Expression of RNautophagy/DNautophagy-related genes is regulated under control of an innate immune receptor.

Double-stranded RNA (dsRNA) is a molecular pattern uniquely produced in cells infected with various viruses as a product or byproduct of replication. Cells detect such molecules, which indicate non-self invasion, and induce diverse immune responses to eliminate them. The degradation of virus-derived molecules can also play a role in the removal of pathogens and suppression of their replication. RNautophagy and DNautophagy are cellular degradative pathways in which RNA and DNA are directly imported into a hydrolytic organelle, the lysosome. Two lysosomal membrane proteins, SIDT2 and LAMP2C, mediate nucleic acid uptake via this pathway. Here, we showed that the expression of both SIDT2 and LAMP2C is selectively upregulated during the intracellular detection of poly(I:C), a synthetic analog of dsRNA that mimics viral infection. The upregulation of these two gene products upon poly(I:C) introduction was transient and synchronized. We also observed that the induction of SIDT2 and LAMP2C expression by poly(I:C) was dependent on MDA5, a cytoplasmic innate immune receptor that directly recognizes poly(I:C) and induces various antiviral responses. Finally, we showed that lysosomes can target viral RNA for degradation via RNautophagy and may suppress viral replication. Our results revealed a novel degradative pathway in cells as a downstream component of the innate immune response and provided evidence suggesting that the degradation of viral nucleic acids via RNautophagy/DNautophagy contributes to the suppression of viral replication.

Experimental inoculation of a tissue homogenate containing porcine circovirus type 3 obtained after two in vivo passages in NIBS miniature pigs.

Porcine circovirus type 3 (PCV3) is a novel porcine circovirus that has been detected in pigs showing various clinical and pathological conditions, as well as in many asymptomatic pigs. The pathogenesis of PCV3 infection in pigs remains unclear. To evaluate the in vivo growth and pathogenicity of PCV3, we performed two experiments on PCV3 infection in laboratory-grade miniature pigs with strictly controlled genetic backgrounds and microbiological status. A PCV3 passage experiment confirmed PCV3 genome detection in the sera and multiple organs via in vivo serial passage generations. PCV3 was successively passaged in miniature pigs by inoculating tissue homogenates from infected pigs supporting Koch's principles. In the PCV3 infection experiment, viremia was observed in all the inoculated pigs, and transient neurological signs were observed in one of the three pigs. Histopathologically, all three pigs in the PCV3 inoculation group exhibited lung disorders such as interstitial pneumonia and lymphoplasmacytic perivasculitis. In addition, one pig with neurological signs in the PCV3 inoculation group showed focal thrombosis in the meninges of the cerebellum. Vascular lesions in both the lungs and brain suggest that PCV3 may cause injury to vascular tissues. In situ hybridization (ISH)-RNA analysis demonstrated that the PCV3 genome was localized in the lymph nodes of pigs inoculated with PCV3. The PCV3 in vivo passage system in NIBS miniature pigs will help investigate the pathogenicity of PCV3.

Diffuse pleural thickening and thoracic contraction: An indistinguishable case from malignant pleural mesothelioma.

The differential diagnosis of reactive mesothelial hyperplasia and mesothelioma is difficult. We present a rare case of diffuse pleural thickening with thoracic contraction that was indistinguishable from mesothelioma. A 66-year-old woman with no history of asbestos exposure visited our hospital with a complaint of dyspnea. The clinical findings included circumferential pleural thickening on chest computed tomography image and a high concentration of hyaluronic acid in the pleural fluid. Pleural biopsies obtained by thoracoscopy under local anesthesia were pathologically consistent with mesothelioma, but the patient refused to take any kind of mesothelioma treatments. Four months later, she consented to a surgical pleural biopsy under general anesthesia to obtain larger tissue samples, which included typical proliferating polygonal cells positive for CAM5.2, calretinin, WT-1, D2-40, CK5/6, epithelial membrane antigen, and glucose transporter-1 and negative for carcinoembryonic antigen, BerEP4, and MOC31. The analysis was consistent with diagnosis of epithelioid mesothelioma. Fluorescence in situ hybridization, however, showed the presence of p16 gene, and the expression of BRCA1-associated protein-1 was detected by immunohistochemistry. Our final diagnosis was diffuse pleural thickening unrelated to asbestos exposure. Differential diagnosis of diffuse pleural thickening and malignant mesothelioma is thus difficult and routine immunohistochemical examinations are often insufficient for accurate diagnosis. Multiple diagnostic methods are required for correct diagnosis in a clinically marginal case.

