The functional assembly of the mammalian minichromosome maintenance complex

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and is essential for DNA replication. Mis-regulation of its assembly and function can lead to genomic instability, replication stress and genome re-replication, which are powerful drivers toward the acquisition of mutations and the formation of cancer. I have produced and purified recombinant human MCM2-7 (hMCM) in Escherichia coli (E. coli). Recombinant hMCM has ATP hydrolysis and helicase activity, when assayed in the absence of post-translational modifications or accessory proteins. Using electron microscopy asymmetric single particle reconstruction, I have produced 23 Å resolution structures. The structures reveal that recombinant hMCM forms an asymmetric complex that changes conformation in the presence of a forked DNA substrate. By applying serial extractions to mammalian cells synchronised by release from quiescence, I have revealed dynamic changes in the sub-nuclear compartmentalisation of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear matrix. This suggests that functional MCM2-7 loading takes place at the nuclear matrix. Using this information I have developed a system to study the regulated assembly of recombinant hMCM, to support molecular dissection of the loading process. Using cellular extracts, I show regulated binding of recombinant hMCM, which translates into initiation of DNA replication and is stimulated by cyclin E/CDK2. Furthermore, I have demonstrated that both cyclin A/CDK2 and Dbf4/Drf1-dependant kinase (DDK) are required for inducing a conformational change in hMCM that is associated with S phase of the cell cycle. Understanding of the assembly of hMCM will serve as a foundation for analysis of corruption of the process, its effect on genome instability and gene expression, and how these events lead to the ‘birth’ of cancer cells. Very early events in the development of cancer cells are most likely to yield new and effective opportunities for detection and treatment of a broad range of cancers

Variation in life history and flight morphological traits in Speckled Wood (Pararge aegeria) butterflies infected with a baculovirus.

Sub-lethal impacts are known to affect the insect-host relationship and have an important role in describing host dynamics. The impact of sub-lethal infections of pathogens on life history traits of affected hosts has been understudied in natural or semi-natural systems. The Speckled Wood (Pararge aegeria) is a satyrine butterfly that is common in temperate zones and has been extensively used as a model system for evolutionary ecology studies. It is known that the deployment of the immune system within this species, as with other invertebrates, is energetically costly and may result in trade-offs with fitness-related traits. In this study, we investigated the sub-lethal effect of exposure to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) on life history and flight morphological traits of P. aegeria. Larvae were inoculated with increasing doses of AcMNPV and measurements made of life history and flight morphology traits. Generally, larvae exposed to virus took longer to develop to pupae and larval mass acquisition per day was significantly reduced in viral exposed larvae. However, viral exposed larvae were able to attain the same pupal mass and their duration as pupae was the same as controls. Forewing length, forewing aspect ratio, dry thorax mass and forewing loading were related to sex and bioassay differences but there was no evidence of any viral impact on these measures. Adult male butterflies had significantly less basal wing melanisation when exposed to virus compared to control males but there was no difference between females. Implications for population dynamics of P. aegeria are discussed

New pleiotropic effects of eliminating a rare tRNA from Streptomyces coelicolor, revealed by combined proteomic and transcriptomic analysis of liquid cultures

Background: In Streptomyces coelicolor, bldA encodes the only tRNA for a rare leucine codon, UUA. This tRNA is unnecessary for growth, but is required for some aspects of secondary metabolism and morphological development. We describe a transcriptomic and proteomic analysis of the effects of deleting bldA on cellular processes during submerged culture: conditions relevant to the industrial production of antibiotics. Results: At the end of rapid growth, a co-ordinated transient up-regulation of about 100 genes, including many for ribosomal proteins, was seen in the parent strain but not the ΔbldA mutant. Increased basal levels of the signal molecule ppGpp in the mutant strain may be responsible for this difference. Transcripts or proteins from a further 147 genes classified as bldA-influenced were mostly expressed late in culture in the wild-type, though others were significantly transcribed during exponential growth. Some were involved in the biosynthesis of seven secondary metabolites; and some have probable roles in reorganising metabolism after rapid growth. Many of the 147 genes were "function unknown", and may represent unknown aspects of Streptomyces biology. Only two of the 147 genes contain a TTA codon, but some effects of bldA could be traced to TTA codons in regulatory genes or polycistronic operons. Several proteins were affected posttranslationally by the bldA deletion. There was a statistically significant but weak positive global correlation between transcript and corresponding protein levels. Different technical limitations of the two approaches were a major cause of discrepancies in the results obtained with them. Conclusion: Although deletion of bldA has very conspicuous effects on the gross phenotype, the bldA molecular phenotype revealed by the "dualomic" approach has shown that only about 2% of the genome is affected; but this includes many previously unknown effects at a variety of different levels, including post translational changes in proteins and global cellular physiology

Combining high-resolution cryo-electron microscopy and mutagenesis to develop cowpea mosaic virus for bionanotechnology

Particles of cowpea mosaic virus (CPMV) have enjoyed considerable success as nanoparticles. The development of a system for producing empty virus-like particles (eVLPs) of the virus, which are non-infectious and have the potential to be loaded with heterologous material, has increased the number of possible applications for CPMV-based particles. However, for this potential to be realised, it was essential to demonstrate that eVLPs were accurate surrogates for natural virus particles, and this information was provided by high-resolution cryo-EM studies of eVLPs. This demonstration has enabled the approaches developed for the production of modified particles developed with natural CPMV particles to be applied to eVLPs. Furthermore, a combination of cryo-EM and mutagenic studies allowed the development of particles which are permeable but which could still assemble efficiently. These particles were shown to be loadable with cobalt, indicating that they can, indeed, be used as nano-containers

