The African child

No Abstrac

On-demand delivery of single DNA molecules using nanopipettes

Understanding the behavioral properties of single molecules or larger scale populations interacting with single molecules is currently a hotly pursued topic in nanotechnology. This arises from the potential such techniques have in relation to applications such as targeted drug delivery, early stage detection of disease, and drug screening. Although label and label-free single molecule detection strategies have existed for a number of years, currently lacking are efficient methods for the controllable delivery of single molecules in aqueous environments. In this article we show both experimentally and from simulations that nanopipets in conjunction with asymmetric voltage pulses can be used for label-free detection and delivery of single molecules through the tip of a nanopipet with “on-demand” timing resolution. This was demonstrated by controllable delivery of 5 kbp and 10 kbp DNA molecules from solutions with concentrations as low as 3 pM

The immune system of the liver: 50 years of strangeness

Editoria

CMV and the Art of Memory Maintenance

The CD8+ T cell responses to CMV gradually increase in magnitude over time—so-called memory “inflation.” In this issue of Immunity, Snyder et al. (2008) examine the dynamics of memory inflation and demonstrate continuous turnover of inflating T cells, drawing on both memory cells and naive cells to replace them

On-Demand Delivery of Single DNA Molecules Using Nanopipets

Understanding the behavioral properties of single molecules or larger scale populations interacting with single molecules is currently a hotly pursued topic in nanotechnology. This arises from the potential such techniques have in relation to applications such as targeted drug delivery, early stage detection of disease, and drug screening. Although label and label-free single molecule detection strategies have existed for a number of years, currently lacking are efficient methods for the controllable delivery of single molecules in aqueous environments. In this article we show both experimentally and from simulations that nanopipets in conjunction with asymmetric voltage pulses can be used for label-free detection and delivery of single molecules through the tip of a nanopipet with “on-demand” timing resolution. This was demonstrated by controllable delivery of 5 kbp and 10 kbp DNA molecules from solutions with concentrations as low as 3 pM

Exceptional Heterogeneity in Viral Evolutionary Dynamics Characterises Chronic Hepatitis C Virus Infection.

The treatment of HCV infection has seen significant progress, particularly since the approval of new direct-acting antiviral drugs. However these clinical achievements have been made despite an incomplete understanding of HCV replication and within-host evolution, especially compared with HIV-1. Here, we undertake a comprehensive analysis of HCV within-host evolution during chronic infection by investigating over 4000 viral sequences sampled longitudinally from 15 HCV-infected patients. We compare our HCV results to those from a well-studied HIV-1 cohort, revealing key differences in the evolutionary behaviour of these two chronic-infecting pathogens. Notably, we find an exceptional level of heterogeneity in the molecular evolution of HCV, both within and among infected individuals. Furthermore, these patterns are associated with the long-term maintenance of viral lineages within patients, which fluctuate in relative frequency in peripheral blood. Together, our findings demonstrate that HCV replication behavior is complex and likely comprises multiple viral subpopulations with distinct evolutionary dynamics. The presence of a structured viral population can explain apparent paradoxes in chronic HCV infection, such as rapid fluctuations in viral diversity and the reappearance of viral strains years after their initial detection.status: publishe

The upper and lower airway microbiome in severe asthma

Introduction: Severe asthma is complex and immunologically heterogenous; airways infection and innate immune dysregulation may confer inhaled corticosteroid resistance and remains clinically challenging. We hypothesise that marked airway microbiome compositional shifts in severe asthma represent a ‘treatable trait’, with dominance of pathogenic species, warranting targeted therapy. Additionally, these changes are thought to be distinct in the paucibacillary lower airway vs. heavily colonised nasopharynx. Methods: We performed Oxford Nanopore metagenomic sequencing, and RT-qPCR of induced sputum (n=67), bronchoalveolar lavage (BAL;n=71) and nasal lavage (NL;n=28 paired with sputum) samples from the Wessex Severe Asthma Cohort encompassing mild/moderate/severe asthma and health. Findings were integrated with clinical and cytokine data. Key Results: A dominant pathogenic organism (H.influenzae, S.pneumoniae, M.catarrhalis) was identified in sputua of 21% of patients with severe asthma and accompanied by sputum neutrophilia and elevated type-1 cytokines (including IL-1β, IL-6, IL-8, TNF;p<0.01, unpaired t-test, Benjamini-Hochberg correction). From BAL, airways infection was identified infrequently (Severe asthma, n=1/21 H.influenzae; Mild-Moderate asthma,n=2/25 T.whipplei). Metagenomic analysis of NL demonstrated a distinct microbiome; presence of pathogenic organisms in the upper airways did not predict concurrent presence in sputum of severe asthmatics. Conclusion: Airways infection is reliably identified in sputum using metagenomic sequencing and is associated with neutrophilic inflammation. The microbiome in the upper and lower airway are distinct and ongoing work is exploring its impact on mucosal immune responses at these sites

A Dominant Role for the Immunoproteasome in CD8+ T Cell Responses to Murine Cytomegalovirus

Murine cytomegalovirus (MCMV) is an important animal model of human cytomegalovirus (HCMV), a β-Herpesvirus that infects the majority of the world's population and causes disease in neonates and immunocompromised adults. CD8+ T cells are a major part of the immune response to MCMV and HCMV. Processing of peptides for presentation to CD8+ T cells may be critically dependent on the immunoproteasome, expression of which is affected by MCMV. However, the overall importance of the immunoproteasome in the generation of immunodominant peptides from MCMV is not known. We therefore examined the role of the immunoproteasome in stimulation of CD8+ T cell responses to MCMV – both conventional memory responses and those undergoing long-term expansion or “inflation”. We infected LMP7−/− and C57BL/6 mice with MCMV or with newly-generated recombinant vaccinia viruses (rVVs) encoding the immunodominant MCMV protein M45 in either full-length or epitope-only minigene form. We analysed CD8+ T cell responses using intracellular cytokine stain (ICS) and MHC Class I tetramer staining for a panel of MCMV-derived epitopes. We showed a critical role for immunoproteasome in MCMV affecting all epitopes studied. Interestingly we found that memory “inflating” epitopes demonstrate reduced immunoproteasome dependence compared to non-inflating epitopes. M45-specific responses induced by rVVs remain immunoproteasome-dependent. These results help to define a critical restriction point for CD8+ T cell epitopes in natural cytomegalovirus (CMV) infection and potentially in vaccine strategies against this and other viruses