Immunological features of a lung cancer patient achieving an objective response with anti-programmed death-1 blockade therapy.

The role of immune checkpoint inhibitors in metastatic lung cancer has been established in recent years and the pretherapeutic profiles of the tumor microenvironment in responders have been increasingly reported. The role of salvage surgery and the immune profiles of the posttherapeutic specimens in patients achieving an objective response have rarely been studied. We report a case of metastatic lung cancer treated by anti-programmed death-1 Ab followed by surgical resection. The immune status of the tumor was assessed, showing germinal center formation, memory B cell infiltration, and a high frequency of interferon gamma -secreting T cells.

Classification of lung adenocarcinoma transcriptome subtypes from pathological images using deep convolutional networks.

PURPOSE: Convolutional neural networks have become rapidly popular for image recognition and image analysis because of its powerful potential. In this paper, we developed a method for classifying subtypes of lung adenocarcinoma from pathological images using neural network whose that can evaluate phenotypic features from wider area to consider cellular distributions. METHODS: In order to recognize the types of tumors, we need not only to detail features of cells, but also to incorporate statistical distribution of the different types of cells. Variants of autoencoders as building blocks of pre-trained convolutional layers of neural networks are implemented. A sparse deep autoencoder which minimizes local information entropy on the encoding layer is then proposed and applied to images of size [Formula: see text]. We applied this model for feature extraction from pathological images of lung adenocarcinoma, which is comprised of three transcriptome subtypes previously defined by the Cancer Genome Atlas network. Since the tumor tissue is composed of heterogeneous cell populations, recognition of tumor transcriptome subtypes requires more information than local pattern of cells. The parameters extracted using this approach will then be used in multiple reduction stages to perform classification on larger images. RESULTS: We were able to demonstrate that these networks successfully recognize morphological features of lung adenocarcinoma. We also performed classification and reconstruction experiments to compare the outputs of the variants. The results showed that the larger input image that covers a certain area of the tissue is required to recognize transcriptome subtypes. The sparse autoencoder network with [Formula: see text] input provides a 98.9% classification accuracy. CONCLUSION: This study shows the potential of autoencoders as a feature extraction paradigm and paves the way for a whole slide image analysis tool to predict molecular subtypes of tumors from pathological features.

First detection of porcine circovirus type 3 in Japan.

Porcine circovirus associated diseases (PCVAD) have multiple manifestations that have been attributed to porcine circovirus type 2 (PCV2). Recently, a novel porcine circovirus, PCV type 3 (PCV3), was identified in pigs with systemic inflammation of unknown etiology. In this study, we tried to detect the PCV3 genome in tissue samples collected from Japanese pig herds in 2016. The PCV3 genome was detected by PCR in 7 of 73 samples. The homology between each Japanese strain was 99.5% for the full-length sequence and 98.9 to 99.2% for the open reading frame 2. These results suggest that PCV3 has already invaded Japanese pig farms.

An integrative network-based approach to identify novel disease genes and pathways: a case study in the context of inflammatory bowel disease.

BACKGROUND: There are different and complicated associations between genes and diseases. Finding the causal associations between genes and specific diseases is still challenging. In this work we present a method to predict novel associations of genes and pathways with inflammatory bowel disease (IBD) by integrating information of differential gene expression, protein-protein interaction and known disease genes related to IBD. RESULTS: We downloaded IBD gene expression data from NCBI's Gene Expression Omnibus, performed statistical analysis to determine differentially expressed genes, collected known IBD genes from DisGeNet database, which were used to construct a IBD related PPI network with HIPPIE database. We adapted our graph-based clustering algorithm DPClusO to cluster the disease PPI network. We evaluated the statistical significance of the identified clusters in the context of determining the richness of IBD genes using Fisher's exact test and predicted novel genes related to IBD. We showed 93.8% of our predictions are correct in the context of other databases and published literatures related to IBD. CONCLUSIONS: Finding disease-causing genes is necessary for developing drugs with synergistic effect targeting many genes simultaneously. Here we present an approach to identify novel disease genes and pathways and discuss our approach in the context of IBD. The approach can be generalized to find disease-associated genes for other diseases.

Purification and characterization of immunogenic recombinant virus-like particles of porcine circovirus type 2 expressed in silkworm pupae.

Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm-baculovirus expression vector system (silkworm-BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine.

Efficacy of genogroup 1 based porcine epidemic diarrhea live vaccine against genogroup 2 field strain in Japan.

