Population-level comparisons of gene regulatory networks modeled on high-throughput single-cell transcriptomics data.

Single-cell technologies enable high-resolution studies of phenotype-defining molecular mechanisms. However, data sparsity and cellular heterogeneity make modeling biological variability across single-cell samples difficult. Here we present SCORPION, a tool that uses a message-passing algorithm to reconstruct comparable gene regulatory networks from single-cell/nuclei RNA-sequencing data that are suitable for population-level comparisons by leveraging the same baseline priors. Using synthetic data, we found that SCORPION outperformed 12 existing gene regulatory network reconstruction techniques. Using supervised experiments, we show that SCORPION can accurately identify differences in regulatory networks between wild-type and transcription factor-perturbed cells. We demonstrate SCORPION's scalability to population-level analyses using a single-cell RNA-sequencing atlas containing 200,436 cells from colorectal cancer and adjacent healthy tissues. The differences between tumor regions detected by SCORPION are consistent across multiple cohorts as well as with our understanding of disease progression, and elucidate phenotypic regulators that may impact patient survival.

Ablation of long noncoding RNA MALAT1 activates antioxidant pathway and alleviates sepsis in mice.

The metastasis-associated lung adenocarcinoma transcript1 (MALAT1) is a long noncoding RNA (lncRNA) and is known for its role in cancer development and prognosis. In this study, we report that MALAT1 plays an important role in regulating acute inflammatory responses in sepsis. In patient samples, MALAT1 expression was positively correlated with severity of sepsis. In cultured macrophages, LPS treatment significantly induced MALAT1 expression, while genetic ablation of MALAT1 greatly reduced proinflammatory cytokine levels. Furthermore, MALAT1-ablated mice had significantly increased survival rates in cecal ligation and puncture (CLP)-induced sepsis and LPS-induced endotoxemia. One novel and salient feature of MALAT1-ablated mice is greatly reduced ROS level in macrophages and other cell types and increased glutathione/oxidized glutathione (GSH/GSSG) ratio in macrophages, suggesting an increased antioxidant capacity. We showed a mechanism for MALAT1 ablation leading to enhanced antioxidant capacity is through activation of methionine cycle by epitranscriptomical regulation of methionine adenosyltransferase 2A (MAT2A). MAT2A 3'UTR can be methylated by METTL16 which was known to directly bind to MALAT1. MALAT1 ablation was found to reduce methylation in MAT2A hairpin1 and increase MAT2A protein levels. Our results suggest a MALAT1-METTL16-MAT2A interactive axis which may be targeted for treatments of sepsis.

Single-cell RNA Sequencing Reveals How the Aryl Hydrocarbon Receptor Shapes Cellular Differentiation Potency in the Mouse Colon.

Despite recent progress recognizing the importance of aryl hydrocarbon receptor (Ahr)-dependent signaling in suppressing colon tumorigenesis, its role in regulating colonic crypt homeostasis remains unclear. To assess the effects of Ahr on intestinal epithelial cell heterogeneity and functional phenotypes, we utilized single-cell transcriptomics and advanced analytic strategies to generate a high-quality atlas for colonic intestinal crypts from wild-type and intestinal-specific Ahr knockout mice. Here we observed the promotive effects of Ahr deletion on Foxm1-regulated genes in crypt-associated canonical epithelial cell types and subtypes of goblet cells and deep crypt-secretory cells. We also show that intestinal Ahr deletion elevated single-cell entropy (a measure of differentiation potency or cell stemness) and RNA velocity length (a measure of the rate of cell differentiation) in noncycling and cycling Lgr5+ stem cells. In general, intercellular signaling cross-talk via soluble and membrane-bound factors was perturbed in Ahr-null colonocytes. Taken together, our single-cell RNA sequencing analyses provide new evidence of the molecular function of Ahr in modulating putative stem cell driver genes, cell potency lineage decisions, and cell-cell communication in vivo. PREVENTION RELEVANCE: Our mouse single-cell RNA sequencing analyses provide new evidence of the molecular function of Ahr in modulating colonic stemness and cell-cell communication in vivo. From a cancer prevention perspective, Ahr should be considered a therapeutic target to recalibrate remodeling of the intestinal stem cell niche.

Regulatory Network of PD1 Signaling Is Associated with Prognosis in Glioblastoma Multiforme.

