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Background: The infection of a participant with norovirus during the adaptive study of interleukin-2 dose on regulatory T cells in type 1 diabetes (DILT1D) allowed a detailed insight into the cellular and cytokine immune responses to this prevalent gastrointestinal pathogen. Methods: Serial blood, serum and peripheral blood mononuclear cell (PBMC) samples were collected pre-, and post-development of the infection. To differentiate between the immune response to norovirus and to control for the administration of a single dose of aldesleukin (recombinant interleukin-2, rIL-2) alone, samples from five non-infected participants administered similar doses were analysed in parallel. Results: Norovirus infection was self-limited and resolved within 24 hours, with the subsequent development of anti-norovirus antibodies. Serum pro- and anti-inflammatory cytokine levels, including IL-10, peaked during the symptomatic period of infection, coincident with increased frequencies of monocytes and neutrophils. At the same time, the frequency of regulatory CD4+ T cell (Treg), effector T cell (Teff) CD4+ and CD8+ subsets were dynamically reduced, rebounding to baseline levels or above at the next sampling point 24 hours later. NK cells and NKT cells transiently increased CD69 expression and classical monocytes expressed increased levels of CD40, HLA-DR and SIGLEC-1, biomarkers of an interferon response. We also observed activation and mobilisation of Teffs, where increased frequencies of CD69+ and Ki-67+ effector memory Teffs were followed by the emergence of memory CD8+ Teff expressing the mucosal tissue homing markers CD103 and β7 integrin. Treg responses were coincident with the innate cell, Teff and cytokine response. Key Treg molecules FOXP3, CTLA-4, and CD25 were upregulated following infection, alongside an increase in frequency of Tregs with the capacity to home to tissues. Conclusions: The results illustrate the innate, adaptive and counter-regulatory immune responses to norovirus infection. Low-dose IL-2 administration induces many of the Treg responses observed during infection.

Microbes, including viruses, influence type 1 diabetes (T1D) development, but many such influences remain undefined. Previous work on underlying immune mechanisms has focussed on cytokines and T cells. Here, we compared two nonobese diabetic (NOD) mouse colonies, NODlow and NODhigh, differing markedly in their cumulative T1D incidence (22% vs. 90% by 30 weeks in females). NODhigh mice harbored more complex intestinal microbiota, including several pathobionts; both colonies harbored segmented filamentous bacteria (SFB), thought to suppress T1D. Young NODhigh females had increased B-cell activation in their mesenteric lymph nodes. These phenotypes were transmissible. Co-housing of NODlow with NODhigh mice after weaning did not change T1D development, but T1D incidence was increased in female offspring of co-housed NODlow mice, which were exposed to the NODhigh environment both before and after weaning. These offspring also acquired microbiota and B-cell activation approaching those of NODhigh mice. In NODlow females, the low rate of T1D was unaffected by cyclophosphamide but increased by PD-L1 blockade. Thus, environmental exposures that are innocuous later in life may promote T1D progression if acquired early during immune development, possibly by altering B-cell activation and/or PD-L1 function. Moreover, T1D suppression in NOD mice by SFB may depend on the presence of other microbial influences. The complexity of microbial immune regulation revealed in this murine model may also be relevant to the environmental regulation of human T1D.

BACKGROUND:DNA methylation is a stable epigenetic mark that is frequently altered in tumors. DNA methylation features are attractive biomarkers for disease states given the stability of DNA methylation in living cells and in biologic specimens typically available for analysis. Widespread accumulation of methylation in regulatory elements in some cancers (specifically the CpG island methylator phenotype, CIMP) can play an important role in tumorigenesis. High resolution assessment of CIMP for the entire genome, however, remains cost prohibitive and requires quantities of DNA not available for many tissue samples of interest. Genome-wide scans of methylation have been undertaken for large numbers of tumors, and higher resolution analyses for a limited number of cancer specimens. Methods for analyzing such large datasets and integrating findings from different studies continue to evolve. An approach for comparison of findings from a genome-wide assessment of the methylated component of tumor DNA and more widely applied methylation scans was developed. METHODS:Methylomes for 76 primary endometrial cancer and 12 normal endometrial samples were generated using methylated fragment capture and second generation sequencing, MethylCap-seq. Publically available Infinium HumanMethylation 450 data from The Cancer Genome Atlas (TCGA) were compared to MethylCap-seq data. RESULTS:Analysis of methylation in promoter CpG islands (CGIs) identified a subset of tumors with a methylator phenotype. We used a two-stage approach to develop a 13-region methylation signature associated with a "hypermethylator state." High level methylation for the 13-region methylation signatures was associated with mismatch repair deficiency, high mutation rate, and low somatic copy number alteration in the TCGA test set. In addition, the signature devised showed good agreement with previously described methylation clusters devised by TCGA. CONCLUSION:We identified a methylation signature for a "hypermethylator phenotype" in endometrial cancer and developed methods that may prove useful for identifying extreme methylation phenotypes in other cancers.

