transfected with a human APP SP-inducible expression vector. There was no difference in the average particle size or particle concentration of EV collected from T-REx AspALP cells and T-REx Mock cells. When the SP content in the EV was examined by mass spectrometry, the C-terminal fragment of APP SP was identified in the exosomes (SEV) of T-REx AspALP cells. In our preparation of SEV fractions, no ER-specific proteins were detected; therefore, SPs may be included in SEV but not in the debris of degraded ER. This is the first indication that SPs are secreted from cells via EV.

Frankia communities at revegetating sites in Mt. Ontake, Japan.

In 1984 at Mt. Ontake in Japan, an earthquake caused a devastating landslide, and as a result, the vegetation on the south slope of the mountain was completely eliminated. In higher elevation (2000 m) areas, revegetation has not yet been completed even 30 years after the landslide. Revegetation progress throughout the area was heterogeneous. In the partially revegetated areas, actinorhizal plant species such as Alnus maximowiczii and Alnus matsumurae have been found. In the present study, we investigated the Frankia communities in the higher-elevation area using sequence analysis of the amplified nifH (dinitrogenase reductase) gene from nodule and soil samples collected in the disturbed region, undisturbed forest, and in the boundary between the disturbed region and the undisturbed forest. Phylogenetic analysis of partial nifH sequences revealed the presence of six clusters, each of which consisted of highly similar (> 99%) sequences. Four clusters showed significant sequence similarity to Frankia (three Alnus- and a Casuarina-infecting strains). Diversity in the Frankia community was relatively low-only one or two clusters were detected in a site. At most of the sampling sites, a dominant cluster in a nodule coincided with that in rhizosphere soil, indicating that community structure in the rhizosphere is a primary factor that determines occupancy in a nodule. No significant difference in community structure was observed between plant species. Diversity in the Frankia community varied depending on revegetation progress. Cluster A, which was the most dominant in the disturbed region, was likely to have invaded from undisturbed forest.

Inducible nitric oxide synthase during the late phase of sepsis is associated with hypothermia and immune cell migration.

Hypothermia is a significant sign of sepsis, which is associated with poor prognosis, but few mechanisms underlying the regulation of hypothermia are known. Inducible nitric oxide synthase (iNOS) is a key inflammatory mediator of sepsis. However, the therapeutic benefit of iNOS inhibition in sepsis is still controversial, and requires elucidation in an accurate model system. In this study, wild-type (WT) mice showed temperature drops in a biphasic manner at the early and late phase of sepsis, and all mice died within 48 h of sepsis. In contrast, iNOS-knockout (KO) mice never showed the second temperature drop and exhibited improved mortality. Plasma nitric oxide (NO) levels of WT mice increased in the late phase of sepsis and correlated to hypothermia. The results indicate that iNOS-derived NO during the late phase of sepsis caused vasodilation-induced hypothermia and a lethal hypodynamic state. The expression of the iNOS mRNA was high in the lung of WT mice with sepsis, which reflects the pathology of acute respiratory distress syndrome (ARDS). We obtained the results in a modified keyhole-type cecal ligation and puncture model of septic shock induced by minimally invasive surgery. In this accurate and reproducible model system, we transplanted the bone marrow cells of GFP transgenic mice into WT and iNOS-KO mice, and evaluated the role of increased pulmonary iNOS expression in cell migration during the late phase of sepsis. We also investigated the quantity and type of bone marrow-derived cells (BMDCs) in the lung. The number of BMDCs in the lung of iNOS-KO mice was less than that in the lung of WT mice. The major BMDCs populations were CD11b-positive, iNOS-negative cells in WT mice, and Gr-1-positive cells in iNOS-KO mice that expressed iNOS. These results suggest that sustained hypothermia may be a beneficial guide for future iNOS-targeted therapy of sepsis, and that iNOS modulated the migratory efficiency and cell type of BMDCs in septic ARDS.

LC-MS/MS imaging with thermal film-based laser microdissection.

