CXCL12 targets the primary cilium cAMP/cGMP ratio to regulate cell polarity during migration.

Directed cell migration requires sustained cell polarisation. In migrating cortical interneurons, nuclear movements are directed towards the centrosome that organises the primary cilium signalling hub. Primary cilium-elicited signalling, and how it affects migration, remain however ill characterised. Here, we show that altering cAMP/cGMP levels in the primary cilium by buffering cAMP, cGMP or by locally increasing cAMP, influences the polarity and directionality of migrating interneurons, whereas buffering cAMP or cGMP in the apposed centrosome compartment alters their motility. Remarkably, we identify CXCL12 as a trigger that targets the ciliary cAMP/cGMP ratio to promote sustained polarity and directed migration. We thereby uncover cAMP/cGMP levels in the primary cilium as a major target of extrinsic cues and as the steering wheel of neuronal migration.

Subcellular second messenger networks drive distinct repellent-induced axon behaviors.

Second messengers, including cAMP, cGMP and Ca2+ are often placed in an integrating position to combine the extracellular cues that orient growing axons in the developing brain. This view suggests that axon repellents share the same set of cellular messenger signals and that axon attractants evoke opposite cAMP, cGMP and Ca2+ changes. Investigating the confinement of these second messengers in cellular nanodomains, we instead demonstrate that two repellent cues, ephrin-A5 and Slit1, induce spatially segregated signals. These guidance molecules activate subcellular-specific second messenger crosstalk, each signaling network controlling distinct axonal morphology changes in vitro and pathfinding decisions in vivo.

Pathogenic TRIO variants associated with neurodevelopmental disorders perturb the molecular regulation of TRIO and axon pathfinding in vivo.

The RhoGEF TRIO is known to play a major role in neuronal development by controlling actin cytoskeleton remodeling, primarily through the activation of the RAC1 GTPase. Numerous de novo mutations in the TRIO gene have been identified in individuals with neurodevelopmental disorders (NDDs). We have previously established the first phenotype/genotype correlation in TRIO-associated diseases, with striking correlation between the clinical features of the individuals and the opposite modulation of RAC1 activity by TRIO variants targeting different domains. The mutations hyperactivating RAC1 are of particular interest, as they are recurrently found in patients and are associated with a severe form of NDD and macrocephaly, indicating their importance in the etiology of the disease. Yet, it remains unknown how these pathogenic TRIO variants disrupt TRIO activity at a molecular level and how they affect neurodevelopmental processes such as axon outgrowth or guidance. Here we report an additional cohort of individuals carrying a pathogenic TRIO variant that reinforces our initial phenotype/genotype correlation. More importantly, by performing conformation predictions coupled to biochemical validation, we propose a model whereby TRIO is inhibited by an intramolecular fold and NDD-associated variants relieve this inhibition, leading to RAC1 hyperactivation. Moreover, we show that in cultured primary neurons and in the zebrafish developmental model, these gain-of-function variants differentially affect axon outgrowth and branching in vitro and in vivo, as compared to loss-of-function TRIO variants. In summary, by combining clinical, molecular, cellular and in vivo data, we provide compelling new evidence for the pathogenicity of novel genetic variants targeting the TRIO gene in NDDs. We report a novel mechanism whereby the fine-tuned regulation of TRIO activity is critical for proper neuronal development and is disrupted by pathogenic mutations.

von Willebrand factor links primary hemostasis to innate immunity.

The plasma multimeric glycoprotein von Willebrand factor (VWF) plays a critical role in primary hemostasis by tethering platelets to exposed collagen at sites of vascular injury. Recent studies have identified additional biological roles for VWF, and in particular suggest that VWF may play an important role in regulating inflammatory responses. However, the molecular mechanisms through which VWF exerts its immuno-modulatory effects remain poorly understood. In this study, we report that VWF binding to macrophages triggers downstream MAP kinase signaling, NF-κB activation and production of pro-inflammatory cytokines and chemokines. In addition, VWF binding also drives macrophage M1 polarization and shifts macrophage metabolism towards glycolysis in a p38-dependent manner. Cumulatively, our findings define an important biological role for VWF in modulating macrophage function, and thereby establish a novel link between primary hemostasis and innate immunity.