In a prospective longitudinal study the authors examined the associations between parent locus of control of reinforcement (LOCR), measured before the birth of a child, and behavioral-emotional outcomes in that child at age 7 years. A total of 307 couples completed questionnaires regarding their emotional status and LOCR at their first prenatal care appointment. When their children turned 7 years old, teachers completed questionnaires regarding each participating child's behavior. Findings indicate significant associations between fathers' prenatal LOCR and child outcomes, particularly hyperactivity in sons. Hyperactivity and behavioral-emotional problems in girls, in contrast, were better predicted by maternal prenatal emotional distress. Results provide evidence that paternal and maternal characteristics that predate the birth of a child relate to later behavioral outcomes in that child. Implications for prevention of child psychopathology are discussed. (Contains 3 tables and 1 note.)

In this article, we describe an observational approach, "looking and listening-in," that we have used to try to understand the experience of an infant in an Australian family day-care home. The article is drawn from a larger study of infants' experiences of early childhood education and care settings. In keeping with the mosaic methodology of the larger study, "looking and listening-in" encompasses constructs drawn from diverse (phenomenological, socio-cultural, social cognition) theoretical perspectives. In the context in which we are using it, looking and listening-in has dual utility: as a methodological approach for helping us to edge closer to understanding the infant's experience, and as a way of describing how the infant made meaning of his experience. The infant's looking and listening-in is illustrated and analysed through a visual narrative. (Contains 2 notes and 1 table.)

Autochthonous blockfield mantles may indicate alpine surfaces that have not been glacially eroded. These surfaces may therefore serve as markers against which to determine Quaternary erosion volumes in adjacent glacially eroded sectors. To explore these potential utilities, chemical weathering features, erosion rates, and regolith residence durations of mountain blockfields are investigated in the northern Swedish Scandes. This is done, firstly, by assessing the intensity of regolith chemical weathering along altitudinal transects descending from three blockfield-mantled summits. Clay / silt ratios, secondary mineral assemblages, and imaging of chemical etching of primary mineral grains in fine matrix are each used for this purpose. Secondly, erosion rates and regolith residence durations of two of the summits are inferred from concentrations of in situ-produced cosmogenic 10Be and 26Al in quartz at the blockfield surfaces. An interpretative model is adopted that includes temporal variations in nuclide production rates through surface burial by glacial ice and glacial isostasy-induced elevation changes of the blockfield surfaces. Together, our data indicate that these blockfields are not derived from remnants of intensely weathered Neogene weathering profiles, as is commonly considered. Evidence for this interpretation includes minor chemical weathering in each of the three examined blockfields, despite consistent variability according to slope position. In addition, average erosion rates of ~16.2 and ~6.7 mm ka−1, calculated for the two blockfield-mantled summits, are low but of sufficient magnitude to remove present blockfield mantles, of up to a few metres in thickness, within a late Quaternary time frame. Hence, blockfield mantles appear to be replenished by regolith formation through, primarily physical, weathering processes that have operated during the Quaternary. The persistence of autochthonous blockfields over multiple glacial–interglacial cycles confirms their importance as key markers of surfaces that were not glacially eroded through, at least, the late Quaternary. However, presently blockfield-mantled surfaces may potentially be subjected to large spatial variations in erosion rates, and their Neogene regolith mantles may have been comprehensively eroded during the late Pliocene and early Pleistocene. Their role as markers by which to estimate glacial erosion volumes in surrounding landscape elements therefore remains uncertain.