Mass spectrometry (MS) imaging is a useful tool for direct and simultaneous visualization of specific molecules. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to evaluate the abundance of molecules in tissues using sample homogenates. To date, however, LC-MS/MS has not been utilized as an imaging tool because spatial information is lost during sample preparation. Here we report a new approach for LC-MS/MS imaging using a thermal film-based laser microdissection (LMD) technique. To isolate tissue spots, our LMD system uses a 808-nm near infrared laser, the diameter of which can be freely changed from 2.7 to 500 µm; for imaging purposes in this study, the diameter was fixed at 40 µm, allowing acquisition of LC-MS/MS images at a 40-µm resolution. The isolated spots are arranged on a thermal film at 4.5-mm intervals, corresponding to the well spacing on a 384-well plate. Each tissue spot is handled on the film in such a manner as to maintain its spatial information, allowing it to be extracted separately in its individual well. Using analytical LC-MS/MS in combination with the spatial information of each sample, we can reconstruct LC-MS/MS images. With this imaging technique, we successfully obtained the distributions of pilocarpine, glutamate, γ-aminobutyric acid, acetylcholine, and choline in a cross-section of mouse hippocampus. The protocol we established in this study is applicable to revealing the neurochemistry of pilocarpine model of epilepsy. Our system has a wide range of uses in fields such as biology, pharmacology, pathology, and neuroscience. Graphical abstract Schematic Indication of LMD-LC-MS/MS imaging.

Protective effect of INI-0602, a gap junction inhibitor, on dopaminergic neurodegeneration of mice with unilateral 6-hydroxydopamine injection.

INI-0602, a novel gap junction hemichannel inhibitor, was administered to hemi-Parkinsonism mice generated by striatal 6-hydroxydopamine injection. INI-0602 prevented the toxic activation of microglia, such as the increased number of the activated form, enlargement of cell bodies and induction of proinflammatory cytokines, such as IL-1ß and TNFα, in the ipsilateral striatum. On the other hand, INI-0602 induced the expression of neurotrophic factors, such as brain-derived neurotrophic factor and NT-4/5, in the 6-hydroxydopamine-treated striatum. INI-0602 treatment blocked not only dopaminergic loss in both the striatum and substantia nigra, but also apomorphine-induced rotational behavior.

Glutamate release from astrocyte cell-line GL261 via alterations in the intracellular ion environment.

Astrocytes modify and maintain neural activity and functions via gliotransmitter release such as, glutamate. They also change their properties and functions in response to alterations of ion environment resulting from neurotransmission; however, the direct evidence for whether intracellular ion alteration in astrocytes triggers gliotransmitter release is not indicated. Recent studies have reported that channelrhodopsin-2 (ChR2) is useful for alteration of intracellular ion environment in several types of cells with blue light exposure. Here, we show that ChR2-expressing GL261 (GLChR2) cells, clonal astrocytes, change their properties by photo-activation. Increased intracellular sodium and calcium ion concentrations and an altered membrane potential were observed in GLChR2 cells with blue light exposure. Alterations in the intracellular ion environment caused intracellular acidification and the inhibition of proliferation. In addition, it triggered glutamate release from GLChR2 cells. Glutamate from GLChR2 cells acted on N18 cells, clonal neuronal cells, as both a transmitter and neurotoxin depending on photo-activation. Our results show that the properties of ChR2-expressing astrocytes can be controlled by blue light exposure, and cation influx through photo-activated ChR2 might trigger functional cation influx via endogenous channels and result in the increase of glutamate release. Further, our results suggest that ChR2-expressing glial cells could become a useful tool in understanding the roles of glial cell activation and neural communication in the regulation of brain functions.

Photosensitizer-singlet oxygen sensor conjugated silica nanoparticles for photodynamic therapy and bioimaging.

Intracellular singlet oxygen (1O2) generation and detection help optimize the outcome of photodynamic therapy (PDT). Theranostics programmed for on-demand phototriggered 1O2 release and bioimaging have great potential to transform PDT. We demonstrate an ultrasensitive fluorescence turn-on sensor-sensitizer-RGD peptide-silica nanoarchitecture and its 1O2 generation-releasing-storing-sensing properties at the single-particle level or in living cells. The sensor and sensitizer in the nanoarchitecture are an aminomethyl anthracene (AMA)-coumarin dyad and a porphyrin or CdSe/ZnS quantum dots (QDs), respectively. The AMA in the dyad quantitatively quenches the fluorescence of coumarin by intramolecular electron transfer, the porphyrin or QD moiety generates 1O2, and the RGD peptide facilitates intracellular delivery. The small size, below 200 nm, as verified by scanning electron microscopy and differential light scattering measurements, of the architecture within the 1O2 diffusion length enables fast and efficient intracellular fluorescence switching by the tandem ultraviolet (UV)-visible or visible-near-infrared (NIR) photo-triggering. While the red emission and 1O2 generation by the porphyrin are continually turned on, the blue emission of coumarin is uncaged into 230-fold intensity enhancement by on-demand photo-triggering. The 1O2 production and release by the nanoarchitecture enable spectro-temporally controlled cell imaging and apoptotic cell death; the latter is verified from cytotoxic data under dark and phototriggering conditions. Furthermore, the bioimaging potential of the TCPP-based nanoarchitecture is examined in vivo in B6 mice.