Enhanced flavour profiles through radicicol induced genomic variation in the lager yeasts, Saccharomyces pastorianus.

The yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and have acquired traits from the combined parental genomes such as ability to ferment a range of sugars at low temperatures and to produce aromatic flavour compounds, allowing for the production of lager beers with crisp, clean flavours. The polyploid strains are sterile and have reached an evolutionary bottleneck for genetic variation. Here we describe an accelerated evolution approach to obtain lager yeasts with enhanced flavour profiles. As the relative expression of orthologous alleles is a significant contributor to the transcriptome during fermentation, we aimed to induce genetic variation by altering the S. cerevisiae to S. eubayanus chromosome ratio. Aneuploidy was induced through the temporary inhibition of the cell's stress response and strains with increased production of aromatic amino acids via the Shikimate pathway were selected by resistance to amino acid analogues. Genomic changes such as gross chromosomal rearrangements, chromosome loss and chromosome gain were detected in the characterised mutants, as were single-nucleotide polymorphisms in ARO4, encoding for DAHP synthase, the catalytic enzyme in the first step of the Shikimate pathway. Transcriptome analysis confirmed the upregulation of genes encoding enzymes in the Ehrlich pathway and the concomitant increase in the production of higher alcohols and esters such as 2-phenylethanol, 2-phenylethyl acetate, tryptophol, and tyrosol. We propose that the polyploid nature of S. pastorianus genomes is an advantageous trait supporting opportunities for genetic alteration in otherwise sterile strains.

Inhibition of the IFN-α JAK/STAT Pathway by MERS-CoV and SARS-CoV-1 Proteins in Human Epithelial Cells.

Coronaviruses (CoVs) have caused several global outbreaks with relatively high mortality rates, including Middle East Respiratory Syndrome coronavirus (MERS)-CoV, which emerged in 2012, and Severe Acute Respiratory Syndrome (SARS)-CoV-1, which appeared in 2002. The recent emergence of SARS-CoV-2 highlights the need for immediate and greater understanding of the immune evasion mechanisms used by CoVs. Interferon (IFN)-α is the body's natural antiviral agent, but its Janus kinase/signal transducer and activators of transcription (JAK/STAT) signalling pathway is often antagonized by viruses, thereby preventing the upregulation of essential IFN stimulated genes (ISGs). Therapeutic IFN-α has disappointingly weak clinical responses in MERS-CoV and SARS-CoV-1 infected patients, indicating that these CoVs inhibit the IFN-α JAK/STAT pathway. Here we show that in lung alveolar A549 epithelial cells expression of MERS-CoV-nsp2 and SARS-CoV-1-nsp14, but not MERS-CoV-nsp5, increased basal levels of total and phosphorylated STAT1 & STAT2 protein, but reduced IFN-α-mediated phosphorylation of STAT1-3 and induction of MxA. While MERS-CoV-nsp2 and SARS-CoV-1-nsp14 similarly increased basal levels of STAT1 and STAT2 in bronchial BEAS-2B epithelial cells, unlike in A549 cells, they did not enhance basal pSTAT1 nor pSTAT2. However, both viral proteins reduced IFN-α-mediated induction of pSTAT1-3 and ISGs (MxA, ISG15 and PKR) in BEAS-2B cells. Furthermore, even though IFN-α-mediated induction of pSTAT1-3 was not affected by MERS-CoV-nsp5 expression in BEAS-2B cells, downstream ISG induction was reduced, revealing that MERS-CoV-nsp5 may use an alternative mechanism to reduce antiviral ISG induction in this cell line. Indeed, we subsequently discovered that all three viral proteins inhibited STAT1 nuclear translocation in BEAS-2B cells, unveiling another layer of inhibition by which these viral proteins suppress responses to Type 1 IFNs. While these observations highlight cell line-specific differences in the immune evasion effects of MERS-CoV and SARS-CoV-1 proteins, they also demonstrate the broad spectrum of immune evasion strategies these deadly coronaviruses use to stunt antiviral responses to Type IFN.

Aneuploidy influences the gene expression profiles in Saccharomyces pastorianus group I and II strains during fermentation.

