Viral reprogramming of host transcription initiation.

Viruses are master remodelers of the host cell environment in support of infection and virus production. For example, viruses typically regulate cell gene expression through modulating canonical cell promoter activity. Here, we show that Epstein Barr virus (EBV) replication causes 'de novo' transcription initiation at 29674 new transcription start sites throughout the cell genome. De novo transcription initiation is facilitated in part by the unique properties of the viral pre-initiation complex (vPIC) that binds a TATT[T/A]AA, TATA box-like sequence and activates transcription with minimal support by additional transcription factors. Other de novo promoters are driven by the viral transcription factors, Zta and Rta and are influenced by directional proximity to existing canonical cell promoters, a configuration that fosters transcription through existing promoters and transcriptional interference. These studies reveal a new way that viruses interact with the host transcriptome to inhibit host gene expression and they shed light on primal features driving eukaryotic promoter function.

Sistema de monitorización del plan de estudio, en función del perfil de egreso, como un mecanismo de contribución a la mejora de la calidad / Study plan monitoring system, based on the graduation profile, as a contribution mechanism to quality improvement

Introducción: El desarrollo universitario y el aumento de la oferta en educación superior han generado la necesidad de incorporar sistemas para monitorizar y asegurar la calidad de las instituciones, las carreras y los programas educativos. Esto ha motivado el interés de generar y fortalecer mecanismos de control de calidad de los procesos formativos. Material y métodos: Se diseñó un mecanismo que utiliza en una primera etapa encuestas que se aplican a docentes y estudiantes para monitorizar el cumplimiento del perfil de egreso de la carrera de Odontología de la Universidad Diego Portales. Posteriormente, en una segunda etapa se realizan grupos focales para enriquecer la información y validar los resultados. Resultados: Los resultados obtenidos avalan que el plan de estudio permite el desarrollo de las competencias declaradas en el perfil de egreso; existe correspondencia interna entre los contenidos, las metodologías, los criterios y los instrumentos de evaluación; se utilizan variadas metodologías educativas; existen criterios de evaluación; y se evalúan la mayoría de los aprendizajes, que se consideran pertinentes. Docentes y estudiantes reconocen parcialmente el nivel de preparación que adquieren después de cursar asignaturas monitorizadas. Conclusiones: Esta monitorización contribuye al diagnóstico de brechas y falencias en el plan de estudio. Permite realizar ajustes de forma oportuna, avalando que estos sistemas son necesarios. La naturaleza del mecanismo de monitorización permite utilizarse por otras carreras.(AU)

Introduction: The academic development and the increase in the offer in higher education, have generated the need to incorporate tools to monitor and to ensure the quality of institutions, careers, and educational programs. Which have motivated the interest in generating quality control mechanisms for these purposes. Materials and methods: it was designed a monitoring tool that uses in a first stage, surveys to teachers and students to monitor the compliance of the graduation profile of the Diego Portales University Dentistry career. Subsequently, in a second stage, focus groups were held to enrich the information and validate the results. Results: The results showed that the study plan allows the development of the competences declared in the graduation profile; correspondence between the contents, methodologies, criteria and evaluation instruments are presents; various educational methodologies are used; evaluation criteria exists and most of the learning is evaluated and considering it as relevant. Teachers and students partially recognize the level of preparation they acquire after completing the monitored subjects. Conclusions: This monitoring contributes to the diagnosis of gaps and shortcomings in the Study Plan. It allows adjustments in a timely manner, and it shows that these monitoring tools are a necessity. The essence of the questionnaire allows it to be used by other careers as well.(AU)

Parental protective and risk factors regarding cannabis use in adolescence: A national sample from the Chilean school population.