Singlet-oxygen-sensitizing near-infrared-fluorescent multimodal nanoparticles.

Nanoprobes based on quantum clusters (QC) with near-infrared fluorescence, magnetic-resonance-imaging contrast, and singlet-oxygen-sensitized intracellular fluorescence are studied. The generation of singlet oxygen and singlet-oxygen-sensitized fluorescence uncaging by magnetic and NIR-emitting nanoparticles are exploited for multimodal bioimaging in vitro.

Toward Feature-Preserving Vector Field Compression.

The objective of this work is to develop error-bounded lossy compression methods to preserve topological features in 2D and 3D vector fields. Specifically, we explore the preservation of critical points in piecewise linear and bilinear vector fields. We define the preservation of critical points as, without any false positive, false negative, or false type in the decompressed data, (1) keeping each critical point in its original cell and (2) retaining the type of each critical point (e.g., saddle and attracting node). The key to our method is to adapt a vertex-wise error bound for each grid point and to compress input data together with the error bound field using a modified lossy compressor. Our compression algorithm can be also embarrassingly parallelized for large data handling and in situ processing. We benchmark our method by comparing it with existing lossy compressors in terms of false positive/negative/type rates, compression ratio, and various vector field visualizations with several scientific applications.

Calmodulin as a Key Regulator of Exosomal Signal Peptides.

Signal peptides (SPs) and their fragments play important roles as biomarkers and substances with physiological functions in extracellular fluid. We previously reported that SP fragments were released into extracellular fluid via exosomes and bound to calmodulin (CaM), an exosomal component, in a cell-free system. However, it currently remains unclear whether CaM intracellularly interacts with SP fragments or is involved in the trafficking of these fragments to exosomes. Therefore, the present study examined the binding of CaM to SP fragments in T-REx AspALP cells, transformed HEK293 cells expressing amyloid precursor protein (APP) SP flanking a reporter protein, and their exosomes. APP SP fragments were detected in exosomes from T-REx AspALP cells in the absence of W13, a CaM inhibitor, but were present in lower amounts in exosomes from W13-treated cells. Cargo proteins, such as Alix, CD63, and CD81, were increased in W13-treated T-REx AspALP cells but were decreased in their exosomes. Furthermore, CaM interacted with heat shock protein 70 and CD81 in T-REx AspALP cells and this increased in the presence of W13. APP SP fragments were detected in intracellular CaM complexes in the absence of W13, but not in its presence. These results indicate that CaM functions as a key regulator of the transport of SP fragments into exosomes and plays novel roles in the sorting of contents during exosomal biogenesis.

Efficient compressed database of equilibrated configurations of ring-linear polymer blends for MD simulations.

To effectively archive configuration data during molecular dynamics (MD) simulations of polymer systems, we present an efficient compression method with good numerical accuracy that preserves the topology of ring-linear polymer blends. To compress the fraction of floating-point data, we used the Jointed Hierarchical Precision Compression Number - Data Format (JHPCN-DF) method to apply zero padding for the tailing fraction bits, which did not affect the numerical accuracy, then compressed the data with Huffman coding. We also provided a dataset of well-equilibrated configurations of MD simulations for ring-linear polymer blends with various lengths of linear and ring polymers, including ring complexes composed of multiple rings such as polycatenane. We executed 109 MD steps to obtain 150 equilibrated configurations. The combination of JHPCN-DF and SZ compression achieved the best compression ratio for all cases. Therefore, the proposed method enables efficient archiving of MD trajectories. Moreover, the publicly available dataset of ring-linear polymer blends can be employed for studies of mathematical methods, including topology analysis and data compression, as well as MD simulations.

