1,721,013 research outputs found
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
The role of viral genome dinucleotide content on replication, gene expression, and interferon evasion
Get PDFSignificant suppression of CpG dinucleotides has been observed in some DNA and RNA viruses. Based on this observation, it has long been suggested that dinucleotide frequency may play a role in host defenses by allowing the host cell to distinguish between self and non-self RNA. Recently, the interferon-stimulated Zinc-finger Antiviral Protein (ZAP) has been shown to target viral RNA containing high numbers of CpG dinucleotides for degradation.
Previous work from the Grey lab determined that ZAP is a human cytomegalovirus (HCMV) host restriction factor. HCMV displays a striking pattern of CpG dinucleotides, with suppression limited to the immediate-early (IE) gene, IE1. Previously, the lab demonstrated that suppressed CpG levels in IE1 enabled evasion of ZAP in the context of a co-plasmid expression system. To test whether CpG dinucleotides are important in the context of virus infection, we created a high CpG IE1 mutant HCMV. Based on growth kinetics following reconstitution of the recombinant virus, increased CpG levels in IE1 attenuated the virus. While ZAP depletion increased replication of the high CpG IE1 virus, similar results were observed for the control virus.
Subsequent sequence analysis showed that virus stocks contained the parental virus in addition to the high CpG recombinant, suggesting small amounts of parental virus outcompeted the high CpG virus due to the associated attenuation.
In addition to investigating ZAP evasion, we investigated the role of CpG dinucleotide content in viral gene expression. Nucleotide bias has previously been shown to influence gene expression in a splicing-dependent manner. To determine the effect of CpG levels on unspliced herpesviruses genes, we created reduced CpG constructs of an early gene, pp52, by way of synonymous mutations. We showed that reducing CpG levels in pp52 drastically reduced gene expression efficiency, but also resulted in a decrease in RNA levels. Further studies are needed to investigate how transcription may be affected, but together these experiments suggest that the high CpG frequencies of these genes may be maintained as a means to increase viral fitness.
Finally, herpesviruses express a family of RNA binding proteins that increase the export of unspliced transcripts. The best-studied of these are ICP27 of the alphaherpesvirus Herpes Simplex Virus-1 (HSV-1), and ORF57 of the gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). Using GFP constructs that contain synonymous mutations, we investigated whether these RNA binding proteins recognise transcripts with the same dinucleotide composition as their respective subfamilies. While preliminary, our data suggest that both ICP27 and ORF57 increased expression of a GFP construct with high CpG frequencies, while only ORF57 increased the expression of a low CpG construct. These studies suggest that dinucleotide content plays a fundamental role in herpesviruses biology, including immune evasion and viral gene expression
Systematic identification of host factors influencing human cytomegalovirus replication
Get PDFA high-throughput two-step siRNA screen was performed to identify novel host factors required during human cytomegalovirus (HCMV) replication. Almost 7,000 cellular genes were targeted by the siRNA library and 81 candidates, of which gene depletion led to a 50% decrease or 25% increase in virus production, were identified. Functional annotation clustering by STRING revealed functional clusters in mediator complex, proteasome, ubiquitin-dependent protein degradation and histone modification.
Focused characterisation of asparagine synthetase (ASNS) demonstrated a strict requirement for asparagine for HCMV replication. Depletion of intracellular asparagine levels, as a result of ASNS knockdown, led to a reduced expression of viral immediate-early protein IE2 but not IE1. The reduction on protein expression was not due to a general hindrance of protein synthesis since replication of herpes simplex virus-1 or influenza A virus was not affected. Furthermore, mTOR signalling was maintained during ASNS knockdown, and asparagine supplementation during the course of infection can completely rescue virus replication, suggesting a potential signalling pathway regulated by asparagine required for HCMV replication.
