Schwarzschild Black Hole from Perturbation Theory to All Orders.

Applying the quantum field theoretic perturbiner approach to Einstein gravity, we compute the metric of a Schwarzschild black hole order by order in perturbation theory. Using recursion, this calculation can be carried out in de Donder gauge to all orders in Newton's constant. The result is a geometric series which is convergent outside a disk of finite radius, and it agrees within its region of convergence with the known de Donder gauge metric of a Schwarzschild black hole. It thus provides a first all-order perturbative computation in Einstein gravity with a matter source, and this series converges to the known nonperturbative expression in the expected range of convergence.

The landscape of PBMC methylome in canine mammary tumors reveals the epigenetic regulation of immune marker genes and its potential application in predicting tumor malignancy.

BACKGROUND: Genome-wide dysregulation of CpG methylation accompanies tumor progression and characteristic states of cancer cells, prompting a rationale for biomarker development. Understanding how the archetypic epigenetic modification determines systemic contributions of immune cell types is the key to further clinical benefits. RESULTS: In this study, we characterized the differential DNA methylome landscapes of peripheral blood mononuclear cells (PBMCs) from 76 canines using methylated CpG-binding domain sequencing (MBD-seq). Through gene set enrichment analysis, we discovered that genes involved in the growth and differentiation of T- and B-cells are highly methylated in tumor PBMCs. We also revealed the increased methylation at single CpG resolution and reversed expression in representative marker genes regulating immune cell proliferation (BACH2, SH2D1A, TXK, UHRF1). Furthermore, we utilized the PBMC methylome to effectively differentiate between benign and malignant tumors and the presence of mammary gland tumors through a machine-learning approach. CONCLUSIONS: This research contributes to a better knowledge of the comprehensive epigenetic regulation of circulating immune cells responding to tumors and suggests a new framework for identifying benign and malignant cancers using genome-wide methylome.

ABCA9, an ER cholesterol transporter, inhibits breast cancer cell proliferation via SREBP-2 signaling.

The association between cholesterol metabolism and cancer development and progression has been recently highlighted. However, the role and function of many cholesterol transporters remain largely unknown. Here, we focused on the ATP-binding cassette subfamily A member 9 (ABCA9) transporter given that its expression is significantly downregulated in both canine mammary tumors and human breast cancers, which in breast cancer patients correlates with poor prognosis. We found that ABCA9 is mainly present in the endoplasmic reticulum (ER) and is responsible for promoting cholesterol accumulation in this structure. Accordingly, ABCA9 inhibited sterol-regulatory element binding protein-2 (SREBP-2) translocation from the ER to the nucleus, a crucial step for cholesterol synthesis, resulting in the downregulation of cholesterol synthesis gene expression. ABCA9 expression in breast cancer cells attenuated cell proliferation and reduced their colony-forming abilities. We identified ABCA9 expression to be regulated by Forkhead box O1 (FOXO1). Inhibition of PI3K induced enhanced ABCA9 expression through the activation of the PI3K-Akt-FOXO1 pathway in breast cancer cells. Altogether, our study suggests that ABCA9 functions as an ER cholesterol transporter that suppresses cholesterol synthesis via the inhibition of SREBP-2 signaling and that its restoration halts breast cancer cell proliferation. Our findings provide novel insight into the vital role of ABCA9 in breast cancer progression.

Analysis of ocular fluid in patients with ranibizumab-recalcitrant neovascular age-related macular degeneration who have serum anti-ranibizumab antibodies.

PURPOSE: To evaluate whether anti-drug antibodies (ADAs) are present in the ocular fluid of patients with ranibizumab-recalcitrant neovascular age-related macular degeneration (nAMD). METHODS: Two serum ADA-positive ranibizumab-recalcitrant patients and two serum ADA-negative controls were recruited from patients with nAMD treated with ranibizumab monotherapy. Recalcitrance was defined as persistent fluid after ≥6 monthly ranibizumab injections. Serum and aqueous humor ADAs were detected by enzyme-linked immunosorbent assay and immunoprecipitation, respectively. RESULTS: Two of 156 ranibizumab-treated patients were ADA-positive. The patients received six and 14 ranibizumab injections, respectively, up to 4 weeks prior to blood collection. The serum ADA concentration was estimated to be approximately 50,000 ng/mL. Neutralizing ADAs were confirmed in both samples. A specific band was detected by immunoprecipitation only in ADA-positive samples, consistent with the results of enzyme-linked immunosorbent assay. Based on an assessment of the degree of sensitivity of commercially available anti-ranibizumab antibodies, it was estimated that the immunoprecipitation method could detect ADA levels >30 ng. Nevertheless, ADAs were not detected in the aqueous humor of either the experimental or control group. CONCLUSION: In the aqueous humor, ADAs are either not present or are present at a lower concentration than that which can be detected by immunoprecipitation. This presumably reflects the fact that blood ADA is the product of systemic circulation clearance through anterior elimination of intravitreal ranibizumab. Based on our results, ADAs do not return to the eye in sufficient quantities to interfere with the action of ranibizumab in the vitreous cavity.