Contemporary approaches to the digital transformation of practice in university research and teaching sometimes assume a convergence between the digital and openness. This assumption has led to the idea of ‘digital open scholarship,’ which aims to open up scholarship to participants from outside academic scholarly communities. But scholarship, digitality and openness exist in tension with each other – we can see the individual features of each, but we cannot make sense of the whole picture. It resembles an ‘impossible triangle’. Particularly confounding is the tension between digital scholarship and open knowledge, where the former is focused on the creation by specialist communities of knowledge of a stable and enduring kind, whilst the latter is characterised by encyclopaedic knowledge and participation that is unbounded by affiliation or location. However, we need not be permanently thwarted by the apparent impossibility of this triangle. It is a stimulus to look critically at the contexts of practice in which a relationship between scholarship, digitality and openness is sought. Constructive examples of such critique can be found in the emerging research field of literacy and knowledge practice in the digital university.

Emerging from the artist’s constructed photographs and walking projects in the north, this paper considers the tension between the photograph as a fixed composition of the world and the dynamic image constructed from data. Whereas arguably, the traditional photograph exhibits a stable relationship between the world and the image, the constructed photograph shifts the focus onto the underlying algorithmic processes of production. This focus on the relational nature of the constructed photograph shifts our gaze from the horizon to the underlying systems in operation as we consider the relational nature of data as a photograph.

This paper considers Art’s engagement with the socio-ecological systems in operation in the world. Drawing from both environmental and art history, this paper employs the Deleuzean concept of the fold to suggest three overlapping periods of ecological-systems awareness over the past sixty years. This paper demonstrates how we have shifted our attention from a material engagement with the Earth to a primary engagement with systems which describe and simulate the Earth. This shift in attention to secondary information and an enfoldment within systems, defined as the Post-Systems Condition, manifests in the aesthetic quality of the eerie and a profound sense of saudade or longing for a lost nature.

The lattice parameters and misfit of the γ and γ′ phases in a series of model quaternary Ni-based superalloys with and without Mo additions have been determined using neutron diffraction between room temperature and 700 °C. Despite the fact that Mo is typically expected to partition almost exclusively to the γ phase and thereby increase the lattice parameter of that phase alone, the lattice parameters of both the γ and γ′ phases were observed to increase with Mo addition. Nevertheless, the effect on the γ lattice parameter was more pronounced, leading to an overall decrease in the lattice misfit with increasing Mo content. Alloys with the lowest Mo content were found to be positively misfitting, whilst additions of 5 at.% Mo produced a negative lattice misfit. A general decrease in the lattice misfit with increasing temperature was also observed.

The terahertz time-domain spectroscopy (THz-TDS) technique has been used to obtain transmission THz-radiation spectra of polymer nanocomposites containing a controlled amount of exfoliated graphene. Graphene nanocomposites (1 wt%) that were used in this work were based on poly(ethylene terephthalate-ethylene dilinoleate) (PET-DLA) matrix and were prepared via a kilo-scale (suitable for research and development, and prototyping) in-situ polymerization. This was followed by compression molding into 0.3-mm-thick and 0.9-mm-thick foils. Transmission electron microscopy (TEM) and Raman studies were used to confirm that the graphene nanoflakes dispersed in a polymer matrix consisted of a few-layer graphene. The THz-radiation transients were generated and detected using a low-temperature⁻grown GaAs photoconductive emitter and detector, both excited by 100-fs-wide, 800-nm-wavelength optical pulses, generated at a 76-MHz repetition rate by a Ti:Sapphire laser. Time-domain signals transmitted through the nitrogen, neat polymer reference, and 1-wt% graphene-polymer nanocomposite samples were recorded and subsequently converted into the spectral domain by means of a fast Fourier transformation. The spectral range of our spectrometer was up to 4 THz, and measurements were taken at room temperature in a dry nitrogen environment. We collected a family of spectra and, based on Fresnel equations, performed a numerical analysis, that allowed us to extract the THz-frequency-range refractive index and absorption coefficient and their dependences on the sample composition and graphene content. Using the Clausius-Mossotti relation, we also managed to estimate the graphene effective dielectric constant to be equal to ~7 ± 2. Finally, we extracted from our experimental data complex conductivity spectra of graphene nanocomposites and successfully fitted them to the Drude-Smith model, demonstrating that our graphene nanoflakes were isolated in their polymer matrix and exhibited highly localized electron backscattering with a femtosecond relaxation time. Our results shed new light on how the incorporation of exfoliated graphene nanoflakes modifies polymer electrical properties in the THz-frequency range. Importantly, they demonstrate that the complex conductivity analysis is a very efficient, macroscopic and non-destructive (contrary to TEM) tool for the characterization of the dispersion of a graphene nanofiller within a copolyester matrix.

