Characterisation of PP71 homologues encoded by mammalian cytomegaloviruses

Human cytomegalovirus (HCMV) is a human pathogen that can cause severe disease in immunocompromised or immunosupressed individuals and also in newborns infected in utero. Transcription of the viral genome occurs by a process in which three classes of HCMV genes, immediate early, early and late are expressed in a regulated temporal cascade. The HCMV protein pp71, encoded by gene UL82, is located in the tegument of the HCMV virion and is delivered to cells immediately upon infection. This protein has been identified as a transactivator of viral immediate early gene expression. It also stimulates expression from a number of heterologous promoters by a mechanism that is not promoter sequence specific. Protein pp71 has multiple properties; it can increase the infectivity of transfected viral DNA, modulate the cell cycle and interact with the retinoblastoma family of proteins. Within the cell nucleus, pp71 co-localises with the cellular proteins PML and hDaxx at sub-cellular structures named nuclear domain 10 (ND10). The interaction of pp71 with hDaxx is believed to promote the degradation of hDaxx, resulting in relief of repression at the HCMV major immediate early promoter. Protein pp71 has also been reported to have the unusual property of mediating long-term expression of reporter genes cloned into a herpes simplex virus type 1 (HSV 1) vector. This study describes a comparison of pp71 with the non-human UL82 homologues from simian CMV, baboon CMV, rhesus CMV and chimpanzee CMV, named S82, B82, RH82 and Ch82, respectively. Plasmids expressing all of the UL82 homologues as enhanced yellow fluorescent protein (EYFP) or myc-tagged proteins were constructed and analysed for expression by transfection into HFFF2 cells. The EYFP-tagged UL82 homologues all directed b-gal expression in short-term assays, while only pp71 directed both short-term and long-term gene expression. Only myc tagged pp71 was observed to direct gene expression in both the short-term and long-term assays. The EYFP-tagged proteins and myc tagged pp71 and Ch82 were cloned into a mutated HSV-1 vector to produce recombinant viruses. Functional assays in human glioblastoma (U373) cells confirmed that all of the EYFP-tagged and myc tagged non-human UL82 homologues were able to direct short-term expression but only EYFP- and myc-tagged pp71 directed long-term gene expression, confirming results obtained in transfection assays. In agreement with previous reports, pp71 was shown to promote the resumption of gene expression from quiescent HSV-1 genomes. Comparison of the pp71 and Ch82 homologues indicated that pp71 is unique in its ability to do so. No reactivation was observed in cells infected with an HSV-1 recombinant that expressed EYFP-tagged Ch82. In order to establish the region of pp71 responsible for mediating long-term gene expression six plasmids encoding EYFP-tagged hybrid proteins were constructed. The C-terminus, N-terminus and mid-regions of pp71 were substituted for the equivalent Ch82 regions using homologous restriction sites in both coding sequences. All EYFP-tagged hybrids mediated short-term gene expression, while only one protein, with the mid region of pp71 inserted between the C- and N-terminal regions of the Ch82 homologue, appeared to stimulate long-term gene expression. However, levels of expression were significantly lower than that achieved by pp71. A HSV-1 recombinant expressing the hybrid protein was used to confirm results from transfection assays, suggesting that the mid-region of pp71 may be involved in mediating its long-term properties. Given the significantly lower degree of gene expression directed by the hybrid protein in short-term assays it was concluded that alterations to pp71 may result in structural changes that prevent normal function of the protein. Immunofluorescence studies revealed further differences between the non-human UL82 homologues and pp71. In confirmation of previously published findings, in the majority of HFFF2 cells infected with a HSV-1 recombinant expressing EYFP tagged pp71, this protein localised to discrete punctate ND10 foci at all times tested. In cells infected with a HSV-1 recombinant expressing S82 a pattern distinct from that of pp71 was observed. S82 exhibited a punctate/diffuse pattern of fluorescence, which became increasingly diffuse at later times post-infection. The remaining non-human UL82 homologues, despite localising to the discrete punctate foci characteristic of pp71 at early times post infection, all showed nuclear distribution patterns akin to that of S82 at later times, in cells infected with the HSV-1 recombinants expressing EYFP-tagged Ch82, B82 and Rh82. All non-human UL82 homologues, like pp71, co-localised with the endogenous cellular proteins hDaxx and PML at the times tested. Interestingly, however, at later times post-infection, the S82 protein appeared to disperse hDaxx throughout the nucleus, a feature that was not observed with the remaining UL82 homologues. Examination of the hybrid protein observed to stimulate long-term gene expression revealed that, like pp71, it localised to discrete punctate foci, and co-localised with both PML and hDaxx at all times post-infection. In contrast to other published studies, it was not possible to demonstrate pp71-mediated hDaxx degradation, by either pp71 or the non-human UL82 homologues. The work presented in this thesis confirms the previous observation that pp71 directs long-term gene expression, reactivates quiescent genomes and co localises in the nucleus with hDaxx and PML. It also characterises the non-human UL82 homologues of pp71. This study shows that, while each non human UL82 homologue shares some characteristics with pp71, subtle functional differences exist between these proteins

