Amplification of the Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 lytic origin of DNA replication is dependent upon a cis-acting AT-rich region and an ORF50 response element and the trans-acting factors ORF50 (K-Rta) and K8 (K-bZIP)

AbstractKaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), has significant sequence homology to Epstein–Barr virus (EBV). In cell culture, HHV8 is primarily latent, and viral genes associated with lytic replication are not expressed. Two lytic origins of DNA replication (oriLyt) are present within the HHV8 genome and are composed of an AT-rich region adjacent to GC-rich DNA sequences. We have now identified essential cis- and trans-acting elements required for oriLyt-dependent DNA replication. The transient replication assay was used to show that two AT-rich elements, three consensus AP1 transcription factor-binding sites, an ORF50 response element (RE), and a consensus TATA box motif are essential for efficient origin-dependent DNA replication. Transient transfection of luciferase reporter constructs indicated that the downstream region of the HHV8 oriLyt responds to ORF50 and suggests that part of the oriLyt may be an enhancer/promoter. In addition, a transient cotransfection–replication assay elucidated the set of trans-acting factors required for lytic DNA replication. These factors consist of homologues to the core replication proteins: ORF6 (ssDNA binding protein), ORF9 (DNA polymerase), ORF40-41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor) common to all herpesviruses along with ORF50 (K-Rta) and K8 (K-bZIP)

Comparación ultrasonográfica transvaginal y transrectal de la dinámica folicular en ondas sucesivas de llamas (Lama glama)

The ovarian follicular dynamics monitored by transrectal (TR) and transvaginal (TV) routes in successive waves was studied in 10 llamas older than 4 years, multiparous, without calves at foot, body condition of 3.0, during a period of 90 days in the reproductive season. The Sonostar SS8® ultrasound equipment was used with a linear transducer for transrectal examination and endocavitary microconvex transducer for transvaginal examination, both with a frequency of 6.5 MHz. The inter-wave interval was 20 ± 4.4 and 22.5 ± 5.3 via TR and VT, respectively; the number of follicles per cohort was 5.21 ± 0.92 and 6.38 ± 1.26 for TR and TV, respectively (p=0.0011); the maximum diameter of follicles was 12.49 ± 3.57 mm for TR and 13.56 ± 3.91 mm for TV; the growth rate was 0.70 ± 0.35 and 0.67 ± 0.32 mm/day for TR and TV, respectively. The regression equation for the ratio of the maximum follicular diameter and duration of follicular wave via TR was y=0.89766x+6.5554 (R²=0.423) and for the TV route y=0.8466x+10.184 (R²=0.479). The duration of the follicular phases was similar between both techniques except for the dominance phase (10.2±3.19 days for TR and 8.67±2.46 for TV, p=0.0418). The relationship between the follicular diameter and the number of follicles detected showed a low negative correlation (R²=-0.0827 for TV and R²=-0.0876 for TR). In conclusion, the transvaginal endocavitary transducer makes it possible to determine with greater precision the characteristics of llama follicular dynamics in their recruitment, dominance and regression phases with overlapping of follicular waves as an important characteristic.La dinámica folicular ovárica monitoreada por vía transrectal (TR) y transvaginal (TV) en ondas sucesivas en llamas fue estudiada en 10 llamas con más de 4 años, multíparas, sin cría, estado corporal de 3.0, durante un periodo de 90 días en la época reproductiva. Se utilizó el ecógrafo Sonostar SS8® con un transductor lineal para el examen transrectal y transductor microconvexo endocavitario para el examen transvaginal, ambos con frecuencia de 6.5 MHz. El intervalo inter-onda fue de 20±4.4 y 22.5±5.3 por vía TR y TV, respectivamente; el número de folículos por cohorte fue 5.21±0.92 y 6.38±1.26 para TR y TV, respectivamente (p=0.0011); el diámetro máximo de folículos fue 12.49±3.57 mm para TR y 13.56±3.91 mm para TV; la tasa de crecimiento fue de 0.70±0.35 y 0.67±0.32 mm/día para TR y TV, respectivamente. La ecuación de regresión para relación del diámetro máximo folicular y duración de onda folicular vía TR fue de y=0.89766x+6.5554 (R²=0.423) y para la vía TV de y=0.8466x+10.184 (R²=0.479). La duración de las fases foliculares fue similar entre ambas técnicas con excepción de la fase de dominancia (10.2±3.19 días para TR y 8.67±2.46 para TV, p=0.0418). La relación entre el diámetro folicular y número de folículos detectados presentó una correlación negativa baja (R²=-0.0827 para TV y R²=-0.0876 para TR). En conclusión, el transductor endocavitario transvaginal permite determinar con mayor precisión las características de dinámica folicular en llamas en sus fases de reclutamiento, dominancia y regresión con sobreposición de ondas foliculares como característica importante.The ovarian follicular dynamics monitored by transrectal (TR) and transvaginal (TV) routes in successive waves was studied in 10 llamas older than 4 years, multiparous, without calves at foot, body condition of 3.0, during a period of 90 days in the reproductive season. The Sonostar SS8® ultrasound equipment was used with a linear transducer for transrectal examination and endocavitary microconvex transducer for transvaginal examination, both with a frequency of 6.5 MHz. The inter-wave interval was 20 ± 4.4 and 22.5 ± 5.3 via TR and VT, respectively; the number of follicles per cohort was 5.21 ± 0.92 and 6.38 ± 1.26 for TR and TV, respectively (p=0.0011); the maximum diameter of follicles was 12.49 ± 3.57 mm for TR and 13.56 ± 3.91 mm for TV; the growth rate was 0.70 ± 0.35 and 0.67 ± 0.32 mm/day for TR and TV, respectively. The regression equation for the ratio of the maximum follicular diameter and duration of follicular wave via TR was y=0.89766x+6.5554 (R²=0.423) and for the TV route y=0.8466x+10.184 (R²=0.479). The duration of the follicular phases was similar between both techniques except for the dominance phase (10.2±3.19 days for TR and 8.67±2.46 for TV, p=0.0418). The relationship between the follicular diameter and the number of follicles detected showed a low negative correlation (R²=-0.0827 for TV and R²=-0.0876 for TR). In conclusion, the transvaginal endocavitary transducer makes it possible to determine with greater precision the characteristics of llama follicular dynamics in their recruitment, dominance and regression phases with overlapping of follicular waves as an important characteristic