Background: Research has increased our understanding of the parental factors associated with the initiation and development of cannabis use disorder in adolescents, but few studies about this have been performed in middle- or low-income countries.Objective: First, to examine whether perceived past parental drug use, parental monitoring, and attitude toward adolescent cannabis use are associated with general and problematic cannabis use in Chilean adolescents. Second, to explore whether perceived past parental drug use weakens the associations of protective factors with general and problematic adolescent cannabis use.Methods: Regression analyses were performed on cross-sectional data from a multistage probabilistic sample stratified by clusters (municipalities, school and grade) of 43,060 students (47% male, mean age 15.5 years) from grades 8 to 12, which was collected from the Chilean National School Survey on Drug Use (2013).Results: Perceived past parental drug use increased the likelihood of adolescent cannabis use in general, but not its problematic use. Parental monitoring of adolescents' whereabouts and parental opposition to adolescent cannabis use decreased the likelihood of adolescent cannabis use in general, as well as problematic use. Perceived past parental drug use only interacted with parental monitoring of school activities.Conclusions: In line with research from the United States, the Netherlands and Spain, parental monitoring of adolescents' whereabouts and a strong parental opposition to cannabis use appear to be protective factors, irrespective of past parental use. However, the effectiveness of monitoring adolescents' school activities seems to decrease when parents are perceived as having used drugs in the past.

Circular RNAs add diversity to androgen receptor isoform repertoire in castration-resistant prostate cancer.

Deregulated expression of circular RNAs (circRNAs) is associated with various human diseases, including many types of cancer. Despite their growing links to cancer, there has been limited characterization of circRNAs in metastatic castration-resistant prostate cancer, the major cause of prostate cancer mortality. Here, through the analysis of an exome-capture RNA-seq dataset from 47 metastatic castration-resistant prostate cancer samples and ribodepletion and RNase R RNA-sequencing of patient-derived xenografts (PDXs) and cell models, we identified 13 circRNAs generated from the key prostate cancer driver gene-androgen receptor (AR). We validated and characterized the top four most abundant, clinically relevant AR circRNAs. Expression of these AR circRNAs was upregulated during castration-resistant progression of PDXs. The upregulation was not due to global increase of circRNA formation in these tumors. Instead, the levels of AR circRNAs correlated strongly with that of the linear AR transcripts (both AR and AR variants) in clinical samples and PDXs, indicating a transcriptional mechanism of regulation. In cultured cells, androgen suppressed the expression of these AR circRNAs and the linear AR transcripts, and the suppression was attenuated by an antiandrogen. Using nuclear/cytoplasmic fractionation and RNA in-situ hybridization assays, we demonstrated predominant cytoplasmic localization of these AR circRNAs, indicating likely cytoplasmic functions. Overall, this is the first comprehensive characterization of circRNAs arising from the AR gene. With greater resistance to exoribonuclease compared to the linear AR transcripts and detectability of AR circRNAs in patient plasma, these AR circRNAs may serve as surrogate circulating markers for AR/AR-variant expression and castration-resistant prostate cancer progression.

The Epstein Barr virus circRNAome.

Our appreciation for the extent of Epstein Barr virus (EBV) transcriptome complexity continues to grow through findings of EBV encoded microRNAs, new long non-coding RNAs as well as the more recent discovery of over a hundred new polyadenylated lytic transcripts. Here we report an additional layer to the EBV transcriptome through the identification of a repertoire of latent and lytic viral circular RNAs. Utilizing RNase R-sequencing with cell models representing latency types I, II, and III, we identified EBV encoded circular RNAs expressed from the latency Cp promoter involving backsplicing from the W1 and W2 exons to the C1 exon, from the EBNA BamHI U fragment exon, and from the latency long non-coding RPMS1 locus. In addition, we identified circular RNAs expressed during reactivation including backsplicing from exon 8 to exon 2 of the LMP2 gene and a highly expressed circular RNA derived from intra-exonic backsplicing within the BHLF1 gene. While expression of most of these circular RNAs was found to depend on the EBV transcriptional program utilized and the transcription levels of the associated loci, expression of LMP2 exon 8 to exon 2 circular RNA was found to be cell model specific. Altogether we identified over 30 unique EBV circRNAs candidates and we validated and determined the structural features, expression profiles and nuclear/cytoplasmic distributions of several predominant and notable viral circRNAs. Further, we show that two of the EBV circular RNAs derived from the RPMS1 locus are detected in EBV positive clinical stomach cancer specimens. This study increases the known EBV latency and lytic transcriptome repertoires to include viral circular RNAs and it provides an essential foundation and resource for investigations into the functions and roles of this new class of EBV transcripts in EBV biology and diseases.

A comprehensive approach to expression of L1 loci.