HIV/AIDS awareness and risk behaviour among pregnant women in Semey, Kazakhstan, 2007

<p>Abstract</p> <p>Background</p> <p>Central Asia has one of the most rapidly increasing HIV prevalence in the world. The aim of this study was to evaluate current knowledge, risk behaviour and attitudes to voluntary counselling and testing concerning HIV/AIDS among pregnant women in Semey, Kazakhstan.</p> <p>Methods</p> <p>We collected 226 questionnaires in a consecutive sample from a population on 520 pregnant women. The results were related to ethnicity, age and education level.</p> <p>Results</p> <p>Ninety-six percent had heard about HIV.</p> <p>Positive findings were that 89% and 86% of the women were aware of the two main routes of transmission: sexual intercourses without a condom and sharing needles while injecting drugs. The women had first heard about HIV/AIDS through the media with, 52%, and at school with 40%. Only 46% and 68% of the women pointed out breastfeeding and mother-to-child transmission during pregnancy or delivery as routes of transmission. Eighty-three percent were prepared not to breastfeed their baby if they were found to be HIV positive. Slightly more, 86%, accepted the need to take medicine, but fewer women, 68%, were positive to Caesarean section. Negative findings were that only 28% answered that there are ways to protect oneself against sexually transmitted HIV/AIDS and specified that this was condom use.</p> <p>Conclusion</p> <p>The pregnant women in Semey have poor knowledge about specific mother-to-child HIV transmission and do not know about the means of reducing mother-to-child HIV infection. The information in the public health program needs to be improved. However, most of the women in Semey were positive to prevention strategies for mother-to-child transmission after hearing about it.</p

Crystal structure and proteomics analysis of empty virus-like particles of Cowpea mosaic virus

Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C-terminus of the small (S) subunit. The intact C-terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C-termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed

An umbrella review of the benefits and risks associated with youths’ interactions with electronic screens

The influence of electronic screens on the health of children and adolescents and their education is not well understood. In this prospectively registered umbrella review (PROSPERO identifier CRD42017076051), we harmonized effects from 102 meta-analyses (2,451 primary studies; 1,937,501 participants) of screen time and outcomes. In total, 43 effects from 32 meta-analyses met our criteria for statistical certainty. Meta-analyses of associations between screen use and outcomes showed small-to-moderate effects (range: r = –0.14 to 0.33). In education, results were mixed; for example, screen use was negatively associated with literacy (r = –0.14, 95% confidence interval (CI) = –0.20 to –0.09, P ≤ 0.001, k = 38, N = 18,318), but this effect was positive when parents watched with their children (r = 0.15, 95% CI = 0.02 to 0.28, P = 0.028, k = 12, N = 6,083). In health, we found evidence for several small negative associations; for example, social media was associated with depression (r = 0.12, 95% CI = 0.05 to 0.19, P ≤ 0.001, k = 12, N = 93,740). Limitations of our review include the limited number of studies for each outcome, medium-to-high risk of bias in 95 out of 102 included meta-analyses and high heterogeneity (17 out of 22 in education and 20 out of 21 in health with I2 > 50%). We recommend that caregivers and policymakers carefully weigh the evidence for potential harms and benefits of specific types of screen use

Genome-wide transcriptomic analysis of the response to nitrogen limitation in Streptomyces coelicolor A3(2)

<p>Abstract</p> <p>Background</p> <p>The present study represents a genome-wide transcriptomic analysis of the response of the model streptomycete <it>Streptomyces coelicolor </it>A3(2) M145 to fermentor culture in Modified Evans Media limited, respectively, for nitrogen, phosphate and carbon undertaken as part of the ActinoGEN consortium to provide a publicly available reference microarray dataset.</p> <p>Findings</p> <p>A microarray dataset using samples from two replicate cultures for each nutrient limitation was generated. In this report our analysis has focused on the genes which are significantly differentially expressed, as determined by Rank Products Analysis, between samples from matched time points correlated by growth phase for the three pairs of differently limited culture datasets. With a few exceptions, genes are only significantly differentially expressed between the N6/N7 time points and their corresponding time points in the C and P-limited cultures, with the vast majority of the differentially expressed genes being more highly expressed in the N-limited cultures. Our analysis of these genes indicated expression of several members of the GlnR regulon are induced upon nitrogen limitation, as assayed for by [NH₄⁺] measurements, and we are able to identify several additional genes not present in the GlnR regulon whose expression is induced in response to nitrogen limitation. We also note SCO3327 which encodes a small protein (32 amino acid residues) unusually rich in the basic amino acids lysine (31.25%) and arginine (25%) is significantly differentially expressed in the nitrogen limited cultures. Additionally, we investigate the expression of known members of the GlnR regulon and the relationship between gene organization and expression for the SCO2486-SCO2487 and SCO5583-SCO5585 operons.</p> <p>Conclusions</p> <p>We provide a list of genes whose expression is differentially expressed in low nitrogen culture conditions, including a putative nitrogen storage protein encoded by SCO3327. Our list includes several genes whose expression patterns are similar to up-regulated members of the GlnR regulon and are induced in response to nitrogen limitation. These genes represent likely targets for future studies into the nitrogen starvation response in <it>Streptomyces coelicolor</it>.</p