Contactless Interface Using Exhaled Breath and Thermal Imaging.

A new type of interface using a conduction hot spot reflecting the user's intention is presented. Conventional methods using fingertips to generate conduction hot points cannot be applied to those who have difficulty using their hands or cold hands. In order to overcome this problem, an exhaling interaction using a hollow rod is proposed and extensively analyzed in this paper. A preliminary study on exhaling interaction demonstrated the possibility of the method. This paper is an attempt to develop and extend the concept and provide the necessary information for properly implementing the interaction method. We have repeatedly performed conduction hot-point-generation experiments on various materials that can replace walls or screens to make wide use of the proposed interfaces. Furthermore, a lot of experiments have been conducted in different seasons, considering that the surface temperature of objects also changes depending on the season. Based on the results of an extensive amount of experiments, we provide key observations on important factors such as material, season, and user condition, which should be considered for realizing contactless exhaling interfaces.

LONGITUDINAL ASSESSMENT OF CHOROIDAL STRUCTURE IN PATIENTS WITH MACULAR NEOVASCULARIZATION.

PURPOSE: To investigate morphologic changes of choroidal structure through chronologic aspect in progression of macular neovascularization (MNV) with pachychoroid features. METHODS: One hundred seventy-one MNV participants above 50 years old with or without pachychoroid features were included in the analysis. Age-matched 132 normal patients were analyzed as control group. The total choroidal area and ratio of Sattler's layer area to total choroidal area, derived by summing 25 horizontal raster scans of the 30° × 20° scan area on enhanced depth imaging optical coherence tomography, were calculated to compare the difference among the normal eyes and the MNV eyes with/without pachychoroid features. RESULTS: The mean ratio of Sattler's layer area to total choroidal area is maintained at around 40% in normal eyes and MNV eyes without pachychoroid features. In MNV with pachychoroid features, the ratio of Sattler's layer area to total choroidal area changes according to the disease activity. Ratio of Sattler's layer area to total choroidal area is 34.1 ± 4.4% at the time of onset, 37.2 ± 4.8% at the time of remission, and decreases during recurrence from 36.8 ± 3.8% to 33.4 ± 3.8% (all P < 0.001). CONCLUSION: MNV with pachychoroid features is a disease whose development and progression are related to a change in the choroidal interlayer area ratio following the relatively larger dilation of Haller's layer vessels.

Surgical Outcomes of Non-Small Cell Lung Cancer in Single-Zone N2 in the Aortopulmonary Zone.

OBJECTIVES: We evaluate the surgical outcome of clinically single-zone N2 lung cancer limited to aortopulmonary zone (AP zone; lymph node #5 or #6). PATIENTS AND METHODS: We performed a retrospective analysis of patients with non-small cell lung cancer, in whom mediastinal lymph node metastasis was confined to AP zone. RESULTS: A total of 84 patients who underwent upfront surgery were included in final analysis. Among these patients, pathological nodal outcomes were pN0-1 in 27 patients (32.1%), pN2a in 31 (36.9%), and pN2b in 26 (31.0%). In multivariate analysis, adenocarcinoma (p = 0.005) and staging workup without endobronchial ultrasound-transbronchial needle aspiration (p = 0.002) were independent risk factors for unexpected pN2b. The 5-year overall survival (OS) and disease-free survival (DFS) were 55.9 and 54.4%, respectively. There was no survival difference among patients with pN0-1, pN2a, and pN2b (p = 0.717). In survival analysis, there were no significant risk factors for OS. However, female sex and the ratio of positive lymph nodes to removed lymph nodes were significant risk factors for DFS in multivariate analysis (p = 0.032 and p = 0.009). CONCLUSION: In this study, cN2a in the AP zone with current diagnostic tool exhibited a relatively high false-positive rate (cN2/pN0-1; 32.1%). However, despite the possibility of pN2b, there were no significant differences in survival outcome according to the pathologic nodal stage.