BACKGROUND: Porcine epidemic diarrhea (PED) is a lethal infectious disease in suckling piglets with symptoms including watery diarrhea caused by PED virus (PEDV). Since the late 1990's, live vaccines based on genogroup 1 virus have been used in Japan, and a significant amount of the vaccine has been used even after new genogroups invaded in 2013. In this study, we evaluated the effect of a conventional PED live vaccine on a newly prevalent genogroup 2 field strain in experimental and field situations. METHODS: Two pregnant sows were administered twice the live vaccine before farrowing. A pregnant sow was served as a negative control. All newborn piglets were challenged with the genogroup 2 virus, and clinical signs were monitored for 7 days post challenge. PEDV-specific immune responses in serum and milk of the sows were assayed by virus neutralization assay. The efficacy of PED live vaccine in vaccinated or non-vaccinated farms was evaluated by comparing the mortality rate of suckling piglets after the onset of PED. RESULTS: The challenged piglets exhibited watery diarrhea with or without vaccination. However, the clinical score of piglets born from vaccinated sows significantly improved after the 4th day of the challenge. The survival rate of piglets in the vaccinated group at the end of the experimental period was 80%, whereas in the control group was 0%. Neutralizing antibody titers in serum and milk of control sow was negative throughout the experimental period, whereas high titers were observed in the vaccinated sows. The vaccinated farms significantly reduced the mortality rate of suckling piglets after the onset of PED, compared to farms not vaccinated. CONCLUSIONS: The conventional PED live vaccine induced the lactogenic immunity to vaccinated sows and showed partial protection against the genogroup 2 virus both under the experimental and field conditions.

Successful removal of porcine circovirus-1 from immunoglobulin G formulated in glycine solution using nanofiltration.

Porcine circovirus (PCV) is a potentially harmful virus that has been shown to contaminate biological products. The virus is resistant to many inactivation and/or removal procedures performed during manufacturing. Anion exchange chromatography has been shown to be useful for PCV type 1 (PCV1) removal; however, reduction of PCV1 using methods such as heat inactivation, low pH, nanofiltration, UV-C, and gamma irradiation has not been successful. Therefore, in this study, we evaluate various conditions for process solutions during nanofiltration using PCV1. The results indicated that PCV could be effectively removed from glycine solution at 0.1-0.3 M, pH 4.0 without IgG, using a nanofilter with a pore size of 19 nm (19-nm filter); log reduction values (LRVs) of ≥4.5 and ≥ 5.0, respectively, were obtained. In contrast, PCV1 was significantly removed (LRV: 2.2) in glycine solution at 0.3 M, pH 6.0 with 1.0% IgG using the 19-nm filter, but some virus genomes were detected in the filtrates. In summary, the use of a 19-nm filter in glycine solution with/without IgG is an appropriate condition for PCV removal.

Comparison of Airway Measurements for Tracheobronchial Stenosis Between Stereoscopic Bronchoscope and MD-CT.

BACKGROUND: Stereoscopic bronchoscopy is a new diagnostic tool to measure the diameter and cross-sectional area of the airway. The stereoscopic bronchoscope, which operates the same as a standard bronchoscope, utilizes 2 lenses to measure the airway using the principles of triangulation. Furthermore, the stereoscopic bronchoscope has the capability to measure the size of the airway during intervention in real-time, including variable stenosis. MATERIALS AND METHODS: To prospectively compare preoperative stereoscopic and multidetector computed tomography (MD-CT) images to select the appropriate stent size for airway stenosis. Stereoscopic and MD-CT images were then measured to confirm the correct placement of the stent. RESULTS: Airway stenting was performed on 21 consecutive patients of whom, 15 were diagnosed with malignant and 6 with benign diseases. In total, 165 measurements were taken (134 healthy; 31 affected). For the diameter, Bland-Altman plots were used to measure data from 165 matched stereoscopic and MD-CT measurement sites (bias, 0.40±2.86 mm SD; percentage error, 33%), 134 healthy sites (bias, 0.554±2.83 mm SD; percentage error, 34%), and 31 affected sites (bias, 1.20±2.67 mm SD; percentage error, 52%). For the cross-sectional area, matched stereoscopic and MD-CT measurements were analyzed for 65 sites (bias, -10.53±92.85 mm SD; percentage error, 89%), 49 healthy sites (bias, -9.88±39.00 mm SD; percentage error, 32%), and 16 affected sites (bias, -13.12±48.81 mm SD; percentage error, 92%). CONCLUSION: Stereoscopic bronchoscopy was able to accurately measure the size of the airway during intervention, to assist in selecting the appropriate size of the stent.

Real-time prediction of cell division timing in developing zebrafish embryo.