The lager yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and are divided into two broad groups, Group I and II. The two groups evolved from at least one common hybridisation event but have subsequently diverged with Group I strains losing many S. cerevisiae chromosomes while the Group II strains retain both sub-genomes. The complex genomes, containing orthologous alleles from the parental chromosomes, pose interesting questions regarding gene regulation and its impact on the fermentation properties of the strains. Superimposed on the presence of orthologous alleles are complexities of gene dosage due to the aneuploid nature of the genomes. We examined the contribution of the S. cerevisiae and S. eubayanus alleles to the gene expression patterns of representative Group I and II strains during fermentation. We show that the relative expression of S. cerevisiae and S. eubayanus orthologues is positively correlated with gene copy number. Despite the reduced S. cerevisiae content in the Group I strain, S. cerevisiae orthologues contribute to biochemical pathways upregulated during fermentation which may explain the retention of specific chromosomes in the strain. Conversely, S. eubayanus genes are significantly overrepresented in the upregulated gene pool in the Group II strain. Comparison of the transcription profiles of the strains during fermentation identified both common and unique gene expression patterns, with gene copy number being a dominant contributory factor. Thus, the aneuploid genomes create complex patterns of gene expression during fermentation with gene dosage playing a crucial role both within and between strains.

CRISPR/Cas9-mediated SARM1 knockout and epitope-tagged mice reveal that SARM1 does not regulate nuclear transcription, but is expressed in macrophages.

SARM1 is a toll/interleukin-1 receptor -domain containing protein, with roles proposed in both innate immunity and neuronal degeneration. Murine SARM1 has been reported to regulate the transcription of chemokines in both neurons and macrophages; however, the extent to which SARM1 contributes to transcription regulation remains to be fully understood. Here, we identify differential gene expression in bone-marrow-derived macrophages (BMDMs) from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice compared with wild type (WT). However, we found that passenger genes, which are derived from the 129 donor strain of mice that flank the Sarm1 locus, confound interpretation of the results, since many of the identified differentially regulated genes come from this region. To re-examine the transcriptional role of SARM1 in the absence of passenger genes, here we generated three Sarm1-/- mice using CRISPR/Cas9. Treatment of neurons from these mice with vincristine, a chemotherapeutic drug causing axonal degeneration, confirmed SARM1's function in that process; however, these mice also showed that lack of SARM1 has no impact on transcription of genes previously shown to be affected such as chemokines. To gain further insight into SARM1 function, we generated an epitope-tagged SARM1 mouse. In these mice, we observed high SARM1 protein expression in the brain and brainstem and lower but detectable levels in macrophages. Overall, the generation of these SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages yet has no general role in macrophage transcriptional regulation and has provided important new models to further explore SARM1 function.

cAMP-Dependent Co-stabilization of Axonal Arbors from Adjacent Developing Neurons.

Axonal arbors in many neuronal networks are exuberant early during development and become refined by activity-dependent competitive mechanisms. Theoretical work proposed non-competitive interactions between co-active axons to co-stabilize their connections, but the demonstration of such interactions is lacking. Here, we provide experimental evidence that reducing cyclic AMP (cAMP) signaling in a subset of retinal ganglion cells favors the elimination of thalamic projections from neighboring neurons, pointing to a cAMP-dependent interaction that promotes axon stabilization.

SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation.

Calcium is a second messenger crucial to a myriad of cellular processes ranging from regulation of metabolism and cell survival to vesicle release and motility. Current strategies to directly manipulate endogenous calcium signals lack cellular and subcellular specificity. We introduce SpiCee, a versatile and genetically encoded chelator combining low- and high-affinity sites for calcium. This scavenger enables altering endogenous calcium signaling and functions in single cells in vitro and in vivo with biochemically controlled subcellular resolution. SpiCee paves the way to investigate local calcium signaling in vivo and directly manipulate this second messenger for therapeutic use.

A population of proinflammatory T cells coexpresses αß and γδ T cell receptors in mice and humans.

