Expression of Retroelements in Cervical Cancer and Their Interplay with HPV Infection and Host Gene Expression.

Retroelements are expressed in diverse types of cancer and are related to tumorigenesis and to cancer progression. We characterized the expression of retroelements in cervical cancer and explored their interplay with HPV infection and their association with expression of neighboring genes. Forty biopsies of invasive cervical carcinoma (squamous cell carcinomas and adenocarcinomas) with genotyped HPV were selected and analyzed for human endogenous retrovirus (HERV) and long interspersed nuclear element 1 (L1) expression through RNA-seq data. We found 8060 retroelements expressed in the samples and a negative correlation of DNA methyltransferase 1 expression with the two most expressed L1 elements. A total of 103 retroelements were found differentially expressed between tumor histological types and between HPV types, including several HERV families (HERV-K, HERV-H, HERV-E, HERV-I and HERV-L). The comparison between HPV mono- and co-infections showed the highest proportion of differentially expressed L1 elements. The location of retroelements affected neighboring gene expression, such as shown for the interleukin-20 gene family. Three HERVs and seven L1 were located close to this gene family and two L1 showed a positive association with IL20RB expression. This study describes the expression of retroelements in cervical cancer and shows their association with HPV status and host gene expression.

Development and Validation of a Combined Hypoxia and Immune Prognostic Classifier for Head and Neck Cancer.

PURPOSE: Intratumoral hypoxia and immunity have been correlated with patient outcome in various tumor settings. However, these factors are not currently considered for treatment selection in head and neck cancer (HNC) due to lack of validated biomarkers. Here we sought to develop a hypoxia-immune classifier with potential application in patient prognostication and prediction of response to targeted therapy. EXPERIMENTAL DESIGN: A 54-gene hypoxia-immune signature was constructed on the basis of literature review. Gene expression was analyzed in silico using the The Cancer Genome Atlas (TCGA) HNC dataset (n = 275) and validated using two independent cohorts (n = 130 and 123). IHC was used to investigate the utility of a simplified protein signature. The spatial distribution of hypoxia and immune markers was examined using multiplex immunofluorescence staining. RESULTS: Unsupervised hierarchical clustering of TCGA dataset (development cohort) identified three patient subgroups with distinct hypoxia-immune phenotypes and survival profiles: hypoxialow/immunehigh, hypoxiahigh/immunelow, and mixed, with 5-year overall survival (OS) rates of 71%, 51%, and 49%, respectively (P = 0.0015). The prognostic relevance of the hypoxia-immune gene signature was replicated in two independent validation cohorts. Only PD-L1 and intratumoral CD3 protein expression were associated with improved OS on multivariate analysis. Hypoxialow/immunehigh and hypoxiahigh/immunelow tumors were overrepresented in "inflamed" and "immune-desert" microenvironmental profiles, respectively. Multiplex staining demonstrated an inverse correlation between CA-IX expression and prevalence of intratumoral CD3+ T cells (r = -0.5464; P = 0.0377), further corroborating the transcription-based classification. CONCLUSIONS: We developed and validated a hypoxia-immune prognostic transcriptional classifier, which may have clinical application to guide the use of hypoxia modification and targeted immunotherapies for the treatment of HNC.

Characterization of HPV integration, viral gene expression and E6E7 alternative transcripts by RNA-Seq: A descriptive study in invasive cervical cancer.

Scarce data are available on the expression of papillomavirus genome and the frequency of alternatively spliced E6E7 mRNAs in invasive cervical cancer. We carried out a comprehensive characterization of HPV expression by RNA-Seq analysis in 22 invasive cervical cancer with HPV16 or HPV18, characterizing the presence of integrated/episomal viral DNA, the integration sites in human genome and the proportion of alternative splicing products of E6 and E7 genes. The expression patterns suggested the presence of episomal and/or integrated viral DNA, with integration detected in most tumors, frequently occurring within human genes in HPV18+ and in intergenic regions in HPV16+ tumors. Alternative splicing of E6E7 transcripts showed E6*I as the most frequent isoform for both viral types, followed by E6*II and E6/E7 (unspliced) transcripts in HPV16+, and by E6/E7 in HPV18+ tumors. Previously described E6*VI and E6*V transcript isoforms for HPV16, and E6*X for HPV18, were rare or not detected.