CXCL5 secreted from macrophages during cold exposure mediates white adipose tissue browning.

Adipose tissue affects metabolic-related diseases because it consists of various cell types involved in fat metabolism and adipokine release. CXC ligand 5 (CXCL5) is a member of the CXC chemokine family and is highly expressed by macrophages in white adipose tissue (WAT). In this study, we generated and investigated the function of CXCL5 in knockout (KO) mice using CRISPR/Cas9. The male KO mice did not show significant phenotype differences in normal conditions. However, proteomic analysis revealed that many proteins involved in fatty acid beta-oxidation and mitochondrial localization were enriched in the inguinal WAT (iWAT) of Cxcl5 KO mice. Cxcl5 KO mice also showed decreased protein and transcript expression of genes associated with thermogenesis, including uncoupling protein 1 (UCP1), a well-known thermogenic gene, and increased expression of genes associated with inflammation. The increase in UCP1 expression in cold conditions was significantly retarded in Cxcl5 KO mice. Finally, we found that CXCL5 treatment increased the expression of transcription factors that mediate Ucp1 expression and Ucp1 itself. Collectively, our data show that Ucp1 expression is induced in adipocytes by CXCL5, which is secreted upon ß-adrenergic stimulation by cold stimulation in M1 macrophages. Our data indicate that CXCL5 plays a crucial role in regulating energy metabolism, particularly upon cold exposure. These results strongly suggest that targeting CXCL5 could be a potential therapeutic strategy for people suffering from disorders affecting energy metabolism.

Spatiotemporal expression of long noncoding RNA Moshe modulates heart cell lineage commitment.

The roles of long non-coding RNA (LncRNA) have been highlighted in various development processes including congenital heart defects (CHD). Here, we characterized the molecular function of LncRNA, Moshe (1010001N08ik-203), one of the Gata6 antisense transcripts located upstream of Gata6, which is involved in both heart development and the most common type of congenital heart defect, atrial septal defect (ASD). During mouse embryonic development, Moshe was first detected during the cardiac mesoderm stage (E8.5 to E9.5) where Gata6 is expressed and continues to increase at the atrioventricular septum (E12.5), which is involved in ASD. Functionally, the knock-down of Moshe during cardiogenesis caused significant repression of Nkx2.5 in cardiac progenitor stages and resulted in the increase in major SHF lineage genes, such as cardiac transcriptional factors (Isl1, Hand2, Tbx2), endothelial-specific genes (Cd31, Flk1, Tie1, vWF), a smooth muscle actin (a-Sma) and sinoatrial node-specific genes (Shox2, Tbx18). Chromatin Isolation by RNA Purification showed Moshe activates Nkx2.5 gene expression via direct binding to its promoter region. Of note, Moshe was conserved across species, including human, pig and mouse. Altogether, this study suggests that Moshe is a heart-enriched lncRNA that controls a sophisticated network of cardiogenesis by repressing genes in SHF via Nkx2.5 during cardiac development and may play an important role in ASD.

Natural Progression of Ground-glass Nodules after Curative Resection for Non-small Cell Lung Cancer.

BACKGROUND: This retrospective study investigated the natural course of synchronous ground-glass nodules (GGNs) that remained after curative resection for non-small-cell lung cancer (NSCLC). METHODS: Prospectively collected retrospective data were reviewed concerning 2,276 patients who underwent curative resection for NSCLC between 2008 and 2017. High-resolution computed tomography or thin-section computed tomography data of 82 patients were included in the study. Growth in size was considered the most valuable outcome, and patients were grouped according to GGN size change. Patient demographic data (e.g., age, sex, and smoking history), perioperative data (e.g., GGN characteristics, histopathology and pathological stage of the resected tumours), and other medical history were evaluated in a risk factor analysis concerning GGN size change. RESULTS: The median duration of follow-up was 36.0 months (interquartile range, 23.0-59.3 months). GGN size decreased in 6 patients (7.3%), was stationary in 43 patients (52.4%), and increased in 33 patients (40.2%). In univariate analysis, male sex, the GGN size on initial CT, part-solid GGN and smoking history (≥ 10 pack-years) were significant risk factors. Among them, multivariate analysis revealed that lager GGN size, part-solid GGN and smoking history were independent risk factors. CONCLUSION: During follow-up, 40.2% of GGNs increased in size, emphasising that patients with larger GGNs, part-solid GGN or with a smoking history should be observed.