Combination of live-imaging and live-manipulation of developing embryos in vivo provides a useful tool to study developmental processes. Identification and selection of target cells for an in vivo live-manipulation are generally performed by experience- and knowledge-based decision-making of the observer. Computer-assisted live-prediction method would be an additional approach to facilitate the identification and selection of the appropriate target cells. Herein we report such a method using developing zebrafish embryos. We choose V2 neural progenitor cells in developing zebrafish embryo as their successive shape changes can be visualized in real-time in vivo. We developed a relatively simple mathematical method of describing cellular geometry of V2 cells to predict cell division-timing based on their successively changing shapes in vivo. Using quantitatively measured 4D live-imaging data, features of V2 cell-shape at each time point prior to division were extracted and a statistical model capturing the successive changes of the V2 cell-shape was developed. By applying sequential Bayesian inference method to the model, we successfully predicted division-timing of randomly selected individual V2 cells while the cell behavior was being live-imaged. This system could assist pre-selecting target cells desirable for real-time manipulation-thus, presenting a new opportunity for in vivo experimental systems.

Memory of cell shape biases stochastic fate decision-making despite mitotic rounding.

Cell shape influences function, and the current model suggests that such shape effect is transient. However, cells dynamically change their shapes, thus, the critical question is whether shape information remains influential on future cell function even after the original shape is lost. We address this question by integrating experimental and computational approaches. Quantitative live imaging of asymmetric cell-fate decision-making and their live shape manipulation demonstrates that cellular eccentricity of progenitor cell indeed biases stochastic fate decisions of daughter cells despite mitotic rounding. Modelling and simulation indicates that polarized localization of Delta protein instructs by the progenitor eccentricity is an origin of the bias. Simulation with varying parameters predicts that diffusion rate and abundance of Delta molecules quantitatively influence the bias. These predictions are experimentally validated by physical and genetic methods, showing that cells exploit a mechanism reported herein to influence their future fates based on their past shape despite dynamic shape changes.

Extracellular Release of Annexin A2 is Enhanced upon Oxidative Stress Response via the p38 MAPK Pathway after Low-Dose X-Ray Irradiation.

The extracellular microenvironment affects cellular responses to various stressors including radiation. Annexin A2, which was initially identified as an intracellular molecule, is also released into the extracellular environment and is known to regulate diverse cell surface events, however, the molecular mechanisms underlying its release are not well known. In this study, we found that in cultured human cancer and non-cancerous cells an extracellular release of annexin A2 was greatly enhanced 1-4 h after a single 20 cGy X-ray dose, but not after exposure to ultraviolet C (UVC) radiation. Extracellular release of annexin A2 was also enhanced after H2O2 and nicotine treatments, which was suppressed by pretreatment with the antioxidant, N-acetyl cysteine. Among the oxidative stress pathway molecules examined in HeLa cells, AMP-activated protein kinase α (AMPKα) and p38 mitogen-activated protein kinase (MAPK) were mostly activated by low-dose X-ray radiation, and the p38 MAPK inhibitor, SB203580, but not compound C (an AMPKα inhibitor), suppressed the enhancement of the annexin A2 extracellular release after low-dose X irradiation. In addition, the enhancement was suppressed in the cells in which p38α MAPK was downregulated by siRNA. HeLa cells and human cultured cells preirradiated with 20 cGy or precultured in media from low-dose X-irradiated cells showed an increase in resistance to radiation-induced cell death, and the increase was suppressed by treatment of the irradiated cell-derived media with anti-annexin A2 antibodies. In addition, extracellularly added recombinant annexin A2 conferred cellular radiation resistance. These results indicate that an oxidative stress-activated pathway via p38 MAPK was involved in the extracellular release of annexin A2, and this pathway was stimulated by low-dose X-ray irradiation. Furthermore, released annexin A2 may function in low-dose ionizing radiation-induced responses, such as radioresistance.

Integrated pathway-based transcription regulation network mining and visualization based on gene expression profiles.

Conventionally, workflows examining transcription regulation networks from gene expression data involve distinct analytical steps. There is a need for pipelines that unify data mining and inference deduction into a singular framework to enhance interpretation and hypotheses generation. We propose a workflow that merges network construction with gene expression data mining focusing on regulation processes in the context of transcription factor driven gene regulation. The pipeline implements pathway-based modularization of expression profiles into functional units to improve biological interpretation. The integrated workflow was implemented as a web application software (TransReguloNet) with functions that enable pathway visualization and comparison of transcription factor activity between sample conditions defined in the experimental design. The pipeline merges differential expression, network construction, pathway-based abstraction, clustering and visualization. The framework was applied in analysis of actual expression datasets related to lung, breast and prostrate cancer.