Epidemiological Characteristics of Poliomyelitis During the 21st Century (2000-2013)

Poliovirus is the pathogenic agent of paralytic poliomyelitis that belongs to the picornaviridae family. Poliomyelitis has an extended history dating over to the Egyptian eighteenth dynasty. It was recognized as distinct disease in the late nineteenth century when the world was ravaged by large number of outbreaks and epidemics in many countries. Paralytic Polio, the rarest but the most severe form of the disease, is characterized by acute flaccid paralysis of any or rarely both of the limbs. Increasing epidemics during the late 19th and 20th centuries lead to the initiation of a worldwide global effort for polio eradication in 1988, super headed by WHO and various other organizations. The launch of Global Polio Eradication Initiative together with the introduction of two polio vaccines resulted in 99% reduction of wild poliovirus cases worldwide while the total number of polio-endemic countries dropped from 24 countries in the year 2000 to only three countries in 2012; Afghanistan, Nigeria and Pakistan. This review will focus on the general biology of poliovirus, some historic and geographic epidemiological aspects of poliomyelitis eradication during the year 2000-2012 and also on the major failing factors associated with the efficiency of the vaccines to eradicate polio in Pakistan

Ewald methods for inverse power-law interactions in tridimensional and quasi-two dimensional systems

In this paper, we derive the Ewald method for inverse power-law interactions in quasi-two dimensional systems. The derivation is done by using two different analytical methods. The first uses the Parry's limit, that considers the Ewald methods for quasi-two dimensional systems as a limit of the Ewald methods for tridimensional systems, the second uses Poisson-Jacobi identities for lattice sums. Taking into account the equivalence of both derivations, we obtain a new analytical Fourier transform intregral involving incomplete gamma function. Energies of the generalized restrictive primitive model of electrolytes ($\eta$-RPM) and of the generalized one component plasma model ($\eta$-OCP) are given for the tridimensional, quasi-two dimensional and monolayers systems. Few numerical results, using Monte-Carlo simulations, for $\eta$-RPM and $\eta$-OCP monolayers systems are reported.Comment: to be published in Journal of Physics A: Mathematical and Theoretical (19 pages, 2 figures and 3 tables

The implications of ambitious decarbonisation of heat and road transport for Britain’s net zero carbon energy systems

Decarbonisation of heating and road transport are regarded as necessary but very challenging steps on the pathway to net zero carbon emissions. Assessing the most efficient routes to decarbonise these sectors requires an integrated view of energy and road transport systems. Here we describe how a national gas and electricity transmission network model was extended to represent multiple local energy systems and coupled with a national energy demand and road transport model. The integrated models were applied to assess a range of technologies and policies for heating and transport where the UK’s 2050 net zero carbon emissions target is met. Overall, annual primary energy use is projected to reduce by between 25% and 50% by 2050 compared to 2015, due to ambitious efficiency improvements within homes and vehicles. However, both annual and peak electricity demands in 2050 are more than double compared with 2015. Managed electric vehicle charging could save 14TWh/year in gas-fired power generation at peak times, and associated emissions, whilst vehicle-to-grid services could provide 10GW of electricity supply during peak hours. Together, managed vehicle charging, and vehicle-to-grid supplies could result in a 16% reduction in total annual energy costs. The provision of fast public charging facilities could reduce peak electricity demand by 17GW and save an estimated £650 million annually. Although using hydrogen for heating and transport spreads the hydrogen network costs between homeowners and motorists, it is still estimated to be more costly overall compared to an all-electric scenario. Bio-energy electricity generation plants with carbon capture and storage are required to drive overall energy system emissions to net zero, utilisation of which is lowest when heating is electrified, and road transport consists of a mix of electric and hydrogen fuel-cell vehicles. The analysis demonstrates the need for an integrated systems approach to energy and transport policies and for coordination between national and local governments

Initial Assessment of a Suicide Prevention Resource for Vermont Primary Care

Suicide is the second leading cause of death for Vermonters ages 10-44. Studies have shown that of those who die from suicide 45% have seen their primary care provider in the month prior to their death and only 20% saw a mental health provider. To help strengthen suicide risk screening in primary care, a group of Larner College of Medicine students partnered with Chittenden Accountable Community for Health to curate a suicide prevention informational resource to be used in Vermont\u27s primary care practices to promote universal suicide screening. We then surveyed users of the tool to assess its utility and identify barriers to suicide screening within their practices.https://scholarworks.uvm.edu/comphp_gallery/1310/thumbnail.jp

Properties of virion transactivator proteins encoded by primate cytomegaloviruses