METTL8 mRNA Methyltransferase Enhances Cancer Cell Migration via Direct Binding to ARID1A.

The association of RNA modification in cancer has recently been highlighted. Methyltransferase like 8 (METTL8) is an enzyme and its role in mRNA m3C modification has barely been studied. In this study, we found that METTL8 expression was significantly up-regulated in canine mammary tumor and investigated its functional roles in the tumor process, including cancer cell proliferation and migration. METTL8 expression was up-regulated in most human breast cancer cell lines tested and decreased by Yin Yang 1 (YY1) transcription factor knockdown, suggesting that YY1 is a regulating transcription factor. The knockdown of METTL8 attenuated tumor cell growth and strongly blocked tumor cell migration. AT-rich interactive domain-containing protein 1A (ARID1A) was identified as a candidate mRNA by METTL8. ARID1A mRNA binds to METTL8 protein. ARID1A mRNA expression was not changed by METTL8 knockdown, but ARID1A protein level was significantly increased. Collectively, our study indicates that METTL8 up-regulated by YY1 in breast cancer plays an important role in cancer cell migration through the mRNA modification of ARID1A, resulting in the attenuation of its translation.

Protein Arginine Methyltransferase 1 Is Essential for the Meiosis of Male Germ Cells.

Protein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells; however, its function in vivo is not well understood due to its early embryonic lethality in null mice exhibiting spontaneous DNA damage, cell cycle delays, and defects in check point activation. Here, we generated germ cell-specific Prmt1 knock-out (KO) mice to evaluate the function of PRMT1 in spermatogenesis. Our findings demonstrate that PRMT1 is vital for male fertility in mice. Spermatogenesis in Prmt1 KO mice was arrested at the zygotene-like stage of the first meiotic division due to an elevated number of DNA double-strand breaks (DSBs). There was a loss of methylation in meiotic recombination 11 (MRE11), the key endonuclease in MRE11/RAD50/NBS 1 (MRN) complex, resulting in the accumulation of SPO11 protein in DSBs. The ATM-mediated negative feedback control over SPO11 was lost and, consequently, the repair pathway of DSBs was highly affected in PRMT1 deficient male germ cells. Our findings provide a novel insight into the role of PRMT1-mediated asymmetric demethylation in mouse spermatogenesis.

Long Non-Coding RNA Associated with Cholesterol Homeostasis and Its Involvement in Metabolic Diseases.

Cholesterol is an essential cell component that functions to create and maintain all kinds of cell membranes and lipoprotein particles. It is crucial to maintain the proper amount of cholesterol at both the cellular and systemic level. Recently, the importance of cholesterol has been reported not only in various cell development processes but also in the development of diseases. Furthermore, the involvement of long non-coding RNAs (lncRNAs), which are regarded as important epigenetic regulators in gene expression, has also been reported in cholesterol homeostasis. It is thus necessary to summarize the research on lncRNAs related to cholesterol with increased interest. This review organized the role of lncRNAs according to the major issues in cholesterol homeostasis: efflux, metabolism and synthesis, and disease process.

ANK2 Hypermethylation in Canine Mammary Tumors and Human Breast Cancer.

Canine mammary tumors (CMT) constitute the most common tumor types found in female dogs. Understanding this cancer through extensive research is important not only for clinical veterinary applications, but also in the scope of comparative oncology. The use of DNA methylation as a biomarker has been noted for numerous cancers in the form of both tissue and liquid biopsies, yet the study of methylation in CMT has been limited. By analyzing our canine methyl-binding domain sequencing (MBD-seq) data, we identified intron regions of canine ANK2 and EPAS1 as differentially methylated regions (DMGs) in CMT. Subsequently, we established quantitative methylation specific PCR (qMSP) of ANK2 and EPAS1 to validate the target hypermethylation in CMT tissue, as well as cell free DNA (cfDNA) from CMT plasma. Both ANK2 and EPAS1 were hypermethylated in CMT and highlighted as potential tissue biomarkers in CMT. ANK2 additionally showed significant hypermethylation in the plasma cfDNA of CMT, indicating that it could be a potential liquid biopsy biomarker as well. A similar trend towards hypermethylation was indicated in HBC at a specific CpG of the ANK2 target on the orthologous human region, which validates the comparative approach using aberrant methylation in CMT.

