RNA-Seq Analysis of Differentiated Keratinocytes Reveals a Massive Response to Late Events during Human Papillomavirus 16 Infection, Including Loss of Epithelial Barrier Function.

The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalized keratinocytes (NIKS) and NIKS stably transfected with HPV16 episomal genomes (NIKS16) were compared using next-generation sequencing (RNA-Seq). HPV16 infection altered expression of 2,862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNA-Seq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log2 = 1.8, 3.5-fold change), 670 genes were downregulated and 296 genes were upregulated. HPV downregulated many genes involved in epithelial barrier function, which involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant CCL20, and proinflammatory cytokines interleukin 1α (IL-1α) and IL-1ß. However, the type I interferon regulator IRF1, kappa interferon (IFN-κ), and viral restriction factors (IFIT1, -2, -3, and -5, OASL, CD74, and RTP4) were upregulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions, and cell adhesion. Quantitative PCR (qRT-PCR) and Western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress.IMPORTANCE HPV genome amplification and capsid formation take place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNA-Seq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3,000 genes were differentially expressed in keratinocytes due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into the virus-host interaction that is crucial for the production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle.

Deep sequencing analysis of defective genomes of parainfluenza virus 5 and their role in interferon induction.

Preparations of parainfluenza virus 5 (PIV5) that are potent activators of the interferon (IFN) induction cascade were generated by high-multiplicity passage in order to accumulate defective interfering virus genomes (DIs). Nucleocapsid RNA from these virus preparations was extracted and subjected to deep sequencing. Sequencing data were analyzed using methods designed to detect internal deletion and "copyback" DIs in order to identify and characterize the different DIs present and to approximately quantify the ratio of defective to nondefective genomes. Trailer copybacks dominated the DI populations in IFN-inducing preparations of both the PIV5 wild type (wt) and PIV5-VΔC (a recombinant virus that does not encode a functional V protein). Although the PIV5 V protein is an efficient inhibitor of the IFN induction cascade, we show that nondefective PIV5 wt is unable to prevent activation of the IFN response by coinfecting copyback DIs due to the interfering effects of copyback DIs on nondefective virus protein expression. As a result, copyback DIs are able to very rapidly activate the IFN induction cascade prior to the expression of detectable levels of V protein by coinfecting nondefective virus.

Developmental effects of over-expression of normal and mutated forms of a Xenopus NF-kappa B homologue.

High level over-expression of XrelA1, a homologue of the p65 sub-unit of NF-kappa B and of Drosophila dorsal, arrests Xenopus development at the gastrula stage, producing a reduction in the levels of expression of various genes of developmental interest without general reduction in transcription or cessation of cell division. There is little Goosecoid expression, even though a dorsal lip forms. At lower levels XrelA1 mRNA primarily produces disruption of the mid-dorsal axis. A dominant interference gene product, delta 222, produces mainly posterior, but also anterior abnormalities. On the basis of these results we postulate that the role of XrelA1 in the vertebrate embryo is unlikely to be in dorsoventral development, but more likely in the formation of the termini.

Somitogenesis in the marsupial frog Gastrotheca riobambae.

This paper reports the first description of somitogenesis in a non-aquatic-developing amphibian, the Andean marsupial frog, Gastrotheca riobambae. This frog develops from an embryonic disk located on top of a large yolky egg, bearing some resemblance to the embryo of the chick. Besides the histological characterization of somite formation, we quantified cell number in the developing somites of Gastrotheca, Xenopus laevis and the chick. Gastrotheca was found to have a mode of somitogenesis which has previously been encountered in the aquatic-developing toad Bombina. Somitic cell number was found to be an order of magnitude higher than that of Xenopus, and approximately double that of the chick. We discuss the possible relation between mode of somitogenesis, somitic cell number, speed of development and egg size.

Adaptation of standard spreadsheet software for the analysis of DNA sequences.

This paper presents use of spreadsheet software to derive various statistics on nucleotide and codon distribution and frequency from gene sequence data, including chi 2 test and their derivatives, and proportion of nucleotides in the third position. The basic principles can be easily extended to other more complex functions. In addition, it can be used to translate a nucleic acid sequence into a protein sequence or to produce a codon usage table. This adaptation permits sequence analysis without expensive dedicated software and easy modification of the analysis according to the user's requirements. It is also ideal for introducing biological science students to the programming potential of spreadsheets.

On Units of Selection in Cultural Evolution.