L1 elements represent the only currently active, autonomous retrotransposon in the human genome, and they make major contributions to human genetic instability. The vast majority of the 500 000 L1 elements in the genome are defective, and only a relatively few can contribute to the retrotransposition process. However, there is currently no comprehensive approach to identify the specific loci that are actively transcribed separate from the excess of L1-related sequences that are co-transcribed within genes. We have developed RNA-Seq procedures, as well as a 1200 bp 5΄ RACE product coupled with PACBio sequencing that can identify the specific L1 loci that contribute most of the L1-related RNA reads. At least 99% of L1-related sequences found in RNA do not arise from the L1 promoter, instead representing pieces of L1 incorporated in other cellular RNAs. In any given cell type a relatively few active L1 loci contribute to the 'authentic' L1 transcripts that arise from the L1 promoter, with significantly different loci seen expressed in different tissues.

Global transcript structure resolution of high gene density genomes through multi-platform data integration.

Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Second generation sequencing and high-density microarray studies have revealed vastly greater herpesvirus transcriptome complexity than is captured by existing annotation. The pervasive nature of overlapping transcription throughout herpesvirus genomes, however, poses substantial problems in resolving transcript structures using these methods alone. We present an approach that combines the unique attributes of Pacific Biosciences Iso-Seq long-read, Illumina short-read and deepCAGE (Cap Analysis of Gene Expression) sequencing to globally resolve polyadenylated isoform structures in replicating Epstein-Barr virus (EBV). Our method, Transcriptome Resolution through Integration of Multi-platform Data (TRIMD), identifies nearly 300 novel EBV transcripts, quadrupling the size of the annotated viral transcriptome. These findings illustrate an array of mechanisms through which EBV achieves functional diversity in its relatively small, compact genome including programmed alternative splicing (e.g. across the IR1 repeats), alternative promoter usage by LMP2 and other latency-associated transcripts, intergenic splicing at the BZLF2 locus, and antisense transcription and pervasive readthrough transcription throughout the genome.

Segal crystallinity index revisited by the simulation of X-ray diffraction patterns of cotton cellulose Iß and cellulose II.

The Segal method estimates the amorphous fraction of cellulose Iß materials simply based on intensity at 18° 2θ in an X-ray diffraction pattern and was extended to cellulose II using 16° 2θ intensity. To address the dependency of Segal amorphous intensity on crystal size, cellulose polymorph, and the degree of polymorphic conversion, we simulated the diffraction patterns of cotton celluloses (Iß and II) and compared the simulated amorphous fractions with the Segal values. The diffraction patterns of control and mercerized cottons, respectively, were simulated with perfect crystals of cellulose Iß (1.54° FWHM) and cellulose II (2.30° FWHM) as well as 10% and 35% amorphous celluloses. Their Segal amorphous fractions were 15% and 31%, respectively. The higher Segal amorphous fraction for control cotton was attributed to the peak overlap. Although the amorphous fraction was set in the simulation, the peak overlap induced by the increase of FWHM further enhanced the Segal amorphous intensity of cellulose Iß. For cellulose II, the effect of peak overlap was smaller; however the lower reflection of the amorphous cellulose scattering in its Segal amorphous location resulted in smaller Segal amorphous fractions. Despite this underestimation, the relatively good agreement of the Segal method with the simulation for mercerized cotton was attributed to the incomplete conversion to cellulose II. The (1-10) and (110) peaks of cellulose Iß remained near the Segal amorphous location of cellulose II for blends of control and mercerized cotton fibers.

Exposure of Mycobacterium marinum to low-shear modeled microgravity: effect on growth, the transcriptome and survival under stress.

Waterborne pathogenic mycobacteria can form biofilms, and certain species can cause hard-to-treat human lung infections. Astronaut health could therefore be compromised if the spacecraft environment or water becomes contaminated with pathogenic mycobacteria. This work uses Mycobacterium marinum to determine the physiological changes in a pathogenic mycobacteria grown under low-shear modeled microgravity (LSMMG). M. marinum were grown in high aspect ratio vessels (HARVs) using a rotary cell culture system subjected to LSMMG or the control orientation (normal gravity, NG) and the cultures used to determine bacterial growth, bacterium size, transcriptome changes, and resistance to stress. Two exposure times to LSMMG and NG were examined: bacteria were grown for ~40 h (short), or 4 days followed by re-dilution and growth for ~35 h (long). M. marinum exposed to LSMMG transitioned from exponential phase earlier than the NG culture. They were more sensitive to hydrogen peroxide but showed no change in resistance to gamma radiation or pH 3.5. RNA-Seq detected significantly altered transcript levels for 562 and 328 genes under LSMMG after short and long exposure times, respectively. Results suggest that LSMMG induced a reduction in translation, a downregulation of metabolism, an increase in lipid degradation, and increased chaperone and mycobactin expression. Sigma factor H (sigH) was the only sigma factor transcript induced by LSMMG after both short and long exposure times. In summary, transcriptome studies suggest that LSMMG may simulate a nutrient-deprived environment similar to that found within macrophage during infection. SigH is also implicated in the M. marinum LSMMG transcriptome response.