Transposable element-mediated structural variation analysis in dog breeds using whole-genome sequencing.

Naturally occurring diseases in dogs provide an important animal model for studying human disease including cancer, heart disease, and autoimmune disorders. Transposable elements (TEs) make up ~ 31% of the dog (Canis lupus familiaris) genome and are one of main drivers to cause genomic variations and alter gene expression patterns of the host genes, which could result in genetic diseases. To detect structural variations (SVs), we conducted whole-genome sequencing of three different breeds, including Maltese, Poodle, and Yorkshire Terrier. Genomic SVs were detected and visualized using BreakDancer program. We identified a total of 2328 deletion SV events in the three breeds compared with the dog reference genome of Boxer. The majority of the genetic variants were found to be TE insertion polymorphism (1229) and the others were TE-mediated deletion (489), non-TE-mediated deletion (542), simple repeat-mediated deletion (32), and other indel (36). Among the TE insertion polymorphism, 286 elements were full-length LINE-1s (L1s). In addition, the 49 SV candidates located in the genic regions were experimentally verified and their polymorphic rates within each breed were examined using PCR assay. Polymorphism analysis of the genomic variants revealed that some of the variants exist polymorphic in the three dog breeds, suggesting that their SV events recently occurred in the dog genome. The findings suggest that TEs have contributed to the genomic variations among the three dog breeds of Maltese, Poodle, and Yorkshire Terrier. In addition, the polymorphic events between the dog breeds indicate that TEs were recently retrotransposed in the dog genome.

Concise approach for screening long non-coding RNAs functionally linked to human breast cancer associated genes.

Cancer research studies using next-generation sequencing have revealed a number of genes of which aberrant expression is associated with various cancers. Recently, long non-coding RNA (lncRNA) has been highlighted due to its tissue-specific expression and cell cancerization functions, such as the regulation of key tumor suppressors. In this study, we suggest a very efficient approach to survey lncRNAs putatively associated with breast cancer. We targeted lncRNAs linked with breast cancer associated genes (BCAGs) and analyzed their expression pattern in human breast cancer cell lines. A total of 337 BCAGs were retrieved from literature review and the existence of 121 lncRNAs were identified from the 15 kb up- and downstream regions of the list of genes. Twenty lncRNAs' expression were detectable in human breast cancer cell lines with different expression patterns. Interestingly, the expression of three lncRNAs, two up-regulated (RAD51C v.4, LOC105371849) and one down-regulated (LOC102724064), were closely correlated with adjacent BCAGs (RAD51C, HEATR6 and BRMS1) in breast cancer cell lines. We thus demonstrated association between the lncRNA and its adjacent BCAG using LOC105371849-HEATR6, of which the function and regulation in breast cancer are still unknown. Knockdown of LOC105371849 by siRNA decreased the expression of HEATR6 mRNA in the MCF7 human breast cancer cell line. In conclusion, this study provides a better understanding about the biological roles of lncRNAs in breast cancer and may be useful in the investigation of proper targets for diagnostic and/or therapeutic breast cancer markers using public databases.

Surgical Outcome of Colon Interposition in Esophageal Cancer Surgery: Analysis of Risk Factors for Conduit-Related Morbidity.

BACKGROUND: We aimed to assess the feasibility, surgical outcomes, and conduit-related complications of colon interposition in patients with esophageal cancer. METHODS: Patients with esophageal cancer who underwent colon interposition for esophageal reconstruction between June 2000 and June 2013 were retrospectively reviewed. RESULTS: A total of 67 consecutive patients (mean age, 62.2 ± 7.9 years) were enrolled. During this time period, 944 patients underwent esophageal reconstruction using gastric conduit. Twelve patients (17.9%) also received neoadjuvant chemoradiotherapy (nCRT). The median follow-up duration was 44 months (range, 1-168 months); median survival duration was 63 months (range, 1-168 months); and 3- and 5-year overall survival rates were 61.6 and 49.4%, respectively. A total of 43 patients (64.2%) experienced at least 1 postoperative morbidity. According to the Clavien-Dindo grading system, 36 patients (54%) experienced postoperative morbidity of higher than Grade III. Pulmonary complications were most commonly observed complications among the patients (18 patients, 26.9%). Anastomosis site leakage developed in 11 patients (16.4%), and 3 of these patients (6.0%) eventually experienced graft failure. On multivariate analysis, nCRT was determined as a significant risk factor for conduit-related complications (leakage, graft failure, fistula, and stricture). CONCLUSION: Colon interposition is associated with relatively high complication rates, whereas nCRT is associated with conduit morbidity.