The authors aimed to examine the possible association between (a) accurately reading emotion in facial expressions and (b) social and academic competence among elementary school-aged children. Participants were 840 7-year-old children who completed a test of the ability to read emotion in facial expressions. Teachers rated children's social and academic behavior using behavioral rating scales. The authors found that children who had more difficulty identifying emotion in faces also were more likely to have more problems overall and, more specifically, with peer relationships among boys and with learning difficulties among girls. Findings suggest that nonverbal receptive skill plays a significant role in children's social and academic adjustment. (Contains 2 tables.)

This article explores the phenomenon of presence and argues that it is a concept worthy of further investigation as a characteristic of adult-child interaction and a key component of quality care. Presence is situated within caring relationships and has a strong affective component that encompasses the heart and soul of professional practice. While cognitive (knowing) and social (engaging with) aspects of early childhood professional practice are important, presence has the potential to significantly impact on children's early emotional wellbeing. A suggested approach to furthering an appreciation of caring presence is to use digital video analysis software as a research tool in practitioner inquiry.

This article extends current understandings of sources of job (dis)satisfaction for childcare staff by investigating the hypothesis that early childhood professionals' satisfaction with regulatory requirements is a predictor of job satisfaction. Findings show that for early childhood professionals in New South Wales, Australia, satisfaction with the regulatory environment is related to the extent these requirements are perceived to support autonomous professional practices conducive to quality care. Considerable dissatisfaction with regulatory requirements was found among study participants. Nonetheless, this level of dissatisfaction did not predict job (dis)satisfaction. The lack of correlation may be indicative of the study's sample, however, and does not preclude a greater connection between the two constructs. Implications of a possible nexus between job satisfaction and satisfaction with regulatory requirements are discussed, and further research is called for.

This article problematises the construction of regulation as an effective manager of risks to children in early childhood education and care (ECEC) services. Adopting a Foucaldian, governmentalist approach to regulation and risk, the authors suggest that governments in Australia have "risk colonised" regulation to meet their own interests rather than make effective use of regulation as a mechanism for quality assurance. They propose that the risk colonising of regulation has not effectively addressed societal risks to children in ECEC services, and has generated its own risks to quality standards through a preoccupation with institutional risk. In these ways, "the laugh of Foucault" resounds in the regulation of ECEC services.

The regulatory environment in which long daycare centres are required to operate plays a key role in determining what early childhood professionals do and how they go about doing it. This paper reports findings from a state-wide survey undertaken in New South Wales, Australia, which shows how early childhood professionals position themselves on four key aspects of the regulatory environment: the premises underlying regulatory requirements; its perceived support of them as professionals to provide quality care; how state regulations and the national accreditation system impact upon their daily practice; and the way these audit systems affect their professional esteem. Implications for job satisfaction are discussed. The paper concludes by asserting that regulatory requirements need to be evaluated in terms of their contribution to structural, process, contextual and work environment dimensions of quality. (Contains 1 table.)

Current cross-campus computing initiatives demand both access and skill in employing technology as a tool for academic success. Consequently, lack of computer skills can affect first-year students' potential for success because many courses assume students are computer literate. In this study, 888 first-year students completed a survey--administered for three years--that examined technology variables. A chi-square statistic and associated p-value indicated that students' access to computers increased over a three-year period, albeit it was income-related. Results showed an improvement in word processing, Internet and library-searching skills, and developing computer-enhanced presentations. However, results from the last year of the study showed that more than 50% of students rated themselves unskilled at library-searching and developing computer-enhanced presentations, with approximately one-third of students rating themselves unskilled at e-mail. Implications for first-year student computer-training support are discussed. (Contains 3 tables and 1 footnote.)

While early childhood professionals in NSW are accountable to a substantial collection of regulatory requirements, little research has explored the outcomes of this regulatory environment, both intended and otherwise. This paper presents findings from a NSW study and shows how early childhood professionals working in long day care centres perceive the regulatory environment to impact on their professional practice. Specific attention is given to the impact of the national Quality Improvement and Accreditation System and the NSW Children's Services Regulation. The notion of the regulatory environment as a "double-edged sword" emerges as a useful conceptual metaphor to understand how early childhood professionals perceive these regulatory frameworks to impact on their practice. Findings show that the benefits afforded by accreditation and the Regulation are offset by unfulfilled intentions and unintended outcomes.