New Noncoding Lytic Transcripts Derived from the Epstein-Barr Virus Latency Origin of Replication, oriP, Are Hyperedited, Bind the Paraspeckle Protein, NONO/p54nrb, and Support Viral Lytic Transcription.

UNLABELLED: We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long noncoding RNAs (vlncRNAs) that are expressed during reactivation. Here we show that the EBV latency origin of replication (oriP) is transcribed bi-directionally during reactivation and that both leftward (oriPtLs) and rightward (oriPtRs) transcripts are largely localized in the nucleus. While the oriPtLs are most likely noncoding, at least some of the oriPtRs contain the BCRF1/vIL10 open reading frame. Nonetheless, oriPtR transcripts with long 5' untranslated regions may partially serve noncoding functions. Both oriPtL and oriPtR transcripts are expressed with late kinetics, and their expression is inhibited by phosphonoacetic acid. RNA sequencing (RNA-seq) analysis showed that oriPtLs and oriPtRs exhibited extensive "hyperediting" at their Family of Repeat (FR) regions. RNA secondary structure prediction revealed that the FR region of both oriPtLs and oriPtRs may form large evolutionarily conserved and thermodynamically stable hairpins. The double-stranded RNA-binding protein and RNA-editing enzyme ADAR was found to bind to oriPtLs, likely facilitating editing of the FR hairpin. Further, the multifunctional paraspeckle protein, NONO, was found to bind to oriPt transcripts, suggesting that oriPts interact with the paraspeckle-based innate antiviral immune pathway. Knockdown and ectopic expression of oriPtLs showed that it contributes to global viral lytic gene expression and viral DNA replication. Together, these results show that these new vlncRNAs interact with cellular innate immune pathways and that they help facilitate progression of the viral lytic cascade. IMPORTANCE: Recent studies have revealed that the complexity of lytic herpesviral transcriptomes is significantly greater than previously appreciated with hundreds of viral long noncoding RNAs (vlncRNAs) being recently discovered. Work on cellular lncRNAs over the past several years has just begun to give us an initial appreciation for the array of functions they play in complex formation and regulatory processes in the cell. The newly identified herpesvirus lncRNAs are similarly likely to play a variety of different functions, although these functions are likely tailored to specific needs of the viral infection cycles. Here we describe novel transcripts derived from the EBV latency origin of replication. We show that they are hyperedited, that they interact with a relatively newly appreciated antiviral pathway, and that they play a role in facilitating viral lytic gene expression. These investigations are a starting point to unraveling the complex arena of vlncRNA function in herpesvirus lytic replication.

Diseño de un sistema para la verificación del desarrollo de una competencia del perfil del egresado / Design of a system for verifying the development of a competence in the profiles of graduates

Introducción: Las universidades determinan el perfil de egreso de sus estudiantes; por tanto, definir programas de estudios conducentes a lograr las competencias de perfil del egresado hace necesario analizar la calidad de éstos. Una forma de alcanzar este objetivo es evidenciando si el plan de estudios de la carrera se organiza de manera que las diferentes asignaturas contribuyen a desarrollar estas competencias gradual y sistemáticamente. Objetivo: Diseñar un sistema que permita verificar la calidad de un programa de carrera mediante un instrumento que evidencia si las asignaturas contribuyen gradual y sistemáticamente a que el estudiante adquiera las competencias declaradas en el perfil del egresado. Sujetos y métodos: Se realizó un estudio analítico-explicativo de diseño mixto. Se seleccionó la competencia a evaluar y se confeccionó una encuesta, que permitió cuantificar las competencias y contenidos desarrollados en cada una de las asignaturas estudiadas y determinar las metodologías educativas-evaluativas más utilizadas para cada competencia. Resultados: Se verificó que el 91% de las competencias y contenidos necesarios para adquirir la competencia del perfil del egresado se desarrollan con los estudiantes en las asignaturas correspondientes. Además, todas las asignaturas utilizan diversas metodologías educativas-evaluativas que contribuyen al logro de las competencias. Conclusiones: Este sistema permite verificar que el plan de estudios de la carrera está alineado con la competencia seleccionada del perfil del egresado y, por tanto, se puede aplicar a otros programas de carrera