Copyright 1998 Academic Press Limited

Comparative cervical profiles of adult and under-18 front-row rugby players: implications for playing policy.

OBJECTIVE: To compare the cervical isometric strength, fatigue endurance and range of motion of adult and under-18 age-grade front-row rugby players to inform the development of a safe age group policy with particular reference to scrummaging. DESIGN: Cross-sectional cohort study. SETTING: 'Field testing' at Murrayfield stadium. PARTICIPANTS: 30 high-performance under-18 players and 22 adult front-row rugby players. OUTCOME MEASURES: Isometric neck strength, height, weight and grip strength. RESULTS: Youth players demonstrated the same height and grip strength as the adult players; however, the adults were significantly heavier and demonstrated substantially greater isometric strength (p<0.001). Only two of the 'elite' younger players could match the adult mean cervical isometric strength value. In contrast to school age players in general, grip strength was poorly associated with neck strength (r=0.2) in front-row players; instead, player weight (r=0.4) and the number of years' experience of playing in the front row (r=0.5) were the only relevant factors in multivariate modelling of cervical strength (R(2)=0.3). CONCLUSIONS: Extreme forces are generated between opposing front rows in the scrum and avoidance of mismatch is important if the risk of injury is to be minimised. Although elite youth front-row rugby players demonstrate the same peripheral strength as their adult counterparts on grip testing, the adults demonstrate significantly greater cervical strength. If older youths and adults are to play together, such findings have to be noted in the development of age group policies with particular reference to the scrum.

Age-related differences in the neck strength of adolescent rugby players: A cross-sectional cohort study of Scottish schoolchildren.

OBJECTIVES: To evaluate the neck strength of school-aged rugby players, and to define the relationship with proxy physical measures with a view to predicting neck strength. METHODS: Cross-sectional cohort study involving 382 rugby playing schoolchildren at three Scottish schools (all male, aged between 12 and 18 years). Outcome measures included maximal isometric neck extension, weight, height, grip strength, cervical range of movement and neck circumference. RESULTS: Mean neck extension strength increased with age (p = 0.001), although a wide inter-age range variation was evident, with the result that some of the oldest children presented with the same neck strength as the mean of the youngest group. Grip strength explained the most variation in neck strength (R(2) = 0.53), while cervical range of movement and neck girth demonstrated no relationship. Multivariable analysis demonstrated the independent effects of age, weight and grip strength, and the resultant model explained 62.1% of the variance in neck strength. This model predicted actual neck strength well for the majority of players, although there was a tendency towards overestimation at the lowest range and underestimation at the highest. CONCLUSION: A wide variation was evident in neck strength across the range of the schoolchild-playing population, with a surprisingly large number of senior players demonstrating the same mean strength as the 12-year-old mean value. This may suggest that current training regimes address limb strength but not neck strength, which may be significant for future neck injury prevention strategies. Age, weight and grip strength can predict around two thirds of the variation in neck strength, however specific assessment is required if precise data is sought.

Small regions of preferential codon usage and their effect on overall codon bias--the case of the plp gene.

Preferential codon usage within synonymous groups (codon bias), is hypothesised to be a consequence of either mutational pressure or translational selection. This paper examines PLP and DM-20, two transcripts differentially spliced from the plp gene, using a variety of previously described indices measuring codon bias as well as a novel index for quantifying the response to directional mutation pressure. The results demonstrate that small regions of extreme codon bias may have appreciable effects on the quantification of total bias, and that this effect may be either an increase or a decrease depending which index is used.

N-acetyl-cysteine causes a late re-specification of the anteroposterior axis in the Xenopus embryo.

N-acetyl cysteine is an agent which has been shown to interrupt signal transduction processes linking a wide range of stimuli to the activation of NF-kappa B in mammalian cells. We have investigated its effect on the early development of Xenopus embryos by injecting it into blastulae, using concentrations comparable to those effective on cultured cells. High concentrations at the late blastula or early gastrula stage suppress posterior and enhance anterior development, yielding embryos with enlarged cement glands and otherwise consisting of little except head in extreme cases. Reducing the amount of N-acetyl cysteine injected leads to progressively more posterior structures developing. Injection into one- or two-cell embryos gives similar phenotypes, but of reduced severity and the cement gland is not so enlarged. Explants of animal cap cells taken several hours after injection develop to give large amounts of cement gland material. We have examined the expression of a number of genes in the anteriorised embryos. Posterior markers and Xsna are reduced. Noggin and Goosecoid mRNA are up-regulated through the gastrula and persist at these levels until at least the late neurula stage, whereas in controls Noggin is much lower and Goosecoid is absent at these stages. The most anteriorised phenotype may be a consequence of this changed expression.