The aim of this study was the preliminary development of a short 10-item version of the CiOQ. First, 10 items were selected on the basis of their high factor loadings in previously published work. Two studies are then reported. In Study 1, the 10 items were subjected to confirmatory factor analysis using data from a sample of college students (n=336) who had experienced adverse events. Study 2 involved a clinical sample (n=40) of people with posttraumatic stress disorder and examined associations of reported changes with posttraumatic stress symptoms and posttraumatic growth. Evidence is provided for a two-factor structure of the CiOQ Short Form, consisting of positive changes and negatives changes, and for the subscales' internal consistency reliability, convergent validity, and differential associations with clinically meaningful outcomes.

Practitioner research is defined as systematic inquiry-based efforts directed towards creating and extending professional knowledge and associated understandings of professional practice. A review of "primary research" articles published in the "Australian Journal of Early Childhood" revealed that only a small number involved early childhood practitioners as researchers. However, changing social and theoretical constructs and a sense of low regard by the community for the profession require early childhood practitioners to be much more articulate about their practices. Collaborative inquiry processes provide opportunities for practitioners to deconstruct some of the taken-for-granted practices found within many early childhood services. One of the benefits of such inquiry is an increased sense of empowerment gained by practitioners through greater insights into their own professional knowledge, their increased capacity to work more effectively with children, and their increased ability to communicate more professionally and effectively with parents and other professionals. (Contains 3 tables.)

This article explores some specific issues involved in online learning and assessment. It draws on data from a postgraduate course for professional educators, delivered globally online, and highlights the relationship between students' online discussion and their written assessed work, arguing that we need to focus on both of these in terms of the writing demands they make on students. In so doing it utilizes a theoretical framework which conceptualizes writing as contextualized social practice. The paper illustrates the complexity of the rhetorical demands being made on students in these new environments of teaching and learning and, in focusing on writing, complements present approaches to online learning which have, to date, tended towards collaborative and constructivist perspectives. The article highlights the relationship between pedagogy, technology and assessment. It concludes with a discussion of the design of an online writing resource to support student writers on this particular masters programme.

A recent study of grandparents who were caring for their young grandchildren on a regular basis has prompted an examination of what appears to be a paradox surrounding the provision of childcare services within Australia. The paradox reflects concerns surrounding the balance between a commitment to high-quality childcare services for children and families and the means by which concerns about what may be considered to be a property view of the child that places childcare within the context of a competitive marketplace. This article explores these issues using terminology drawn from the marketplace to traverse what appears to be an abyss between a business orientation and more humanistic approaches to early childhood education. Following the this may be achieved. The choice that grandparents made to care for their grandchildren in caring and loving environments reflects a humanistic perspective. However, some grandparents raised introduction of the paradox and an examination of market characteristics, the article concludes with questions about who the childcare consumer is, a consideration of approaches to consumer protection and reflections on childcare provision.

The notion of "culture" as an essential attribute of individuals and groups, owed to national or ethnic background, is critiqued in this article as unhelpful to the project of understanding how diverse participants in virtual learning environments (VLEs) individually and jointly construct a culture of interaction. An alternative conceptualisation of culture in VLEs is proposed, which views online discussion as just one of the sites in which the culture of a VLE is negotiated. Other sites are to be found in institutional practices of teaching and learning at a distance, and in the wider cultural narratives of the Internet. Examples from two online masters courses in online and distance education are used to contextualise this concept of culture, exploring the differences in patterns of participation that are produced by contrasting institutional cultures, even though such participation is explicitly valorised as the means and the subject of the learning that goes on in both these courses. Some implications for the understanding and management of student diversity in these environments are considered, in particular the need for emerging cultural narratives around VLEs to reflect all aspects of student engagement in distance education, not just those which relate to online interaction. (Contains 2 figures.)

This paper adopts an academic literacies perspective to argue for a critical approach to the writing practices of the online university classroom. It describes an on-going action research project in an online Masters in Online and Distance Education (MAODE) programme at the UK Open University, which aims to create an online writing resource to support distance learners in developing a critical awareness of the writing practices on the programme. The paper presents the results of an evaluation study of this resource during the 2005 presentation of the MAODE, and discusses the evidence from this study that such a resource can provide a space for students to critique the dominant literacies of the online university.