Introduction: Graduation profile of students is determined by the universities; therefore, defining study programs leading to achieve graduation profile competencies makes necessary for these programs to undergo a quality analysis. One way to achieve this goal is determining if career curriculum is organized so that different subjects gradually and systematically help develop these skills during undergraduate training. Aim: To design a system to verify the career program quality with an instrument that shows up if different subjects gradually and systematically help students to acquire the graduation profile stated competencies. Subjects and methods: An analytical-explanatory, mixed design study was performed in which a graduation profile competition was selected and a survey was designed and applied to quantify the skills and/or contents in each of the subjects studied and besides, help determine what the educational-evaluative methodologies were most commonly used for each competition. Results: We verified that correspondent subjects developed 91% of the skills and/or contents needed to acquire the competence profile selected for graduate students. Additionally, all subjects used various educational-evaluative methodologies, so contributing to competencies achievement. Conclusions: The system verifies curriculum as consistent with selected career competition of graduation profile and therefore can be used in other career programs

Kinetic and structural analysis of fluorescent peptides on cotton cellulose nanocrystals as elastase sensors.

Human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) are serine proteases with destructive proteolytic activity. Because of this activity, there is considerable interest in elastase sensors. Herein we report the synthesis, characterization, and kinetic profiles of tri- and tetrapeptide substrates of elastase as glycine-esterified fluorescent analogs of cotton cellulose nanocrystals (CCN). The degree of substitution of peptide incorporated in CCN was 3-4 peptides per 100 anhydroglucose units. Glycine and peptide-cellulose-nanocrystals revealed crystallinity indices of 79 and 76%, respectively, and a crystallite size of 58.5 Å. A crystallite model of the peptide-cellulose conjugate is shown. The tripeptide conjugate of CCN demonstrated five-fold greater efficiency in HNE than the tripeptide in solution judged by its kcat/Km of 33,515. The sensor limits of detection at 2mg of the tri- and tetrapeptide CCN conjugates over a 10 min reaction time course were 0.03 U/mL PPE and 0.05 U/mL HNE, respectively.

High-throughput RNA sequencing-based virome analysis of 50 lymphoma cell lines from the Cancer Cell Line Encyclopedia project.

UNLABELLED: Using high-throughput RNA sequencing data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human T-lymphotropic virus type 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of murine leukemia virus (MuLV) transcripts was observed in DEL cells, and we identified four transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes, including interleukin-15 (IL-15), IL-6ST, STAT5B, HIVEP1, and IL-9R. Although KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL-10 locus exclusively in the Hodgkin's lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL-6, K4/vIL-8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles. IMPORTANCE: Viruses cause cancer in humans. In lymphomas the Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV) and human T-lymphotropic virus type 1 are major contributors to oncogenesis. We assessed virus-host interactions using a high throughput sequencing method that facilitates the discovery of new virus-host associations and the investigation into how the viruses alter their host environment. We found a previously unknown murine leukemia virus infection in one cell line. We identified cellular genes, including cytokine regulators, that are disrupted by virus integration, and we determined mechanisms through which virus integration causes deregulation of cellular gene expression. Investigation into the KSHV transcriptome in the BCP-1 cell line revealed high-level expression of immune signaling genes. EBV transcriptome analysis showed expression of vIL-10 transcripts in a Hodgkin's lymphoma that was uncoupled from lytic genes. These findings illustrate unique mechanisms of viral gene regulation and to the importance of virus-mediated host immune signaling in lymphomas.

[Sarcoptic mange: report of an outbreak in a family and their pet]. / Sarna sarcóptica: comunicación de un brote en un grupo familiar y su mascota.