Detailed characterisation of another hit, SIN3A, showed a novel DNA damage and repair mechanism required by HCMV. SIN3A forms a complex with histone deacetylase 1 and 2 (HDAC1 and HDAC2), and the complex can exert a range of functions including DNA damage response. SIN3A knockdown had a moderate effect on viral immediate-early and early gene expression, however, production of infectious virions was substantially inhibited. Further studies showed that the expression of a DNA damage marker, γH2AX, is increased and deleterious DNA-RNA hybrids (R-loop) accumulate during HCMV replication when SIN3A is absent.
This study represents the most comprehensive siRNA screen to identify host factors involved in HCMV replication and production to date, and identifies metabolism of non-essential amino acid asparagine and SIN3A/HDAC complex as critical host factors for HCMV
Interaction of host and viral microRNAs with infectious laryngotracheitis virus transcripts
Get PDFInfectious Laryngotracheitis Virus (ILTV) is an Alphaherpesvirus of the domesticated
chicken and other economically important fowl such as pheasants, peafowl and turkeys. It
causes an upper respiratory disease that is clinically characterised by dyspnoea, rales and
expulsion of a thick, sometimes hemorrhagic, tracheal exudate. Incidences of mortality
range from 10 – 70 % whilst morbidity ranges from 50 – 100 %. The disease causes
significant financial losses to the poultry industry through bird death, stunted growth and a
marked decrease in egg production. Due to its economic importance, attenuated live-vaccines
have been developed by serial passage of virus either in eggs or tissue culture.
These have the ability to protect birds against ILTV however they do not stop latent infection
which can result in reactivation of the virus termed ‘vaccinal laryngotracheitis’. The
molecular biology underlying virus-host interactions for ILTV is poorly understood and
there are large gaps in knowledge regarding the pathogenesis of ILTV infection.
MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally regulate gene
expression through targeting of specific mRNAs. Several herpesviruses have been shown to
encode miRNAs that have the ability to regulate both viral and cellular gene expression
which can impact virus-host interactions. Previous work in the literature has shown that
ILTV encodes for 10 miRNAs with sparse data on what they may be regulating.
It was hypothesised that the virus-encoded miRNAs may have an effect upon the
pathogenesis of the virus by targeting both cellular and viral mRNAs. To investigate this
hypothesis initially, the biochemical technique CLASH (Cross-Linking and Sequencing of
Hybrids) was attempted however a lack of suitable reagents such as physiologically relevant
cell lines of chicken origin made this technically challenging and this approach was halted.
Instead, a bioinformatic approach was developed and split into two avenues of research.
Firstly, it was hypothesised that miRNAs encoded by ILTV would target virally derived
transcripts. As the virus genome is poorly annotated, transcripts for all 79 open reading
frames (ORFs) were created manually using an arbitrary system of 1000 bp upstream of the
ATG start site and 50 bp downstream of the designated PolyA tail. These were then fed into
the online algorithm RNA Hybrid alongside sequences for all 10 virus-encoded miRNAs.
Results from the bioinformatic predictions were then sorted and filtered using pre-defined
conditions. This left a total of 227 predicted interactions. These were then filtered again
leaving 28 novel targets that were screened in a reporter based system. Three of the
predicted interactions showed a decrease in luciferase-reporter activity compared to the
siRNA control (UL24, UL29 and UL46/48), however only the latter two showed statistically
significant decreases in activity of 15 % and 20 % respectively. Mutation of the seed
sequences in both UL29 and UL46/48 targets abrogated the effects of the miRNA mimic.
Further work on UL29 and its interaction with ILTV-miR-I2 looked at validating this
interaction by western blotting however these results were inconclusive. Investigations into
the interaction between UL46/48 and ILTV-miR-I6-5p first confirmed by RT-PCR that
UL46 was targeted by ILTV-miR-I6-5p. Validation of the interaction between UL46 and
ILTV-miR-I6-5p by western blotting was inconclusive. Investigations into the interplay
between UL46, UL48 and the ICP4 promoter were also characterised with UL46 able to
negatively modulate the effects of UL48 on ICP4 promoter activity in a reporter-based
system.