Multispecies coalescent analysis of the early diversification of neotropical primates: phylogenetic inference under strong gene trees/species tree conflict.

Neotropical primates (NP) are presently distributed in the New World from Mexico to northern Argentina, comprising three large families, Cebidae, Atelidae, and Pitheciidae, consequently to their diversification following their separation from Old World anthropoids near the Eocene/Oligocene boundary, some 40 Ma. The evolution of NP has been intensively investigated in the last decade by studies focusing on their phylogeny and timescale. However, despite major efforts, the phylogenetic relationship between these three major clades and the age of their last common ancestor are still controversial because these inferences were based on limited numbers of loci and dating analyses that did not consider the evolutionary variation associated with the distribution of gene trees within the proposed phylogenies. We show, by multispecies coalescent analyses of selected genome segments, spanning along 92,496,904 bp that the early diversification of extant NP was marked by a 2-fold increase of their effective population size and that Atelids and Cebids are more closely related respective to Pitheciids. The molecular phylogeny of NP has been difficult to solve because of population-level phenomena at the early evolution of the lineage. The association of evolutionary variation with the distribution of gene trees within proposed phylogenies is crucial for distinguishing the mean genetic divergence between species (the mean coalescent time between loci) from speciation time. This approach, based on extensive genomic data provided by new generation DNA sequencing, provides more accurate reconstructions of phylogenies and timescales for all organisms.

The role of historical barriers in the diversification processes in open vegetation formations during the Miocene/Pliocene using an ancient rodent lineage as a model.

The Neotropics harbors a high diversity of species and several hypotheses have been proposed to account for this pattern. However, while species of forested domains are frequently studied, less is known of species from open vegetation formations occupying, altogether, a larger area than the Amazon Forest. Here we evaluate the role of historical barriers and the riverine hypothesis in the speciation patterns of small mammals by analyzing an ancient rodent lineage (Thrichomys, Hystricomorpha). Phylogenetic and biogeographic analyses were carried out with mitochondrial and nuclear DNA markers to analyze the evolutionary relationships between Thrichomys lineages occurring in dry domains along both banks of the Rio São Francisco. This river is one of the longest of South America whose course and water flow have been modified by inland tectonic activities and climate changes. Molecular data showed a higher number of lineages than previously described. The T. inermis species complex with 2n = 26, FN = 48 was observed in both banks of the river showing a paraphyletic arrangement, suggesting that river crossing had occurred, from east to west. A similar pattern was also observed for the T. apereoides complex. Thrichomys speciation occurred in Late Miocene when the river followed a different course. The current geographic distribution of Thrichomys species and their phylogenetic relationships suggested the existence of frequent past connections between both banks in the middle section of the Rio São Francisco. The extensive palaeodune region found in this area has been identified as a centre of endemism of several vertebrate species and is likely to be a center of Thrichomys diversification.

Positive selection along the evolution of primate mitogenomes.

The mitochondrial genomes of four neotropical primates, Aotus infulatus, Chiropotes israelita, Callimico goeldii and Callicebus lugens were sequenced and annotated. Phylogenetic reconstructions with mitochondrial genes of other 66 primates showed a similar arrangement to a topology based on nuclear genes. Screening for positive selection identified 15 codons in 7 genes along 9 independent lineages, three with two or more genes and five in internal nodes, ruling out false positive estimates. Mitochondrial genes of the electron transport chain (ETC.) complexes evolved with high substitution rates. A study of nuclear ETC. genes might elucidate whether they co-evolved with their mitochondrial counterparts.