Glioblastoma is an aggressive cancer of the brain and spine. While analysis of glioblastoma 'omics data has somewhat improved our understanding of the disease, it has not led to direct improvement in patient survival. Cancer survival is often characterized by differences in gene expression, but the mechanisms that drive these differences are generally unknown. We therefore set out to model the regulatory mechanisms associated with glioblastoma survival. We inferred individual patient gene regulatory networks using data from two different expression platforms from The Cancer Genome Atlas. We performed comparative network analysis between patients with long- and short-term survival. Seven pathways were identified as associated with survival, all of them involved in immune signaling; differential regulation of PD1 signaling was validated to correspond with outcome in an independent dataset from the German Glioma Network. In this pathway, transcriptional repression of genes for which treatment options are available was lost in short-term survivors; this was independent of mutational burden and only weakly associated with T-cell infiltration. Collectively, these results provide a new way to stratify patients with glioblastoma that uses network features as biomarkers to predict survival. They also identify new potential therapeutic interventions, underscoring the value of analyzing gene regulatory networks in individual patients with cancer. SIGNIFICANCE: Genome-wide network modeling of individual glioblastomas identifies dysregulation of PD1 signaling in patients with poor prognosis, indicating this approach can be used to understand how gene regulation influences cancer progression.

Modelado de estructuras-3D de mutaciones raras del receptor-1-melanocortina asociadas al melanismo en el mielero / Modelling of 3d-structures of the rare melanocortin-1-receptor mutations associated to melanism in the bananaquit

ABSTRACT Melanism in plumage color is often associated to the single nucleotide polymorphism of the melanocortin-1-receptor. Despite the striking association between the substitution of a Glutamic-acid by for a Lysine at position 92 on the MC1R protein and a completely black plumage, an in-depth understanding of the effect of missense mutations on the conformational change and behavior of the MC1R in the lipid bilayer caused by the absence of a crystal structure is lacking. We examine the structural basis for receptor activation using DNA sequences from the GenBank to perform in silico protein homology-based modeling. Our tridimensional model shows that the Alanine for a 179-Threonine substitution is a structural complement of the charge-reversing effect associated to the substitution of a Glutamic-acid by for a Lysine at position 92 on the MC1R. We proposed the possibility of gradual evolution in stability and electrostatic properties of the MC1R by the sequential accumulation of these two rare substitutions. These two rare substitutions further perturb physical-chemical properties that may be necessary folding requirements of the constitutively active MC1R forms without altering of ligand binding affinity. The computational coarse-grained molecular dynamics of the MC1R binding affinities to the melanocyte-stimulating hormone predicted the disparity in ligand binding among alleles. We speculate that the disparity in structural constraints and ligand binding among the alleles within heterozygous individuals may contribute as a mechanism to the plumage color variation in the Coereba flaveola.

RESUMEN El melanismo en el color del plumaje se asocia frecuentemente al polimorfismo del receptor melanocortina-1. La ausencia de una estructura cristalográfica de la asociación entre la sustitución del Glutamato por Lisina en la posición 92 de la proteína MC1R y el plumaje completamente negro, no ha permitido tener un mejor entendimiento del efecto de mutaciones no sinónimas en la conformación y en el comportamiento en la membrana del MC1R. Examinamos la estructura asociada a la activación del receptor usando secuencias de ADN obtenidas del GenBank, para un modelamiento in silico de formas homólogas de la proteína. El modelo tridimensional muestra que la sustitución de Alanina por la Treonina en la posición 179 es un complemento estructural al efecto de reversión de carga asociado a la sustitución del Glutamato por Lisina en la posición 92 del MC1R. Proponemos la posibilidad de evolución gradual de la estabilidad y de propiedades electrostáticas del MC1R por la acumulación de estas substituciones. Estas perturban las propiedades fisicoquímicas que podrían ser necesarias para el plegamiento de las formas constitutivamente activas del MC1R sin alterar la afinidad de empalme con el ligando. La modelación computacional de la dinámica molecular de la afinidad de empalme del MC1R a la hormona estimulante de meloncitos predice la disparidad de la unión con el ligando entre alelos. Consideramos que posiblemente la disparidad entre alelos en heterocigotos en cuanto a restricciones estructurales y la unión con el ligando podría contribuir a la variación en el color del plumaje en Coereba flaveola.

Ctdnep1 and Eps8L2 regulate dorsal actin cables for nuclear positioning during cell migration.