Scabies caused by the genus Sarcoptes scabiei var canis is a prevalent infection in dogs and affects abandoned, malnourished and overcrowded animals, causing hair loss and an intensely pruritic crusting dermatitis. In humans the manifestation is a self-limiting pruritic dermatitis, but persistent cases are described. An outbreak of sarcoptic mange is reported in a family group (seven people, including a 5 month infant and his mother). The infective source was their own house dog who was taken from the street. The diagnosis was confirmed by the detection of mites and eggs in the acarotest of the dog and mites of S. scabei in the infant. Sarcoptic mange should be suspected in individuals with allergic dermatitis who have contact with dogs. Treatment in humans is usually symptomatic and may need miticides if the infection persists. The control of the disease requires an appropriate pet treatment.

Sarna sarcóptica: comunicación de un brote en un grupo familiar y su mascota / Sarcoptic mange: report of an outbreak in a family and their pet

Scabies caused by the genus Sarcoptes scabiei var canis is a prevalent infection in dogs and affects abandoned, malnourished and overcrowded animals, causing hair loss and an intensely pruritic crusting dermatitis. In humans the manifestation is a self-limiting pruritic dermatitis, but persistent cases are described. An outbreak of sarcoptic mange is reported in a family group (seven people, including a 5 month infant and his mother). The infective source was their own house dog who was taken from the street. The diagnosis was confirmed by the detection of mites and eggs in the acarotest of the dog and mites of S. scabei in the infant. Sarcoptic mange should be suspected in individuals with allergic dermatitis who have contact with dogs. Treatment in humans is usually symptomatic and may need miticides if the infection persists. The control of the disease requires an appropriate pet treatment.

La sarna producida por el género Sarcoptes scabiei var canis, infección prevalente en perros y de alto potencial zoonótico, afecta a animales abandonados, desnutridos y hacinados y causa alopecia y una dermatitis costrosa intensamente pruriginosa. En el ser humano produce una dermatitis pruriginosa generalmente autolimitada, pero se describen casos persistentes. Se reporta un brote de sarna sarcóptica en un grupo familiar (siete personas, incluidas una lactante y su madre) cuya fuente de infección fue su mascota canina recogida de la calle. El diagnóstico fue confirmado por visualización en el ácarotest de ácaros y huevos en el perro y ácaros de S. scabiei en la lactante. La sarna sarcóptica debe sospecharse en casos de dermatitis alérgica en personas con contacto con perros. El tratamiento en el humano, habitualmente sintomático, puede necesitar acaricidas si el cuadro persiste. El control de la enfermedad requiere el adecuado tratamiento de la mascota.

Global bidirectional transcription of the Epstein-Barr virus genome during reactivation.

Epstein-Barr virus (EBV) reactivation involves the ordered induction of approximately 90 viral genes that participate in the generation of infectious virions. Using strand-specific RNA-seq to assess the EBV transcriptome during reactivation, we found extensive bidirectional transcription extending across nearly the entire genome. In contrast, only 4% of the EBV genome is currently bidirectionally annotated. Most of the newly identified transcribed regions show little evidence of coding potential, supporting noncoding roles for most of these RNAs. Based on previous cellular long noncoding RNA size calculations, we estimate that there are likely hundreds more EBV genes expressed during reactivation than was previously known. Limited 5' and 3' rapid amplification of cDNA ends (RACE) experiments and findings of novel splicing events by RNA-seq suggest that the complexity of the viral genome during reactivation may be even greater. Further analysis of antisense transcripts at some of the EBV latency gene loci showed that they are "late" genes, they are nuclear, and they tend to localize in areas of the nucleus where others find newly synthesized viral genomes. This raises the possibility that these transcripts perform functions such as new genome processing, stabilization, organization, etc. The finding of a significantly more complex EBV transcriptome during reactivation changes our view of the viral production process from one that is facilitated and regulated almost entirely by previously identified viral proteins to a process that also involves the contribution of a wide array of virus encoded noncoding RNAs. Epstein-Barr virus (EBV) is a herpesvirus that infects the majority of the world's population, in rare cases causing serious disease such as lymphoma and gastric carcinoma. Using strand-specific RNA-seq, we have studied viral gene expression during EBV reactivation and have discovered hundreds more viral transcripts than were previously known. The finding of alternative splicing and the prevalence of overlapping transcripts indicate additional complexity. Most newly identified transcribed regions do not encode proteins but instead likely function as noncoding RNA molecules which could participate in regulating gene expression, gene splicing or even activities such as viral genome processing. These findings broaden the scope of what we need to consider to understand the viral manufacturing process. As more detailed studies are undertaken they will likely change the way we view this process as a whole.