Introductory programming: a systematic literature review

As computing becomes a mainstream discipline embedded in the school curriculum and acts as an enabler for an increasing range of academic disciplines in higher education, the literature on introductory programming is growing. Although there have been several reviews that focus on specific aspects of introductory programming, there has been no broad overview of the literature exploring recent trends across the breadth of introductory programming. This paper is the report of an ITiCSE working group that conducted a systematic review in order to gain an overview of the introductory programming literature. Partitioning the literature into papers addressing the student, teaching, the curriculum, and assessment, we explore trends, highlight advances in knowledge over the past 15 years, and indicate possible directions for future research

Mixed methods study protocol for combining stakeholder-led rapid evaluation with near real-time continuous registry data to facilitate evaluations of quality of care in intensive care units [version 1; peer review: awaiting peer review]

BACKGROUND: Improved access to healthcare in low- and middle-income countries (LMICs) has not equated to improved health outcomes. Absence or unsustained quality of care is partly to blame. Improving outcomes in intensive care units (ICUs) requires delivery of complex interventions by multiple specialties working in concert, and the simultaneous prevention of avoidable harms associated with the illness and the treatment interventions. Therefore, successful design and implementation of improvement interventions requires understanding of the behavioural, organisational, and external factors that determine care delivery and the likelihood of achieving sustained improvement. We aim to identify care processes that contribute to suboptimal clinical outcomes in ICUs located in LMICs and to establish barriers and enablers for improving the care processes. METHODS: Using rapid evaluation methods, we will use four data collection methods: 1) registry embedded indicators to assess quality of care processes and their associated outcomes; 2) process mapping to provide a preliminary framework to understand gaps between current and desired care practices; 3) structured observations of processes of interest identified from the process mapping and; 4) focus group discussions with stakeholders to identify barriers and enablers influencing the gap between current and desired care practices. We will also collect self-assessments of readiness for quality improvement. Data collection and analysis will be performed in parallel and through an iterative process across eight countries: Kenya, India, Malaysia, Nepal, Pakistan, South Africa, Uganda and Vietnam. CONCLUSIONS: The results of our study will provide essential information on where and how care processes can be improved to facilitate better quality of care to critically ill patients in LMICs; thus, reduce preventable mortality and morbidity in ICUs. Furthermore, understanding the rapid evaluation methods that will be used for this study will allow other researchers and healthcare professionals to carry out similar research in ICUs and other health services

Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Contains Two Functional Lytic Origins of DNA Replication

We used a transient-transfection replication assay to identify two functional copies of the human herpesvirus 8 (HHV8) lytic origin of DNA replication (oriLyt). BCLB-1 cells were transfected with HHV8 subgenomic fragments containing the putative lytic origin along with a plasmid expressing viral transactivator open reading frame (ORF) 50. The HHV8 left-end oriLyt (oriLyt-L) lies between ORFs K4.2 and K5 and is composed of a region encoding various transcription factor binding sites and an A+T-rich region and a G+C repeat region. The right-end oriLyt (oriLyt-R) maps between ORF 69 and vFLIP, a region similar to the RRV oriLyt, and is an inverted duplication of oriLyt-L

A Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 ORF50 Deletion Mutant Is Defective for Reactivation of Latent Virus and DNA Replication

Kaposi's sarcoma-associated herpesvirus (also called human herpesvirus type 8 [HHV8]) latently infects a number of cell types. Reactivation of latent virus can occur by treatment with the phorbol ester tetradecanoyl phorbol acetate (TPA) or with the transfection of plasmids expressing the lytic switch activator protein K-Rta, the gene product of ORF50. K-Rta expression is sufficient for the activation of the entire lytic cycle and the transactivation of viral genes necessary for DNA replication. In addition, recent evidence has suggested that K-Rta may participate directly in the initiation of lytic DNA synthesis. We have now generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a large deletion within the ORF50 locus. This BAC, BAC36Δ50, failed to produce infectious virus upon treatment with TPA and was defective for DNA synthesis. Expression of K-Rta in trans in BAC36Δ50-containing cells was able to abolish both defects. Real-time PCR revealed that K-bZIP, ORF40/41, and K8.1 were not expressed when BAC36Δ50-containing cells were induced with TPA. However, the mRNA levels of ORF57 were over fivefold higher in TPA-treated BAC36Δ50-containing cells than those observed in similarly treated wild-type BAC-containing cells. In addition, immunohistochemical analysis showed that while the latency-associated nuclear antigen (LANA) was expressed in the mutant BAC-containing cells, ORF59 and K8.1 expression was not detected in TPA-induced BAC36Δ50-containing cells. These results showed that K-Rta is essential for lytic viral reactivation and transactivation of viral genes contributing to DNA replication

Safety of endoscopic removal of self-expandable stents after treatment of benign esophageal diseases

Temporary placement of self-expandable stents has been increasingly used for the management of benign esophageal diseases. To evaluate the safety of endoscopic removal of esophageal self-expandable stents placed for the treatment of benign esophageal diseases. Multicenter retrospective study. Six tertiary care centers in the United States and Europe. A total of 214 patients with benign esophageal diseases undergoing endoscopic stent removal. Endoscopic stent removal. Endoscopic techniques for stent removal, time to stent removal, and adverse events related to stent removal. A total of 214 patients underwent a total of 329 stent extractions. Stents were mainly placed for refractory strictures (49.2%) and fistulae (49.8%). Of the removed stents, 52% were fully covered self-expandable metal stents (FCSEMSs), 28.6% were partially covered self-expandable metal stents (PCSEMSs), and 19.5% were self-expandable plastic stents. A total of 35 (10.6%) procedure-related adverse events were reported, including 7 (2.1%) major adverse events. Multivariate analysis revealed that use of PCSEMSs (P < .001) was a risk factor for adverse events during stent removal. Favorable factors for successful stent removal were FCSEMSs (P ≤ .012) and stent migration (P = .010). No significant associations were found for stent indwelling time (P = .145) and stent embedding (P = .194). Retrospective analysis, only tertiary care centers. With an acceptable major adverse event rate of 2.1%, esophageal stent removal in the setting of benign disease was found to be a safe and feasible procedure. FCSEMSs were more successfully removed than self-expandable plastic stents and PCSEMSs. Adverse events caused by stent removal were not time dependen