T cells are classically recognized as distinct subsets that express αß or γδ TCRs. We identify a novel population of T cells that coexpress αß and γδ TCRs in mice and humans. These hybrid αß-γδ T cells arose in the murine fetal thymus by day 16 of ontogeny, underwent αß TCR-mediated positive selection into CD4+ or CD8+ thymocytes, and constituted up to 10% of TCRδ+ cells in lymphoid organs. They expressed high levels of IL-1R1 and IL-23R and secreted IFN-γ, IL-17, and GM-CSF in response to canonically restricted peptide antigens or stimulation with IL-1ß and IL-23. Hybrid αß-γδ T cells were transcriptomically distinct from conventional γδ T cells and displayed a hyperinflammatory phenotype enriched for chemokine receptors and homing molecules that facilitate migration to sites of inflammation. These proinflammatory T cells promoted bacterial clearance after infection with Staphylococcus aureus and, by licensing encephalitogenic Th17 cells, played a key role in the development of autoimmune disease in the central nervous system.

A comparative analysis of prophylactic antimicrobial use in long-term care facilities in Ireland, 2013 and 2016.

BackgroundLong-term care facilities (LTCFs) are important locations of antimicrobial consumption. Of particular concern is inappropriate prescribing of prophylactic antimicrobials. AimWe aimed to explore factors related to antimicrobial prophylaxis in LTCFs in Ireland. MethodsThe point prevalence surveys of Healthcare-Associated Infections in Long-Term Care Facilities (HALT) were performed in Ireland in May 2013 and 2016. Data were collected on facility (type and stewardship initiatives) and resident characteristics (age, sex, antimicrobial and indication) for those meeting the surveillance definition for a HAI and/or prescribed an antimicrobial. ResultsIn 2013, 9,318 residents (in 190 LTCFs) and in 2016, 10,044 residents (in 224 LTCFs) were included. Of the 10% of residents prescribed antimicrobials, 40% were on prophylaxis, most of which was to prevent urinary tract infection. The main prophylactic agents were: nitrofurantoin (39%) and trimethoprim (41%) for urinary tract (UT); macrolides (47%) for respiratory tract and macrolides and tetracycline (56%) for skin or wounds. More than 50% of the prophylaxis was prescribed in intellectual disability facilities and around 40% in nursing homes. Prophylaxis was recorded more often for females, residents living in LTCFs for more than 1 year and residents with a urinary catheter. No difference in prophylactic prescribing was observed when comparing LTCFs participating and not participating in both years. ConclusionsForty per cent of antimicrobial prescriptions in Irish LTCFs were prophylactic. This practice is not consistent with national antimicrobial prescribing guidelines. Addressing inappropriate prophylaxis prescribing in Irish LTCFs should be a key objective of antimicrobial stewardship initiatives.

Dysregulated mitogen-activated protein kinase signalling as an oncogenic basis for clear cell sarcoma of the kidney.

The oncogenic mechanisms and tumour biology underpinning clear cell sarcoma of the kidney (CCSK), the second commonest paediatric renal malignancy, are poorly understood and currently, therapy depends heavily on doxorubicin with cardiotoxic side-effects. Previously, we characterized the balanced t(10;17)(q22;p13) chromosomal translocation, identified at that time as the only recurrent genetic aberration in CCSK. This translocation results in an in-frame fusion of the genes YWHAE (encoding 14-3-3ϵ) and NUTM2, with a somatic incidence of 12%. Clinico-pathological features of that cohort suggested that this aberration might be associated with higher stage and grade disease. Since no primary CCSK cell line exists, we generated various stably transfected cell lines containing doxycycline-inducible HA-tagged YWHAE-NUTM2, in order to study the effect of expressing this transcript. 14-3-3ϵ-NUTM2-expressing cells exhibited significantly greater cell migration compared to isogenic controls. Gene and protein expression studies were indicative of dysregulated MAPK/PI3K-AKT signalling, and by blocking these pathways using neutralizing antibodies, the migratory advantage conferred by the transcript was abrogated. Importantly, CCSK tumour samples similarly show up-regulation/activation of these pathways. These results support the oncogenic role of 14-3-3ϵ-NUTM2 in CCSK and provide avenues for the exploration of novel therapeutic approaches. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Human factors and non-technical skills: Teamwork.

Making mistakes is part of being human and human error is normal in all areas of life (Bromiley and Mitchell 2009). In some contexts this is of little consequence, but in environments where human safety and well-being are at stake it is vital that such error is minimised. The operating theatre is one such safety critical environment. Research suggests, however, that certain factors predispose to human error. Some or all of these factors may be present in the operating theatre and, therefore, have the potential to compromise patient safety.