Secondly, the same viral transcripts were then used in conjunction with high confidence
chicken miRNAs as per MiRBase (Release 21, Jun 2014). The sorting and filtering of
results mirrored that of the viral transcript study giving a final list of 103 predicted targets.
From the list, three targets were picked that were all targeted by the cellular miRNA ggamiR-
133a-3p and tested using the same reporter system. Two targets, one in UL20 and one
in the coding region of ICP4 showed no statistical difference between the miRNA mimic and
siRNA control. In contrast, one target, located in the 5’UTR of ICP4 and confirmed by RTPCR
to be within the expressed mRNA transcript was found to cause a 55 % reduction in
luciferase activity. This effect was then abrogated upon mutation of the miRNA seed
sequence. Further investigations found that this miRNA can cause an apparent reduction in
virus titer and a statistically significant decrease in plaque size morphology when virus is
harvested from cells transfected with the miRNA mimic and used to infect naïve cells.
Moreover, a combination RT-qPCR and sequencing was used to confirm the sequence of
gga-miR-133a-3p in several tissues of the chicken including the Dorsal Root Ganglia (DRG)
and Harderian gland (HG). These are of importance to ILTV biology as the DRG is a site of
latent infection and the HG is a secondary lymphoid organ (SLO) in the bird which monitors
the upper respiratory tract, the site of lytic replication/clinical symptoms.
Finally, CRISPR-Cas9 genome editing was used to delete a cluster of five miRNAs from the
viral genome. Guide RNAs (sgRNAs) were designed to target the miRNA cluster and shown
to efficiently direct cleavage of target DNA in an in vitro system. Following
transfection/infection of cells, virus was harvested and subsequent sequencing showed that
this approach was successful in creating a recombinant ILTV. This was detectable after
passage of the virus through naïve cells although a pure population of recombinant virus was
not obtained due to a lack of time
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Identification of host ubiquitin modifying and binding enzymes that restrict human cytomegalovirus replication
Get PDFAs obligate, intracellular parasites, viruses rely on host factors for their replication. High throughput siRNA screens are an effective approach for identifying host-virus interactions. We are interested in identifying host-virus interactions in human cytomegalovirus (HCMV), a clinically important herpesvirus. Previously, Valosin-containing protein, a key component of the ubiquitin-proteasome system (UPS), was identified as a host-factor important for HCMV replication using an siRNA screen. To understand the importance of UPS in HCMV infection, we performed an siRNA screen against the ubiquitin-modifying enzymes. From this screen we identified numerous pro- and antiviral host factors that impact HCMV replication. Here we have selected two of the antiviral host factors, Remodelling and spacing factor 1 (RSF1) and F-box and leucin-rich repeat protein 20 (FBLX20), for further investigation.
RSF1 has previously been shown to regulate gene expression through binding to ubiquitinated histones. We have shown that knockdown of RSF1 leads to an increase in expression of the major immediate early (MIE) genes IE1 and IE2, which are critical for driving acute replication of the virus. Furthermore, RSF1 colocalises with the HCMV replication compartment, where viral genome replication and transcription occur, suggesting RSF1 may be directly interacting with the viral genome to regulate the MIE promoter.
FBXL20 is a substrate receptor of a cullin-RING E3 ligase complex. Here we discovered that knocking down FBXL20 leads to a significant decrease in interferon (IFN) production during HCMV infection. This decrease was similar to levels achieved by knocking down cGAS, the major HCMV pathogen recognition receptor, which has previously been shown to be necessary for efficient IFN upregulation triggered by HCMV infection. Decreased levels of FBXL20 also leads to reduced induction of interferon-stimulated genes. This suggests that FBXL20 is involved in regulation of the interferon signalling pathway.
In summary, we have identified two ubiquitin-modifying enzymes that play a major role in restricting HCMV replication through two distinct pathways: via interaction with the incoming genome and through regulation of innate immunity
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