Background: Human cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in situations where the immune system is immature or compromised. HCMV immediate early (IE) gene expression is stimulated by the virion phosphoprotein pp71, encoded by open reading frame (ORF) UL82, and this transactivation activity is important for the efficient initiation of viral replication. It is currently recognized that pp71 acts to overcome cellular intrinsic defences that otherwise block viral IE gene expression, and that interactions of pp71 with the cell proteins Daxx and ATRX are important for this function. A further property of pp71 is the ability to enable prolonged gene expression from quiescent herpes simplex virus type 1 (HSV-1) genomes. Non-human primate cytomegaloviruses encode homologs of pp71, but there is currently no published information that addresses their effects on gene expression and modes of action. Results: The UL82 homolog encoded by simian cytomegalovirus (SCMV), strain Colburn, was identified and cloned. This ORF, named S82, was cloned into an HSV-1 vector, as were those from baboon, rhesus monkey and chimpanzee cytomegaloviruses. The use of an HSV-1 vector enabled expression of the UL82 homologs in a range of cell types, and permitted investigation of their abilities to direct prolonged gene expression from quiescent genomes. The results show that all UL82 homologs activate gene expression, and that neither host cell type nor promoter target sequence has major effects on these activities. Surprisingly, the UL82 proteins specified by non-human primate cytomegaloviruses, unlike pp71, did not direct long term expression from quiescent HSV-1 genomes. In addition, significant differences were observed in the intranuclear localization of the UL82 homologs, and in their effects on Daxx. Strikingly, S82 mediated the release of Daxx from nuclear domain 10 substructures much more rapidly than pp71 or the other proteins tested. All UL82 homologs stimulated the early release of ATRX from nuclear domain 10. Conclusion: All of the UL82 homolog proteins analysed activated gene expression, but surprising differences in other aspects of their properties were revealed. The results provide new information on early events in infection with cytomegaloviruse

Spectrin-beta 2 facilitates the selective accumulation of GABAA receptors at somatodendritic synapses

Fast synaptic inhibition is dependent on targeting specific GABAAR subtypes to dendritic and axon initial segment (AIS) synapses. Synaptic GABAARs are typically assembled from α1-3, β and γ subunits. Here, we isolate distinct GABAARs from the brain and interrogate their composition using quantitative proteomics. We show that α2-containing receptors co-assemble with α1 subunits, whereas α1 receptors can form GABAARs with α1 as the sole α subunit. We demonstrate that α1 and α2 subunit-containing receptors co-purify with distinct spectrin isoforms; cytoskeletal proteins that link transmembrane proteins to the cytoskeleton. β2-spectrin was preferentially associated with α1-containing GABAARs at dendritic synapses, while β4-spectrin was associated with α2-containing GABAARs at AIS synapses. Ablating β2-spectrin expression reduced dendritic and AIS synapses containing α1 but increased the number of synapses containing α2, which altered phasic inhibition. Thus, we demonstrate a role for spectrins in the synapse-specific targeting of GABAARs, determining the efficacy of fast neuronal inhibition

Politicians must heed health effects of climate change.

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Culturally Adapted Motivational Interviewing with Cognitive Behavior Therapy and Mindfulness Based Relapse Prevention for Substance Use Disorder in Pakistan (CAMAIB): Protocol of a feasibility factorial randomised controlled trial.

Background: The use of psychoactive substances significantly impacts the health, social and economic aspects of families, communities and nations. There is a need to develop and test psychological interventions aimed for individuals with substance use disorder (SUD) in lower- and middle-income countries (LMICs), such as in Pakistan. The aim of this exploratory trial is to test the feasibility and acceptability of two culturally adapted psychological interventions in a factorial randomised controlled trial (RCT). Methods: The proposed project will be conducted in three phases. The first phase of the study will focus on cultural adaptation of the interventions through qualitative interviews with key stakeholders. The second phase will be to refine and produce manually assisted interventions. Third and last stage would be to assess the feasibility of the culturally adapted interventions through a factorial RCT. The study will be carried out in Karachi, Hyderabad, Peshawar, Lahore and Rawalpindi, Pakistan. Recruitment of participants will take place from primary care and volunteer organisations/drug rehabilitation centres. A total of 260 individuals diagnosed with SUD (n = 65) in each of the four arms will be recruited. The intervention will be delivered weekly over a period of 12 weeks in both individual and group settings. Assessments will be carried out at baseline, at 12th week (after completion of intervention) and 24th week post-randomisation. The analysis will determine the feasibility of recruitment, randomisation, retention and intervention delivery. Acceptability of intervention will be determined in terms of adherence to intervention, i.e. the mean number of sessions attended, number of home assignments completed, attrition rates, as well as through process evaluation to understand the implementation process, context, participants’ satisfaction, and impact of the study intervention. The health resource use and impact on the quality of life will be established through health economic data. Discussion: This study will provide evidence for feasibility and acceptability of culturally adapted manually assisted psychological interventions for individuals with SUD in the context of Pakistan. The study will have clinical implications if intervention is proven feasible and acceptable. Trial registration: Name of the registry: ClinicalTrials.gov, Trial registration number: NCT04885569, Date of registration: 25th April 2021