Nitrogen-fixing aerobic bacteria have higher genomic GC content than non-fixing species within the same genus.

The genomic GC contents of both nitrogen-fixing and non-fixing members of eight genera of bacteria are investigated. Analysis by t-tests showed that in the two aerobic general investigated (Aquaspirillum and Vibrio) there is a significantly higher GC content in the nitrogen-fixing members of the genus than in those unable to fix nitrogen, whilst in aerobic genera there is either no GC bias, or in the case of two genera (Rhodospirillum and Clostridium) there is a significantly higher GC content in the non-fixing organisms. This suggests that, in many genera, directional mutational pressures are different in nitrogen-fixing and non-fixing organisms. These results are discussed in the light of known mechanisms of mutation pressure and their relation to environmental variables.

Expression of TGF-beta isoforms during first trimester human embryogenesis.

We have studied the expression of the genes encoding transforming growth factors (TGFs) beta 1, beta 2 and beta 3 in human embryos ranging from 32 to 57 days post-coitum, using in situ hybridization. The spatial and temporal pattern of expression of each gene is distinct, though each occasionally overlaps. TGF-beta 1 is expressed in haematopoietic, endothelial and osteogenic tissues. TGF-beta 2 and TGF-beta 3 are expressed in a wide variety of mesenchymal tissues including areas of chondrogenic activity. TGF-beta 2 is also found in several epithelial and in the ventral nervous system. The differential transcript distributions are broadly similar to those seen in mouse embryos suggesting that there is conservation of TGF-beta gene regulatory sequences and developmental function across this species boundary.

Assignment of the gene for dyskeratosis congenita to Xq28.

Dyskeratosis congenita is an X-linked recessive disorder with diagnostic dermatological features, bone marrow hypofunction, and a predisposition to neoplasia in early adult life. Linkage analysis was undertaken in an extensive family with the condition using the Xg blood group and 17 cloned X chromosomal DNA sequences which recognise restriction fragment length polymorphisms (RFLPs). No recombination was observed between the locus for dyskeratosis congenita (DKC) and the RFLPs identified by DXS52 (St14-1) (Zmax = 3.33 at theta max = 0 with 95% confidence limits of 0 to 14cM). Similarly no recombination was observed for the disease locus and F8 (Zmax = 1.23 at theta max = 0) nor for DXS15 (Zmax = 1.62 at theta max = 0), but both of these markers were only informative in part of the family whereas DXS52 was fully informative. DXS52, DXS15, and F8 are known to be tightly linked and have previously been assigned to Xq28. Thus the gene for dyskeratosis congenita can be assigned to Xq28. These DNA sequence polymorphisms will be of clinical value for carrier detection and prenatal diagnosis.

Transforming growth factor betas in mammalian embryogenesis.

Type beta transforming growth factors (TGF beta s) are members of a large superfamily of related proteins, each of which plays a pivotal role in embryonic processes. The TGF beta s per se are at least five in number, though only three isoforms have been identified in mammals. Here we will review the evidence, taken from in vitro studies on bioactivity and histochemical localization of RNAs and encoded proteins in vivo, that TGF beta 1, beta 2 and beta 3 are involved in several mammalian developmental processes, including control of growth, differentiation, tissue inductions and morphogenesis.

The role of TGF-beta s in mammalian development and neoplasia.

To date, three mammalian TGF-beta isoforms have been identified, each encoded by different genetic loci. Through each is very similar in primary amino acid structure, there are clear differences both in the mature bioactive peptide region and in the latency-associated peptide, which could potentially confer differential biological specificity. As one route to investigate differential biological function in vivo we have used gene specific probes for in situ hybridization studies to examine the distribution of RNA transcripts during mammalian embryogenesis. Mouse embryos from 6 to 14.5 gestational age and human embryos from 32 to 57 days post-fertilization have been probed. A general conclusion from these studies is that each TGF-beta gene has a distinct, through overlapping, pattern of transcript distribution and that this pattern, in most cases, is conserved between mouse and man. We have focused on the biological function the TGF-betas play in certain epithelia and in cardiogenesis, which will be discussed in this presentation.