Students with disabilities continue to experience barriers to accessing tertiary (i.e., postsecondary) education in Australia. Using the principles of Universal Design for Learning (UDL) may help educators proactively address barriers through the design of more accessible and inclusive educational experiences. However, at present, references to UDL appear in only a small number of Australian educational policies and tertiary institute websites, and few tertiary educators use UDL in practice. In this article, we describe how a team of educators, learning designers, accessibility advocates, and people with disabilities from multiple institutions across Australia collaborated to co-create a free, accessible eLearning program to build workforce knowledge and skill in UDL. We first describe how the advisory group was established, how the Knowledge to Action cycle was used to guide the activities of the advisory group, and the evaluation framework that was used to assess the outcomes of the eLearning program. We share potential future activities to raise awareness of UDL and influence policy and practice in local contexts and propose directions for future work in this area. [ABSTRACT FROM AUTHOR]

This article describes processes involved in a curriculum mapping exercise that constituted the first phase of a project aimed at furthering the integration of generic skills in a Bachelor of Education (Early Childhood) program. The purpose of the mapping exercise was to identify the generic skills currently fostered in the program, and those that appear to be overlooked. The article draws attention to the complexity of issues associated with curriculum mapping and highlights the need to refine the somewhat simplistic curriculum mapping techniques advocated in much of the existing literature. The centrality of collegial dialogue to curriculum mapping if it is to lead to curriculum change is also emphasized.

Many early childhood practitioners use pedagogical documentation as an important process that enables children's thinking to be represented in a form that can be readily shared with others (Dahlberg et al., 1999). Documentation in the form of a professional portfolio provides a vehicle for reflection and an appreciation of the complexities and ambiguities of professional practice in the human professions. The nursing and teaching professions have also embraced professional portfolios as a means through which accountability to self and to others can be judged. However, it is the fusion of process and product within professional portfolio development that enables those involved in the human professions to examine, reflect on, understand, explain and further develop their professional practice. It is through this interrogation of practice that we can not only gain insight into our capabilities but also the theories, beliefs and values that underpin the wisdom of our professional practices. The process of professional portfolio development can be enriching and empowering. An outline for organizing a professional portfolio is provided.

This paper addresses the nature of literacy practices in online distance learning environments, specifically those involved in text-based collaborative discussion at post-compulsory level. A three-dimensional framework, relating operational, cultural and critical aspects of linguistic interaction online, is used to argue that research in this field needs to take account of wider institutional and social contexts if it is to address issues of student resistance to socialisation into virtual learning communities. Some methodologies for research of this kind are discussed, and sample analyses of participant interaction on UK Open University Masters courses are presented.

ABSTRACT Intrahost genetic diversity and replication error rates are intricately linked to RNA virus pathogenesis, with alterations in viral polymerase fidelity typically leading to attenuation during infections in vivo. We have previously shown that norovirus intrahost genetic diversity also influences viral pathogenesis using the murine norovirus model, as increasing viral mutation frequency using a mutagenic nucleoside resulted in clearance of a persistent infection in mice. Given the role of replication fidelity and genetic diversity in pathogenesis, we have now investigated whether polymerase fidelity can also impact virus transmission between susceptible hosts. We have identified a high-fidelity norovirus RNA-dependent RNA polymerase mutant (I391L) which displays delayed replication kinetics in vivo but not in cell culture. The I391L polymerase mutant also exhibited lower transmission rates between susceptible hosts than the wild-type virus and, most notably, another replication defective mutant that has wild-type levels of polymerase fidelity. These results provide the first experimental evidence that norovirus polymerase fidelity contributes to virus transmission between hosts and that maintaining diversity is important for the establishment of infection. This work supports the hypothesis that the reduced polymerase fidelity of the pandemic GII.4 human norovirus isolates may contribute to their global dominance. IMPORTANCE Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This constitutes the first experimental demonstration that the polymerase fidelity of viruses can impact transmission of infection in their natural hosts. These results provide further insight into potential reasons for the global emergence of pandemic human noroviruses that display alterations in the replication fidelity of their polymerases compared to nonpandemic strains.