Differences in gastric carcinoma microenvironment stratify according to EBV infection intensity: implications for possible immune adjuvant therapy.

Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC - high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.

Halogen bond tunability II: the varying roles of electrostatic and dispersion contributions to attraction in halogen bonds.

In a previous study we investigated the effects of aromatic fluorine substitution on the strengths of the halogen bonds in halobenzene…acetone complexes (halo = chloro, bromo, and iodo). In this work, we have examined the origins of these halogen bonds (excluding the iodo systems), more specifically, the relative contributions of electrostatic and dispersion forces in these interactions and how these contributions change when halogen σ-holes are modified. These studies have been carried out using density functional symmetry adapted perturbation theory (DFT-SAPT) and through analyses of intermolecular correlation energies and molecular electrostatic potentials. It is found that electrostatic and dispersion contributions to attraction in halogen bonds vary from complex to complex, but are generally quite similar in magnitude. Not surprisingly, increasing the size and positive nature of a halogen's σ-hole dramatically enhances the strength of the electrostatic component of the halogen bonding interaction. Not so obviously, halogens with larger, more positive σ-holes tend to exhibit weaker dispersion interactions, which is attributable to the lower local polarizabilities of the larger σ-holes.

Whole-genome sequencing of the Akata and Mutu Epstein-Barr virus strains.

Using a simple viral genome enrichment approach, we report the de novo assembly of the Akata and Mutu Epstein-Barr virus (EBV) genomes from a single lane of next-generation sequencing (NGS) reads. The Akata and Mutu viral genomes are type I EBV strains of approximately 171 kb in length. Evidence for genome heterogeneity was found for the Akata but not for the Mutu strain. A comparative analysis of Akata with another four completely sequenced EBV strains, B95-8/Raji, AG876, Mutu, and GD1, demonstrated that the Akata strain is most closely related to the GD1 strain and exhibits the greatest divergence from the type II strain, AG876. A global comparison of latent and lytic gene sequences showed that the four latency genes, EBNA2, EBNA3A, EBNA3B, and EBNA3C, are uniquely defining of type I and type II strain differences. Within type I strains, LMP1, the latency gene, is among the most divergent of all EBV genes, with three insertion or deletion loci in its CTAR2 and CTAR3 signaling domains. Analysis of the BHLF1 and LF3 genes showed that the reading frames identified in the B95-8/Raji genome are not conserved in Akata (or Mutu, for BHLF1), suggesting a primarily non-protein-coding function in EBV's life cycle. The Akata and Mutu viral-genome sequences should be a useful resource for homology-based functional prediction and for molecular studies, such as PCR, RNA-seq, recombineering, and transcriptome studies. As an illustration, we identified novel RNA-editing events in ebv-miR-BART6 antisense transcripts using the Akata and Mutu reference genomes.

Identification of new viral genes and transcript isoforms during Epstein-Barr virus reactivation using RNA-Seq.

Using an enhanced RNA-Seq pipeline to analyze Epstein-Barr virus (EBV) transcriptomes, we investigated viral and cellular gene expression in the Akata cell line following B-cell-receptor-mediated reactivation. Robust induction of EBV gene expression was observed, with most viral genes induced >200-fold and with EBV transcripts accounting for 7% of all mapped reads within the cell. After induction, hundreds of candidate splicing events were detected using the junction mapper TopHat, including a novel nonproductive splicing event at the gp350/gp220 locus and several alternative splicing events at the LMP2 locus. A more detailed analysis of lytic LMP2 transcripts showed an overall lack of the prototypical type III latency splicing events. Analysis of nuclear versus cytoplasmic RNA-Seq data showed that the lytic forms of LMP2, EBNA-2, EBNA-LP, and EBNA-3A, -3B, and -3C have higher nuclear-to-cytoplasmic accumulation ratios than most lytic genes, including classic late genes. These data raise the possibility that at least some lytic transcripts derived from these latency gene loci may have unique, noncoding nuclear functions during reactivation. Our analysis also identified two previously unknown genes, BCLT1 and BCRT2, that map to the BamHI C-region of the EBV genome. Pathway analysis of cellular gene expression changes following B-cell receptor activation identified an inflammatory response as the top predicted function and ILK and TREM1 as the top predicted canonical pathways.