Cells actively position their nuclei within the cytoplasm for multiple cellular and physiological functions.1-3 Consequently, nuclear mispositioning is usually associated with cell dysfunction and disease, from muscular disorders to cancer metastasis.4-7 Different cell types position their nuclei away from the leading edge during cell migration.8-11 In migrating fibroblasts, nuclear positioning is driven by an actin retrograde flow originated at the leading edge that drives dorsal actin cables away from the leading edge. The dorsal actin cables connect to the nuclear envelope by the linker of nucleoskeleton and cytoskeleton (LINC) complex on transmembrane actin-associated nuclear (TAN) lines.12-14 Dorsal actin cables are required for the formation of TAN lines. How dorsal actin cables are organized to promote TAN lines formation is unknown. Here, we report a role for Ctdnep1/Dullard, a nuclear envelope phosphatase,15-22 and the actin regulator Eps8L223-25 on nuclear positioning and cell migration. We demonstrate that Ctdnep1 and Eps8L2 directly interact, and this interaction is important for nuclear positioning and cell migration. We also show that Ctdnep1 and Eps8L2 are involved in the formation and thickness of dorsal actin cables required for TAN lines engagement during nuclear movement. We propose that Ctdnep1-Eps8L2 interaction regulates dorsal actin cables for nuclear movement during cell migration.

Polarization, migration, and homotypical interactions among prostatic smooth muscle cells in a laminin 111-rich extracellular matrix.

Prostate cancer is a life-threatening condition worldwide. As the tumor progresses, smooth muscle cells (SMCs) become atrophic/dedifferentiated, within a series of stromal changes named stromal reaction. Here, we tested whether a laminin 111-rich extracellular matrix (Lr-ECM) could affect SMCs phenotype and differentiation status. Using time-lapse microscopy, image analyses, quantitative real-time reverse transcription polymerase chain reaction, immunohistochemistry and immunoblotting, and transmission electron microscopy, we showed that SMCs acquires a migratory behavior with a decreased expression of differentiation markers and relocation of focal adhesion kinase. SMCs set homotypic cell junctions and were active in autophagy/phagocytosis. Analysis of the migratory behavior showed that SMCs polarized and migrated toward each other, recognizing long-distance signals such as matrix tensioning. However, half of the cell population were immotile, irrespective of the nearest neighbor distance, suggesting they do not engage in productive interactions, possibly as a result of back-to-back positioning. In conclusion, the Lr-ECM, mimics the effects of the proliferating and infiltrating tumor epithelium, causing SMCs phenotypical change similar to that observed in the stromal reaction, in addition to a hitherto undescribed, stereotyped pattern of cell motility resulting from cell polarization.

Limited Renal Intravascular Lymphoma: A Case Report and Review of the Literature.

Intravascular large B-cell lymphoma (IVLBCL) is a rare subtype of non-Hodgkin lymphoma. It is characterized by the proliferation of cancerous cells into the intraluminal space of the blood vessels. It has a low incidence rate of 0.095 cases per 1,000,000. The clinical presentation is insidious and unspecific, often delaying the diagnosis. IVLBCL can be diagnosed through body images and histopathology analysis. This neoplasm averages a 60% response rate to current chemotherapy treatment, favoring rituximab, and doxorubicin-based regimen if it is diagnosed in time. Here, we present the case of a 56-year-old man admitted to our hospital with a fever who was eventually diagnosed with IVLBCL. He presented to the consultation with anemia, fever, and splenomegaly. An infection panel, a bone marrow biopsy, and a PET-CT scan were performed and ruled out the possibility of infections and neoplasms. The patient later developed edematous syndrome. As a result, a renal biopsy was performed which tested positive for intravascular large B-cell lymphoma. Currently, the patient has been in complete remission for 33 months. Along with presenting this specific case, we also reviewed previously published cases of IVLBCL to illustrate the renal involvement of this pathology.

PIG-1 MELK-dependent phosphorylation of nonmuscle myosin II promotes apoptosis through CES-1 Snail partitioning.

The mechanism(s) through which mammalian kinase MELK promotes tumorigenesis is not understood. We find that the C. elegans orthologue of MELK, PIG-1, promotes apoptosis by partitioning an anti-apoptotic factor. The C. elegans NSM neuroblast divides to produce a larger cell that differentiates into a neuron and a smaller cell that dies. We find that in this context, PIG-1 MELK is required for partitioning of CES-1 Snail, a transcriptional repressor of the pro-apoptotic gene egl-1 BH3-only. pig-1 MELK is controlled by both a ces-1 Snail- and par-4 LKB1-dependent pathway, and may act through phosphorylation and cortical enrichment of nonmuscle myosin II prior to neuroblast division. We propose that pig-1 MELK-induced local contractility of the actomyosin network plays a conserved role in the acquisition of the apoptotic fate. Our work also uncovers an auto-regulatory loop through which ces-1 Snail controls its own activity through the formation of a gradient of CES-1 Snail protein.