A national survey of infection control and antimicrobial stewardship structures in Irish long-term care facilities.

BACKGROUND: Information on infection prevention and control (IPC) and antimicrobial stewardship activities in Irish long-term care facilities (LTCFs) is limited. METHODS: A survey detailing IPC and antimicrobial stewardship activities, including staffing and bed capacity, was circulated to Irish LTCFs. RESULTS: Sixty-nine LTCFs (61 public, 8 private) were surveyed, 56 (81%) of which had an IPC practitioner. Thirty-five (51%) LTCFs had an IPC committee that met on average 5 times (range, 1-10) during the previous year. LTCFs with IPC practitioners based solely in the facility (n = 17) were more likely to have an IPC committee (P = .027). Antimicrobial guidelines were available in 28% (n = 19) and 16% (n = 11) had an antimicrobial stewardship committee in place. Medical care was provided by general practitioners in 51% (n = 35), by physicians employed by the LTCFs in 35% (n = 24), or by both in 14% (n = 10). Medical care and activities were coordinated in 45% (n = 31) of LTCFs. These LTCFs were more likely to have an IPC committee (P < .001), medical staff training (P < .001), and antimicrobial guidelines (P = .005) in place. CONCLUSION: There are significant gaps in Irish LTCFs' IPC and antibiotic stewardship programs and governance structures, highlighting the need for specific LTCF national initiatives.

G-protein-coupled receptor independent, immunomodulatory properties of chemokine CXCL9.

Certain chemokines possess anti-angiogenic and antibacterial activity, in addition to their ability to recruit leukocytes. Herein, we demonstrate that CXCL9/MIG induces the expression, by a monocytic cell line and peripheral blood mononuclear cells, of a variety of chemokines including CXCL8/IL-8, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL2/MCP-1 in a pertussis toxin insensitive manner. Similarly, another cationic chemokine CCL20/MIP-3alpha, but not the non-cationic chemokines CCL2 or CCL3, stimulated monocytic cells to produce substantial amounts of CXCL8 and CCL3. Microarray experiments demonstrated that CXCL9, but not CCL2, induced the expression of hundreds of genes, many of which have known or proposed immunomodulatory functions. Induction of CXCL8 required the p38 and ERK1/2 mitogen-activated protein kinases but not NFkappaB, JAK-STAT or JNK signaling pathways. These results collectively demonstrate that CXCL9 has immunomodulatory functions that are not mediated through a G-protein coupled receptor and may possess additional roles in host defenses against infection.

Challenges of implementing national guidelines for the control and prevention of methicillin-resistant Staphylococcus aureus colonization or infection in acute care hospitals in the Republic of Ireland.

Of the 49 acute care hospitals in Ireland that responded to the survey questionnaire drafted by the Infection Control Subcommittee of the Health Protection Surveillance Centre's Strategy for the Control of Antimicrobial Resistance in Ireland, 43 reported barriers to the full implementation of national guidelines for the control and prevention of methicillin-resistant Staphylococcus aureus infection; these barriers included poor infrastructure (42 hospitals), inadequate laboratory resources (40 hospitals), inadequate staffing (39 hospitals), and inadequate numbers of isolation rooms and beds (40 hospitals). Four of the hospitals did not have an educational program on hand hygiene, and only 17 had an antibiotic stewardship program.

InnateDB: facilitating systems-level analyses of the mammalian innate immune response.

Although considerable progress has been made in dissecting the signaling pathways involved in the innate immune response, it is now apparent that this response can no longer be productively thought of in terms of simple linear pathways. InnateDB (www.innatedb.ca) has been developed to facilitate systems-level analyses that will provide better insight into the complex networks of pathways and interactions that govern the innate immune response. InnateDB is a publicly available, manually curated, integrative biology database of the human and mouse molecules, experimentally verified interactions and pathways involved in innate immunity, along with centralized annotation on the broader human and mouse interactomes. To date, more than 3500 innate immunity-relevant interactions have been contextually annotated through the review of 1000 plus publications. Integrated into InnateDB are novel bioinformatics resources, including network visualization software, pathway analysis, orthologous interaction network construction and the ability to overlay user-supplied gene expression data in an intuitively displayed molecular interaction network and pathway context, which will enable biologists without a computational background to explore their data in a more systems-oriented manner.