Development and mining of a volatile organic compound database.

Volatile organic compounds (VOCs) are small molecules that exhibit high vapor pressure under ambient conditions and have low boiling points. Although VOCs contribute only a small proportion of the total metabolites produced by living organisms, they play an important role in chemical ecology specifically in the biological interactions between organisms and ecosystems. VOCs are also important in the health care field as they are presently used as a biomarker to detect various human diseases. Information on VOCs is scattered in the literature until now; however, there is still no available database describing VOCs and their biological activities. To attain this purpose, we have developed KNApSAcK Metabolite Ecology Database, which contains the information on the relationships between VOCs and their emitting organisms. The KNApSAcK Metabolite Ecology is also linked with the KNApSAcK Core and KNApSAcK Metabolite Activity Database to provide further information on the metabolites and their biological activities. The VOC database can be accessed online.

A Glimpse to Background and Characteristics of Major Molecular Biological Networks.

Recently, biology has become a data intensive science because of huge data sets produced by high throughput molecular biological experiments in diverse areas including the fields of genomics, transcriptomics, proteomics, and metabolomics. These huge datasets have paved the way for system-level analysis of the processes and subprocesses of the cell. For system-level understanding, initially the elements of a system are connected based on their mutual relations and a network is formed. Among omics researchers, construction and analysis of biological networks have become highly popular. In this review, we briefly discuss both the biological background and topological properties of major types of omics networks to facilitate a comprehensive understanding and to conceptualize the foundation of network biology.

Hochu­ekki­to (Bu­zhong­yi­qi­tang), a herbal medicine, enhances cisplatin­induced apoptosis in HeLa cells.

Hochu­ekki­to (HET), a Kampo herbal medicine composed of ten medicinal plants, is traditionally used to improve the general state of patients with malignant diseases such as cancer. Recent studies showed that HET had an anti­cancer effect against several cancer cell lines in vitro by inducing apoptosis. However, high doses of HET may have cytotoxic effects attributed to saponins or detergent­like compounds. Therefore, the present study used low doses of HET (50 µg/ml), which did not affect cell viability, to evaluate its synergistic anti­cancer effects with cisplatin. HeLa cells were cultured for 24 h with 50 µg/ml HET, followed by cisplatin treatment for 24 h at various concentrations. Subsequently, the sensitivity of the cells to cisplatin was assessed using a colony survival and a crystal violet cell viability assay. Furthermore, cisplatin­induced apoptosis was analyzed by flow cytometry. Proteins associated with cell viability and apoptosis, including phosphorylated (p­)Akt, p53, B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax) and active caspase­3 were analyzed by immunoblotting. The present study revealed that cell survival was decreased and apoptosis was increased in HeLa cells pre­treated with HET prior to cisplatin treatment compared with HET­untreated cells. Furthermore, protein expression of p53 and active caspase­3 was increased, while the expression of p­Akt as well as the Bcl­2/Bax ratio, an index of survival activity in cells, were decreased in the HET­pre­treated cells compared with those in HET­untreated cells following incubation with cisplatin. In conclusion, the present study indicated that HET enhanced cisplatin­induced apoptosis of HeLa cells and that the administration of HET may therefore be clinically beneficial alongside apoptosis­inducing chemotherapy.

Targeted Integration of RNA-Seq and Metabolite Data to Elucidate Curcuminoid Biosynthesis in Four Curcuma Species.

Curcuminoids, namely curcumin and its analogs, are secondary metabolites that act as the primary active constituents of turmeric (Curcuma longa). The contents of these curcuminoids vary among species in the genus Curcuma. For this reason, we compared two wild strains and two cultivars to understand the differences in the synthesis of curcuminoids. Because the fluxes of metabolic reactions depend on the amounts of their substrate and the activity of the catalysts, we analyzed the metabolite concentrations and gene expression of related enzymes. We developed a method based on RNA sequencing (RNA-Seq) analysis that focuses on a specific set of genes to detect expression differences between species in detail. We developed a 'selection-first' method for RNA-Seq analysis in which short reads are mapped to selected enzymes in the target biosynthetic pathways in order to reduce the effect of mapping errors. Using this method, we found that the difference in the contents of curcuminoids among the species, as measured by gas chromatography-mass spectrometry, could be explained by the changes in the expression of genes encoding diketide-CoA synthase, and curcumin synthase at the branching point of the curcuminoid biosynthesis pathway.