Chronology of deep nodes in the neotropical primate phylogeny: insights from mitochondrial genomes.

The evolution of Neotropical Primates (NP) is permeated by factors associated with the pattern of diversification and the biogeography of the major lineages. These questions can be better understood by providing a robust estimate of the chronological scenario of NP evolution, a reason why molecular dating methods have been widely applied. One aspect of especial interest is the timing of diversification of the major NP lineages (pitheciids, atelids and cebids), which may have resulted from rapid episodes of adaptive radiation, a question that requires NP divergence time estimates with accurate statistical certainty. In this study, we evaluated the primate timescale focused on the age of nodes of NP radiation. We investigated the performance of complete primate mitochondrial genomes as traditional molecular markers of primate evolution and further including original mitochondrial data from the endangered muriqui, Brachyteles arachnoides (Accession No. JX262672). Comparisons of the age estimates at NP nodes based on mitochondrial genomes with those obtained from a nuclear supermatrix showed similar degrees of uncertainty. Further molecular data and more informative calibration priors are required for a more precise understanding of the early NP diversification.

Molecular evolution of α4 integrin binding site to lentiviral envelope proteins in new world primates.

Integrin epitopes encoded by ITGA4 exons 5 and 6 encompass the α4ß7 binding site to natural ligands and HIV-1 gp120. Functional assays of α4 variants of new world primates (NWP) showed reduced binding of several ligands, including the HIV-1 envelope, probably accounting for restriction phenotypes conferring resistance to lentiviral infection (Darc et al., 2011). In this paper, we have analyzed, by cloning and sequencing, the α4 domain polymorphisms present in 10 NWP species and four old world primates (including human). Analyses of differential selection at codon sites and along evolutionary lineages were carried out. We identified codons under positive selection, including polymorphic variations at codon 201, presumably convergent during NWP radiation and significant positive selection leading to a single allele (SagVar2).

Identification, classification and evolution of owl monkeys (Aotus, Illiger 1811).

BACKGROUND: Owl monkeys, belonging to the genus Aotus, have been extensively used as animal models in biomedical research but few reports have focused on the taxonomy and phylogeography of this genus. Moreover, the morphological similarity of several Aotus species has led to frequent misidentifications, mainly at the boundaries of their distribution. In this study, sequence data from five mitochondrial regions and the nuclear, Y-linked, SRY gene were used for species identification and phylogenetic reconstructions using well characterized specimens of Aotus nancymaae, A. vociferans, A. lemurinus, A. griseimembra, A. trivirgatus, A. nigriceps, A. azarae boliviensis and A. infulatus. RESULTS: The complete MT-CO1, MT-TS1, MT-TD, MT-CO2, MT-CYB regions were sequenced in 18 Aotus specimens. ML and Bayesian topologies of concatenated data and separate regions allowed for the proposition of a tentative Aotus phylogeny, indicating that Aotus diverged some 4.62 Million years before present (MYBP). Similar analyses with included GenBank specimens were useful for assessing species identification of deposited data. CONCLUSIONS: Alternative phylogenetic reconstructions, when compared with karyotypic and biogeographic data, led to the proposition of evolutionary scenarios questioning the conventional diversification of this genus in monophyletic groups with grey and red necks. Moreover, genetic distance estimates and haplotypic differences were useful for species validations.

Evolution of TRIM5alpha B30.2 (SPRY) domain in New World primates.