[Management of patients with bronchial foreign bodies].

The aspiration of foreign bodies into the bronchus frequently occurs in children as well as in elderly people. Foreign bodies in the airway not only cause chronic cough and pneumonia, but also result in life-threatening conditions, such as dyspnea and cyanosis. This report presents the clinical characteristics of 6 patients with bronchial foreign bodies who were treated between 2006 and 2010, including 4 male and 2 female patients. The age of the patients ranged from 8 to 83 years old. Foreign bodies were located in the right bronchial tree in all the patients. Chest X-rays showed pneumonia or atelectasis in 5 out of 6 patients. The foreign bodies were an artificial teeth or a tooth in 5 patients, and a fish bone in 1 patient. Five patients had fiberoptic bronchoscopy under local anesthesia, although an 8-year-old girl required general anesthesia with a laryngeal mask. Surgery was needed in only one case. Bronchial foreign bodies present a large range of symptoms, from trivial symptoms to irreversible damage to the bronchus and the lung, which can be life threatening. Nonspecific respiratory symptoms may be mistakenly attributed to other medical diagnoses unless there is a clear history of aspiration. However, an early diagnosis is very important, because inflammatory granulation due to long-term impaction of foreign bodies makes its removal difficult.

[Surgical treatment for patients with pulmonary sclerosing hemangioma].

Sclerosing hemangioma of the lung, a rare disease, is a low grade malignancy possibly originating from type II pneumocytes or Clara cells. We report the clinical characteristics of 8 patients who underwent surgical resection for sclerosing hemangioma between 2005 and 2010 in our hospital. All cases were female, and the average age was 50 (range: 28-83) years old. The median tumor doubling time was 965 days, suggesting they were slowly growing tumors. In the present cases, five patients had another lung disease: lung cancer in two, metastatic lung tumor in one and atypical adenomatous hyperplasia in two patients. Intraoperative frozen section examinations were performed in seven cases. Five patients were diagnosed correctly, but two patients were diagnosed with adenocarcinoma and organizing pneumonia. As a clinical characteristics, sclerosing hemangioma in the present study showed well-demarcated and slow-growing tumor. The postoperative clinical courses of all cases were uneventful, and no findings of recurrence distant metastasis, lymph node metastasis and local recurrence after surgery were observed in any of the patients.

Evaluation of topoisomerase I/topoisomerase IIalpha status in esophageal cancer.

Human DNA topoisomerases I and IIalpha (Topo-I and -II alpha) are essential for vital cellular processes such as DNA replication, transcription, translation, recombination, and repair. The purpose of this study was to investigate the clinical significance of the expression of Topo-I and Topo-II alpha. Twenty-nine specimens of esophageal squamous cell carcinoma from patients who had been treated by complete resection of the esophageal tumor were studied by an immunohistochemical analysis. High expression of Topo I and II alpha was identified in 48.7% and 55.2% of tumors, respectively. Neither the Topo-I nor -II alpha expression level had any association with clinical characteristics, including differentiation and the depth of tumor invasion, lymph node metastasis, or the patient prognosis. However, a significant positive correlation was observed between the expression levels of Topo-I and Topo-II alpha. Our study results underscore the potential role of topoisomerase expression in esophageal cancer and further exploratory investigation is necessary to evaluate topoisomerase expression as a surrogate marker in chemotherapy with topoisomerase inhibitor for esophageal cancer.

[Clinical analysis of esophageal cancer associated with other primary cancers].

This study aims to investigate the therapeutic and prognostic implications of esophageal cancer in patients with other primary cancer. Between April 1992 and December 2008, in 83 patients underwent surgery for esophageal cancer at our department. Among them, 24 patients (28.9%) had medical history of additional primary cancer. There were 16 metachronous cancers and 8 synchronous cancers. Six patients had antecedent other primary cancers, and subsequent primary cancers developed in 10 patients. The other primary cancers included head and neck cancer in 8 patients, gastric cancer in 8, lung cancer in 6, colorectal cancer in 3, and other cancer in 3. The patients with other primary cancers were both heavy smokers and heavy drinkers in comparison to those without other primary cancers. The post-operative 5-year survival rate in patients with subsequent cancers, antecedent cancers, and synchronous cancers were 100%, 70.0%, and 46.9%. The 5-year survival rate was 33.4% in patients without other primary cancers. A high incidence of multiple primary cancers was observed in patients with esophageal carcinoma but the prognosis of these patients with metachronous cancers are better than that of patient with synchronous cancers and patients without other primary cancers. Post-operative follow up is considered to be necessary for early detection of multiple occurrences of carcinoma, especially in the upper aerodigestive tract.