The Role of Nitrosamine (NNK) in Breast Cancer Carcinogenesis.

Smoking cigarettes is one of the most concerning issues that leads to tobacco-related cancers and can even result in death. Therefore, these issues should be addressed with a great sense of urgency with low-cost and simple approaches. Over the past several years, the scientific community has attempted to find solutions to overcome this issue. Thus, a large number of excellent studies have been reported in this field, and summarizing these results and providing important roadmaps for future studies is currently of great importance. Finding an outstanding solution to address aforementioned issue would be of great value to the community and to the social. Tobacco contains thousands of chemicals, and sixty-nine compounds have been established as human carcinogens; specifically, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the strongest carcinogen among the tobacco-specific nitrosamines. Tobacco carcinogens are also linked to mammary gland pathogenesis and increased risk of developing many cancers, including breast cancer, the most common cancer in women worldwide. This mini-review summarizes the role of NNK and the mechanisms of its receptor, nicotine acetylcholine receptor (nAChR), signaling in breast cancer based on publications identified using the keywords "secondhand smoke (SHS)", "Nitrosamines" and "breast cancer". Furthermore, this review considers the risk of NNK to the public in an effort to reduce exposure to SHS in women and their chances of developing breast cancer.

Genomic landscapes of endogenous retroviruses unveil intricate genetics of conventional and genetically-engineered laboratory mouse strains.

Laboratory strains of mice, both conventional and genetically engineered, have been introduced as critical components of a broad range of studies investigating normal and disease biology. Currently, the genetic identity of laboratory mice is primarily confirmed by surveying polymorphisms in selected sets of "conventional" genes and/or microsatellites in the absence of a single completely sequenced mouse genome. First, we examined variations in the genomic landscapes of transposable repetitive elements, named the TREome, in conventional and genetically engineered mouse strains using murine leukemia virus-type endogenous retroviruses (MLV-ERVs) as a probe. A survey of the genomes from 56 conventional strains revealed strain-specific TREome landscapes, and certain families (e.g., C57BL) of strains were discernible with defined patterns. Interestingly, the TREome landscapes of C3H/HeJ (toll-like receptor-4 [TLR4] mutant) inbred mice were different from its control C3H/HeOuJ (TLR4 wild-type) strain. In addition, a CD14 knock-out strain had a distinct TREome landscape compared to its control/backcross C57BL/6J strain. Second, an examination of superantigen (SAg, a "TREome gene") coding sequences of mouse mammary tumor virus-type ERVs in the genomes of the 46 conventional strains revealed a high diversity, suggesting a potential role of SAgs in strain-specific immune phenotypes. The findings from this study indicate that unexplored and intricate genomic variations exist in laboratory mouse strains, both conventional and genetically engineered. The TREome-based high-resolution genetics surveillance system for laboratory mice would contribute to efficient study design with quality control and accurate data interpretation. This genetics system can be easily adapted to other species ranging from plants to humans.

Repetitive element signature-based visualization, distance computation, and classification of 1766 microbial genomes.

The genomes of living organisms are populated with pleomorphic repetitive elements (REs) of varying densities. Our hypothesis that genomic RE landscapes are species/strain/individual-specific was implemented into the Genome Signature Imaging system to visualize and compute the RE-based signatures of any genome. Following the occurrence profiling of 5-nucleotide REs/words, the information from top-50 frequency words was transformed into a genome-specific signature and visualized as Genome Signature Images (GSIs), using a CMYK scheme. An algorithm for computing distances among GSIs was formulated using the GSIs' variables (word identity, frequency, and frequency order). The utility of the GSI-distance computation system was demonstrated with control genomes. GSI-based computation of genome-relatedness among 1766 microbes (117 archaea and 1649 bacteria) identified their clustering patterns; although the majority paralleled the established classification, some did not. The Genome Signature Imaging system, with its visualization and distance computation functions, enables genome-scale evolutionary studies involving numerous genomes with varying sizes.