Crosslinking activity of non-muscle myosin II is not sufficient for embryonic cytokinesis in C. elegans.

Cytokinesis in animal cells requires the assembly and constriction of a contractile actomyosin ring. Non-muscle myosin II is essential for cytokinesis, but the role of its motor activity remains unclear. Here, we examine cytokinesis in C. elegans embryos expressing non-muscle myosin motor mutants generated by genome editing. Two non-muscle motor-dead myosins capable of binding F-actin do not support cytokinesis in the one-cell embryo, and two partially motor-impaired myosins delay cytokinesis and render rings more sensitive to reduced myosin levels. Further analysis of myosin mutants suggests that it is myosin motor activity, and not the ability of myosin to crosslink F-actin, that drives the alignment and compaction of F-actin bundles during contractile ring assembly, and that myosin motor activity sets the pace of contractile ring constriction. We conclude that myosin motor activity is required at all stages of cytokinesis. Finally, characterization of the corresponding motor mutations in C. elegans major muscle myosin shows that motor activity is required for muscle contraction but is dispensable for F-actin organization in adult muscles.This article has an associated 'The people behind the papers' interview.

Single-cell RNA sequencing of a European and an African lymphoblastoid cell line.

In biomedical research, lymphoblastoid cell lines (LCLs), often established by in vitro infection of resting B cells with Epstein-Barr virus, are commonly used as surrogates for peripheral blood lymphocytes. Genomic and transcriptomic information on LCLs has been used to study the impact of genetic variation on gene expression in humans. Here we present single-cell RNA sequencing (scRNA-seq) data on GM12878 and GM18502-two LCLs derived from the blood of female donors of European and African ancestry, respectively. Cells from three samples (the two LCLs and a 1:1 mixture of the two) were prepared separately using a 10x Genomics Chromium Controller and deeply sequenced. The final dataset contained 7,045 cells from GM12878, 5,189 from GM18502, and 5,820 from the mixture, offering valuable information on single-cell gene expression in highly homogenous cell populations. This dataset is a suitable reference for population differentiation in gene expression at the single-cell level. Data from the mixture provide additional valuable information facilitating the development of statistical methods for data normalization and batch effect correction.

Multiple Pathways Involved in Palmitic Acid-Induced Toxicity: A System Biology Approach.

Inflammation is a complex biological response to injuries, metabolic disorders or infections. In the brain, astrocytes play an important role in the inflammatory processes during neurodegenerative diseases. Recent studies have shown that the increase of free saturated fatty acids such as palmitic acid produces a metabolic inflammatory response in astrocytes generally associated with damaging mechanisms such as oxidative stress, endoplasmic reticulum stress, and autophagic defects. In this aspect, the synthetic neurosteroid tibolone has shown to exert protective functions against inflammation in neuronal experimental models without the tumorigenic effects exerted by sexual hormones such as estradiol and progesterone. However, there is little information regarding the specific mechanisms of tibolone in astrocytes during inflammatory insults. In the present study, we performed a genome-scale metabolic reconstruction of astrocytes that was used to study astrocytic response during an inflammatory insult by palmitate through Flux Balance Analysis methods and data mining. In this aspect, we assessed the metabolic fluxes of human astrocytes under three different scenarios: healthy (normal conditions), induced inflammation by palmitate, and tibolone treatment under palmitate inflammation. Our results suggest that tibolone reduces the L-glutamate-mediated neurotoxicity in astrocytes through the modulation of several metabolic pathways involved in glutamate uptake. We also identified a set of reactions associated with the protective effects of tibolone, including the upregulation of taurine metabolism, gluconeogenesis, cPPAR and the modulation of calcium signaling pathways. In conclusion, the different scenarios studied in our model allowed us to identify several metabolic fluxes perturbed under an inflammatory response and the protective mechanisms exerted by tibolone.

TPXL-1 activates Aurora A to clear contractile ring components from the polar cortex during cytokinesis.