A case of pleuroperitoneal communication in which establishing a laparoscopic pneumoperitoneum was useful for the detection of a fistula.

BACKGROUND: Pleuroperitoneal communication (PPC) is rarely observed, accounting for 1.6% of all patients who undergo continuous ambulatory peritoneal dialysis (CAPD). Although there have been several reports concerning the management of this condition, we have encountered several cases in which control failed. We herein report a valuable case of PPC in which laparoscopic pneumoperitoneum with video-assisted thoracic surgery (VATS) was useful for supporting the diagnosis and treatment. CASE PRESENTATION: The patient was a 58-year-old woman with chronic renal failure due to chronic renal inflammation who was referred to a nephrologist in our hospital to undergo an operation for the induction of CAPD. Post-operatively, she had respiratory failure, and chest X-ray and computed tomography (CT) showed right-sided hydrothorax that decreased when the injection of peritoneal dialysate was interrupted. Therefore, PPC was suspected, and she was referred to our department for surgical repair. We planned surgical treatment via video-assisted thoracic surgery. During the surgery, we failed to detect any lesions with thoracoscopy alone; we therefore added a laparoscopic port at her right-sided abdomen near the navel and infused CO2 gas into the abdominal cavity. On thoracoscopy, bubbles were observed emanating from a small pore at the central tendon of the diaphragm, which was considered to be the lesion responsible for the PPC. We closed it by suturing directly. CONCLUSIONS: VATS with laparoscopic pneumoperitoneum should be considered as an effective method for inspecting tiny pores of the diaphragm, especially when the lesions responsible for PPC are difficult to detect.

Voxel-based simulation of flow and temperature in the human nasal cavity.

The nasal airway is an extremely complex structure, therefore grid generation for numerical prediction of airflow in the nasal cavity is time-consuming. This paper describes the development of a voxel-based model with a Cartesian structured grid, which is characterized by robust and automatic grid generation, and the simulation of the airflow and air-conditioning in an individual human nasal airway. Computed tomography images of a healthy adult nose were used to reconstruct a virtual three-dimensional model of the nasal airway. Simulations of quiet restful inspiratory flow were then performed using a Neumann boundary condition for the energy equation to adequately resolve the flow and heat transfer. General agreements of airflow patterns, which were a high-speed jet posterior to the nasal valve and recirculating flow that occupied the anterior part of the upper cavity, and temperature distributions of the airflow and septum wall were confirmed by comparing in-vivo measurements with numerical simulation results.

The sodium-glucose cotransporter-2 inhibitor Tofogliflozin prevents the progression of nonalcoholic steatohepatitis-associated liver tumors in a novel murine model.

BACKGROUND: Diabetes and obesity contribute to the pathogenesis of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). However, how diabetes and obesity accelerate liver tumorigenesis remains to be fully understood. Moreover, to verify the therapeutic potential of anti-diabetic drugs, there exists a strong need for appropriate animal models that recapitulate human pathophysiology of NASH and HCC. METHODS: We established a novel murine model of NASH-associated liver tumors using genetically obese melanocortin 4 receptor-deficient mice fed on Western diet in combination with a chemical procarcinogen, and verified the validity of our model in evaluating drug efficacy. FINDINGS: Our model developed multiple liver tumors together with obesity, diabetes, and NASH within a relatively short period (approximately 3 months). In this model, sodium glucose cotransporter 2 inhibitor Tofogliflozin prevented the development of NASH-like liver phenotypes and the progression of liver tumors. Tofogliflozin attenuated p21 expression of hepatocytes in non-tumorous lesions in the liver. INTERPRETATION: Tofogliflozin treatment attenuates cellular senescence of hepatocytes under obese and diabetic conditions. This study provides a unique animal model of NASH-associated liver tumors, which is applicable for assessing drug efficacy to prevent or treat NASH-associated HCC.