The tripartite motif 5 protein (TRIM5) has been extensively studied in view of its ability to restrict retroviruses in mammalian hosts. The B30.2 domain, encoded by exon 8 of TRIM5, contains the major restriction determinants. We have analyzed the genetic diversity of the TRIM5 B30.2 domain in a wide range of New World primates (NWP). The TRIM5 region encoding the B30.2 domain of 35 animals, representing all NWP families and 10 genera, was PCR-amplified, sequenced and analyzed at the amino acid level. Comparisons were carried out with available GenBank data; analyses were carried out with a dataset of 44 representative sequences of 32 NWP species and 15 genera, with a human B30.2 sequence as outgroup. A high genetic diversity was observed, both with respect to length and amino acid substitutions, mainly at the three variable regions of this domain associated with the restriction phenotype. Phylogenetic reconstructions based on B30.2 DNA differed from the consensus NWP topology due to positive selection along different lineages and definite codon positions, with robust evidence either with a complete or a pruned dataset. This was especially evident in codons 406 and 496, consistently demonstrated with all methods. Positive selection was virtually absent in all NWP species when analyzing intra-specific polymorphisms except for Saguinus labiatus. Our findings indicated that NWP TRIM5 proteins have been subjected to selection, probably by retroviruses and/or retroelements. We anticipate that the diversity of NWP TRIM5 is indicative of disparate retroviral restriction phenotypes representing a plentiful source of factors countering HIV infection.

Detailed analysis of X chromosome inactivation in a 49,XXXXX pentasomy.

BACKGROUND: Pentasomy X (49,XXXXX) has been associated with a severe clinical condition, presumably resulting from failure or disruption of X chromosome inactivation. Here we report that some human X chromosomes from a patient with 49,XXXXX pentasomy were functionally active following isolation in inter-specific (human-rodent) cell hybrids. A comparison with cytogenetic and molecular findings provided evidence that more than one active X chromosome was likely to be present in the cells of this patient, accounting for her abnormal phenotype. RESULTS: 5-bromodeoxyuridine (BrdU)-pulsed cultures showed different patterns among late replicating X chromosomes suggesting that their replication was asynchronic and likely to result in irregular inactivation. Genotyping of the proband and her mother identified four maternal and one paternal X chromosomes in the proband. It also identified the paternal X chromosome haplotype (P), indicating that origin of this X pentasomy resulted from two maternal, meiotic non-disjunctions. Analysis of the HUMANDREC region of the androgen receptor (AR) gene in the patient's mother showed a skewed inactivation pattern, while a similar analysis in the proband showed an active paternal X chromosome and preferentially inactivated X chromosomes carrying the 173 AR allele. Analyses of 33 cell hybrid cell lines selected in medium containing hypoxanthine, aminopterin and thymidine (HAT) allowed for the identification of three maternal X haplotypes (M1, M2 and MR) and showed that X chromosomes with the M1, M2 and P haplotypes were functionally active. In 27 cell hybrids in which more than one X haplotype were detected, analysis of X inactivation patterns provided evidence of preferential inactivation. CONCLUSION: Our findings indicated that 12% of X chromosomes with the M1 haplotype, 43.5% of X chromosomes with the M2 haplotype, and 100% of the paternal X chromosome (with the P haplotype) were likely to be functionally active in the proband's cells, a finding indicating that disruption of X inactivation was associated to her severe phenotype.

Evolution of cyclophilin A and TRIMCyp retrotransposition in New World primates.

Host cell factors modulate retroviral infections. Among those, cyclophilin A (CypA) promotes virus infectivity by facilitating virus uncoating or capsid unfolding or by preventing retroviral capsid interaction with cellular restriction factors. In Aotus species, a retrotransposed copy of CypA inserted into the tripartite motif 5 (TRIM5) gene encodes a fusion protein which may block human immunodeficiency virus type 1 by targeting the incoming virus to ubiquitin-ligated degradation or by interfering with normal uncoating of the incoming particle, rendering those monkeys resistant to infection. In this study, we have extensively analyzed representative specimens from all New World primate genera and shown that the retrotransposed CypA copy is only present in Aotus. We have shown that this inserted copy diverged from its original counterpart and that this occurred prior to Aotus radiation, although no positive selection was observed. Finally, our data underscores the need for a precise taxonomic identification of primate species used as models for retroviral infections and novel antiviral approaches.