During cytokinesis, a signal from the central spindle that forms between the separating anaphase chromosomes promotes the accumulation of contractile ring components at the cell equator, while a signal from the centrosomal microtubule asters inhibits accumulation of contractile ring components at the cell poles. However, the molecular identity of the inhibitory signal has remained unknown. To identify molecular components of the aster-based inhibitory signal, we developed a means to monitor the removal of contractile ring proteins from the polar cortex after anaphase onset. Using this assay, we show that polar clearing is an active process that requires activation of Aurora A kinase by TPXL-1. TPXL-1 concentrates on astral microtubules coincident with polar clearing in anaphase, and its ability to recruit Aurora A and activate its kinase activity are essential for clearing. In summary, our data identify Aurora A kinase as an aster-based inhibitory signal that restricts contractile ring components to the cell equator during cytokinesis.

L-Type Calcium Channels Modulation by Estradiol.

Voltage-gated calcium channels are key regulators of brain function, and their dysfunction has been associated with multiple conditions and neurodegenerative diseases because they couple membrane depolarization to the influx of calcium-and other processes such as gene expression-in excitable cells. L-type calcium channels, one of the three major classes and probably the best characterized of the voltage-gated calcium channels, act as an essential calcium binding proteins with a significant biological relevance. It is well known that estradiol can activate rapidly brain signaling pathways and modulatory/regulatory proteins through non-genomic (or non-transcriptional) mechanisms, which lead to an increase of intracellular calcium that activate multiple kinases and signaling cascades, in the same way as L-type calcium channels responses. In this context, estrogens-L-type calcium channels signaling raises intracellular calcium levels and activates the same signaling cascades in the brain probably through estrogen receptor-independent modulatory mechanisms. In this review, we discuss the available literature on this area, which seems to suggest that estradiol exerts dual effects/modulation on these channels in a concentration-dependent manner (as a potentiator of these channels in pM concentrations and as an inhibitor in nM concentrations). Indeed, estradiol may orchestrate multiple neurotrophic responses, which open a new avenue for the development of novel estrogen-based therapies to alleviate different neuropathologies. We also highlight that it is essential to determine through computational and/or experimental approaches the interaction between estradiol and L-type calcium channels to assist these developments, which is an interesting area of research that deserves a closer look in future biomedical research.

Estudio de biodisponibilidad de metformina 850 mg tabletas de liberación inmediata / Bioavalability study of Metformin 850 mg immediate-release tablets

Se desarrolló un estudio de biodisponibilidad de metformina 850 mg tabletas recubiertas de liberación inmediata elaboradas por Laboratorios Coaspharma S.A., en 12 voluntarios sanos de ambos sexos, con edades entre 18 y 26 años. Para llevarlo a cabo se validó previamente un método bioanalítico para la determinación de metformina en plasma humano por cromatografía líquida de alta resolución con detector ultravioleta (HPLC-UV), el cual resultó ser selectivo, específico, lineal, exacto y preciso, por lo tanto adecuado para el análisis de las muestras. Estas fueron recolectadas periódicamente en un lapso desde 0 a 24 horas, luego de la administración por vía oral de una única dosis de metformina 850 mg. Posteriormente se determinaron los parámetros farmacocinéticos promedio de los 12 participantes, obteniendo: área bajo la curva, desde tiempo cero hasta el último tiempo de muestreo t (AUC0--->t) 6856,89 ± 2073,8 ng.h/ml, área bajo la curva desde tiempo cero hasta tiempo infinito (AUC0--->∞) 7083,74 ± 2131,52 ng.h/ml, concentración máxima (Cmaxmax) 1299,02 ± 291,90 ng/ml, tiempo máximo (t) 2,33 ± 0,47 h, tiempo de vida media (t1/2) 2,50 ± 0,84 h y constante aparente de eliminación (Ke) de 0,31 ± 0,12 h-1. Los resultados fueron similares en todos los participantes y no se produjeron reacciones adversas.

A bioavailability study was conducted in 12 healthy volunteers of both genders, aged between 18 and 26. Previous to the study, a bioanalytical method for determination of metformin in human plasma by high performance liquid chromatography with ultraviolet detector (HPLCUV) was validated, and proved to be selective, specific, linear, accurate precise, and therefore, suitable for analysis in plasma. Samples were collected from 0 to 24 hours after the oral administration of a single dose of metformin 850 mg immediate-release coated tablets, produced by Coaspharma S.A. Laboratories. Then, average pharmacokinetic parameters of the twelve volunteers were determined: area under the curve from time zero to last sampling time t (AUC0--->t) 6856.89 ± 2073.8 ng.h/mL, area under the curve from time zero to infinite time (AUC0--->∞) 7083.74 ± 2131.52 ng.h/ml, maximum concentration (Cmax) 1299.02 ± 291.90 ng/mL, maximum time (t max) 2.33 ± 0.47 h, half-life (t1/2) 2.50 ± 0.84 h and apparent elimination constant (Ke) of 0.31 ± 0.12 h-1. These results are similar between the volunteers and no adverse effect was observed. Also, the results are in agree with those reported in literature.