Bovine and human cathelicidin cationic host defense peptides similarly suppress transcriptional responses to bacterial lipopolysaccharide.

Genomic approaches can be exploited to expose the complexities and conservation of biological systems such as the immune network across various mammalian species. In this study, temporal transcriptional expression profiles were analyzed in human and bovine monocytic cells in response to the TLR-4 agonist, LPS, in the presence or absence of their respective host defense peptides. The cathelicidin peptides, human LL-37 and bovine myeloid antimicrobial peptide-27 (BMAP-27), are homologs, yet they have diverged notably in terms of sequence similarity. In spite of their low sequence similarities, both of these cathelicidin peptides demonstrated potent, antiendotoxin activity in monocytic cells at low, physiologically relevant concentrations. Microarray studies indicated that 10 ng/ml LPS led to the up-regulation of 125 genes in human monocytes, 106 of which were suppressed in the presence of 5 mug/ml of the human peptide LL-37. To confirm and extend these data, temporal transcriptional responses to LPS were assessed in the presence or absence of the species-specific host defense peptides by quantitative real-time PCR. The transcriptional trends of 20 LPS-induced genes were analyzed in bovine and human monocytic cells. These studies demonstrated conserved trends of gene responses in that both peptides were able to profoundly suppress many LPS-induced genes. Consistent with this, the human and bovine peptides suppressed LPS-induced translocation of NF-kappaB subunits p50 and p65 into the nucleus of monocytic cells. However, there were also distinct differences in responses to LPS and the peptides; for example, treatment with 5 mug/ml BMAP-27 alone tended to influence gene expression (RELA, TNF-alpha-induced protein 2, MAPK phosphatase 1/dual specificity phosphatase 1, IkappaBkappaB, NFkappaBIL1, TNF receptor-associated factor 2) to a greater extent than did the same amount of human LL-37. We hypothesize that the immunomodulatory effects of the species-specific host defense peptides play a critical role in regulating inflammation and represent an evolutionarily conserved mechanism for maintaining homeostasis, although the sequence divergence of these peptides is substantial.

Improving the specificity of high-throughput ortholog prediction.

BACKGROUND: Orthologs (genes that have diverged after a speciation event) tend to have similar function, and so their prediction has become an important component of comparative genomics and genome annotation. The gold standard phylogenetic analysis approach of comparing available organismal phylogeny to gene phylogeny is not easily automated for genome-wide analysis; therefore, ortholog prediction for large genome-scale datasets is typically performed using a reciprocal-best-BLAST-hits (RBH) approach. One problem with RBH is that it will incorrectly predict a paralog as an ortholog when incomplete genome sequences or gene loss is involved. In addition, there is an increasing interest in identifying orthologs most likely to have retained similar function. RESULTS: To address these issues, we present here a high-throughput computational method named Ortholuge that further evaluates previously predicted orthologs (including those predicted using an RBH-based approach) - identifying which orthologs most closely reflect species divergence and may more likely have similar function. Ortholuge analyzes phylogenetic distance ratios involving two comparison species and an outgroup species, noting cases where relative gene divergence is atypical. It also identifies some cases of gene duplication after species divergence. Through simulations of incomplete genome data/gene loss, we show that the vast majority of genes falsely predicted as orthologs by an RBH-based method can be identified. Ortholuge was then used to estimate the number of false-positives (predominantly paralogs) in selected RBH-predicted ortholog datasets, identifying approximately 10% paralogs in a eukaryotic data set (mouse-rat comparison) and 5% in a bacterial data set (Pseudomonas putida - Pseudomonas syringae species comparison). Higher quality (more precise) datasets of orthologs, which we term "ssd-orthologs" (supporting-species-divergence-orthologs), were also constructed. These datasets, as well as Ortholuge software that may be used to characterize other species' datasets, are available at http://www.pathogenomics.ca/ortholuge/ (software under GNU General Public License). CONCLUSION: The Ortholuge method reported here appears to significantly improve the specificity (precision) of high-throughput ortholog prediction for both bacterial and eukaryotic species. This method, and its associated software, will aid those performing various comparative genomics-based analyses, such as the prediction of conserved regulatory elements upstream of orthologous genes.