BackgroundInterleukin-2 (IL-2) has an essential role in the expansion and function of CD4+ regulatory T cells (Tregs). Tregs reduce tissue damage by limiting the immune response following infection and regulate autoreactive CD4+ effector T cells (Teffs) to prevent autoimmune diseases, such as type 1 diabetes (T1D). Genetic susceptibility to T1D causes alterations in the IL-2 pathway, a finding that supports Tregs as a cellular therapeutic target. Aldesleukin (Proleukin; recombinant human IL-2), which is administered at high doses to activate the immune system in cancer immunotherapy, is now being repositioned to treat inflammatory and autoimmune disorders at lower doses by targeting Tregs.Methods and findingsTo define the aldesleukin dose response for Tregs and to find doses that increase Tregs physiologically for treatment of T1D, a statistical and systematic approach was taken by analysing the pharmacokinetics and pharmacodynamics of single doses of subcutaneous aldesleukin in the Adaptive Study of IL-2 Dose on Regulatory T Cells in Type 1 Diabetes (DILT1D), a single centre, non-randomised, open label, adaptive dose-finding trial with 40 adult participants with recently diagnosed T1D. The primary endpoint was the maximum percentage increase in Tregs (defined as CD3+CD4+CD25highCD127low) from the baseline frequency in each participant measured over the 7 d following treatment. There was an initial learning phase with five pairs of participants, each pair receiving one of five pre-assigned single doses from 0.04 × 106 to 1.5 × 106 IU/m2, in order to model the dose-response curve. Results from each participant were then incorporated into interim statistical modelling to target the two doses most likely to induce 10% and 20% increases in Treg frequencies. Primary analysis of the evaluable population (n = 39) found that the optimal doses of aldesleukin to induce 10% and 20% increases in Tregs were 0.101 × 106 IU/m2 (standard error [SE] = 0.078, 95% CI = -0.052, 0.254) and 0.497 × 106 IU/m2 (SE = 0.092, 95% CI = 0.316, 0.678), respectively. On analysis of secondary outcomes, using a highly sensitive IL-2 assay, the observed plasma concentrations of the drug at 90 min exceeded the hypothetical Treg-specific therapeutic window determined in vitro (0.015-0.24 IU/ml), even at the lowest doses (0.040 × 106 and 0.045 × 106 IU/m2) administered. A rapid decrease in Treg frequency in the circulation was observed at 90 min and at day 1, which was dose dependent (mean decrease 11.6%, SE = 2.3%, range 10.0%-48.2%, n = 37), rebounding at day 2 and increasing to frequencies above baseline over 7 d. Teffs, natural killer cells, and eosinophils also responded, with their frequencies rapidly and dose-dependently decreased in the blood, then returning to, or exceeding, pretreatment levels. Furthermore, there was a dose-dependent down modulation of one of the two signalling subunits of the IL-2 receptor, the β chain (CD122) (mean decrease = 58.0%, SE = 2.8%, range 9.8%-85.5%, n = 33), on Tregs and a reduction in their sensitivity to aldesleukin at 90 min and day 1 and 2 post-treatment. Due to blood volume requirements as well as ethical and practical considerations, the study was limited to adults and to analysis of peripheral blood only.ConclusionsThe DILT1D trial results, most notably the early altered trafficking and desensitisation of Tregs induced by a single ultra-low dose of aldesleukin that resolves within 2-3 d, inform the design of the next trial to determine a repeat dosing regimen aimed at establishing a steady-state Treg frequency increase of 20%-50%, with the eventual goal of preventing T1D.Trial registrationISRCTN Registry ISRCTN27852285; ClinicalTrials.gov NCT01827735.

Recent studies suggest that cells make stochastic choices with respect to differentiation or division. However, the molecular mechanism underlying such stochasticity is unknown. We previously proposed that the timing of vertebrate neuronal differentiation is regulated by molecular oscillations of a transcriptional repressor, HES1, tuned by a post-transcriptional repressor, miR-9. Here, we computationally model the effects of intrinsic noise on the Hes1/miR-9 oscillator as a consequence of low molecular numbers of interacting species, determined experimentally. We report that increased stochasticity spreads the timing of differentiation in a population, such that initially equivalent cells differentiate over a period of time. Surprisingly, inherent stochasticity also increases the robustness of the progenitor state and lessens the impact of unequal, random distribution of molecules at cell division on the temporal spread of differentiation at the population level. This advantageous use of biological noise contrasts with the view that noise needs to be counteracted.

Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5′ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP) moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.