Connecting the nucleus to the cytoskeleton for nuclear positioning and cell migration.

The position of the nucleus in the cytoplasm is a highly regulated process and is required for multiple cellular and developmental processes. Defects on different nuclear positioning events are associated with several pathologies such as muscle and nervous system disorders. In this chapter we describe the current knowledge on the mechanism of nuclear positioning. We discuss how the nucleus connects to the cytoskeleton by nesprins and SUN proteins, how this connection is regulated by Samp1, and how this connection is required for proper nuclear positioning. Furthermore, we discuss how nesprins, SUN, and Samp1 form transmembrane actin-associated nuclear (TAN) lines, novel nuclear envelope structures involved in force transduction during nuclear movement. Finally, we describe the recent evidences suggesting a role for the connection between the nucleus and the cytoskeleton in cancer.

Evaluation of Fournier's necrosis in a high complexity hospital.

OBJECTIVES: To determine clinical characteristics of the population with Fournier's necrosis at Hospital Universitario del Valle (HUV) in Cali during the period 2003-2008. METHODS: We performed a retrospective review of patients with the diagnosis of Fournier's necrosis at HUV during the period 2003-2008. We collected information on age,personal history, duration of illness, hospitalization, surgeries performed, time to surgical treatment, isolated germs, and mortality. Univariate descriptive analysis was performed in STATA v 10.1. RESULTS: 42 patients with mean age 51 years and 12 days mean disease duration. 26% had diabetes mellitus and 21.4% urethral trauma before admission. Average time to surgical debridement was 41.4 hours. 62% required suprapubic cystostomy, 14.3% derivative colostomy, 9.5% and 2.4% orchiectomy and penectomy respectively. The average hospital stay was 23 days and 12% required ICU care. Scrotal cultures were positive in 59.5%: 64% a single germ and 36% polymicrobial. Reported mortality was 17%. CONCLUSION: Fournier's necrosis is a life-threatening clinical entity in patients with multiple comorbidities requiring multiple interventions. Broad-spectrum antimicrobial therapy, aggressive debridement of necrotic tissue and comprehensive management of these patients are the mainstays in the treatment of these severely ill patients.

Structural and functional implications of positive selection at the primate angiogenin gene.

BACKGROUND: Angiogenesis, the formation of new blood vessels, is a primordial process in development and its dysregulation has a central role in the pathogenesis of many diseases. Angiogenin (ANG), a peculiar member of the RNase A superfamily, is a potent inducer of angiogenesis involved in many different types of cancer, amyotrophic lateral sclerosis and also with a possible role in the innate immune defense. The evolutionary path of this family has been a highly dynamic one, where positive selection has played a strong role. In this work we used a combined gene and protein level approach to determine the main sites under diversifying selection on the primate ANG gene and analyze its structural and functional implications. RESULTS: We obtained evidence for positive selection in the primate ANG gene. Site specific analysis pointed out 15 sites under positive selection, most of which also exhibited drastic changes in amino acid properties. The mapping of these sites in the ANG 3D-structure described five clusters, four of which were located in functional regions: two in the active site region, one in the nucleolar location signal and one in the cell-binding site. Eight of the 15 sites under selection in the primate ANG gene were highly or moderately conserved in the RNase A family, suggesting a directed event and not a simple consequence of local structural or functional permissiveness. Moreover, 11 sites were exposed to the surface of the protein indicating that they may influence the interactions performed by ANG. CONCLUSION: Using a maximum likelihood gene level analysis we identified 15 sites under positive selection in the primate ANG genes, that were further corroborated through a protein level analysis of radical changes in amino acid properties. These sites mapped onto the main functional regions of the ANG protein. The fact that evidence for positive selection is present in all ANG regions required for angiogenesis may be a good indication that angiogenesis is the process under selection. However, other possibilities to be considered arise from the possible involvement of ANG in innate immunity and the potential influence or co-evolution with its interacting proteins and ligands.