Lipofection with Synthetic mRNA as a Simple Method for T-Cell Immunomonitoring.

The quantification of T-cell immune responses is crucial for the monitoring of natural and treatment-induced immunity, as well as for the validation of new immunotherapeutic approaches. The present study presents a simple method based on lipofection of synthetic mRNA in mononuclear cells as a method to determine in vitro T-cell responses. We compared several commercially available transfection reagents for their potential to transfect mRNA into human peripheral blood mononuclear cells and murine splenocytes. We also investigated the impact of RNA modifications in improving this method. Our results demonstrate that antigen-specific T-cell immunomonitoring can be easily and quickly performed by simple lipofection of antigen-coding mRNA in complex immune cell populations. Thus, our work discloses a convenient solution for the in vitro monitoring of natural or therapy-induced T-cell immune responses

Monitoring pumping test response in a fractured aquifer using ground-penetrating radar

This is the published version. Copyright 2010 by the American Geophysical Union. All rights reserved.Fractured aquifers present a number of problems when attempting to characterize flow on the well scale (less than 100 m). Standard hydraulic testing methods are expensive because of the need for installation of monitoring wells. Geophysical methods may suffer from a lack of resolution and nonunique solutions to data interpretation. We used ground-penetrating radar (GPR) surveying during a pumping test in a well-characterized, fractured, carbonate aquifer to monitor the response of a permeable subhorizontal fracture plane. We observed radar signal amplitude and waveform variations along a fracture reflector and correlated the radar signal response to changes in the water saturation of the fracture. Combining hydraulic measurements with GPR data and electromagnetic modeling, we identified an asymmetric fracture drainage pattern, provided accurate spatial information about the saturation of the fracture, and detected the presence of hydraulic boundaries. This study demonstrates that GPR surveying can be used successfully for real-time monitoring of pumping tests in fractured carbonate aquifers

Disruption of beta cell acetyl-CoA carboxylase-1 in mice impairs insulin secretion and beta cell mass

Aims/hypothesis Pancreatic beta cells secrete insulin to maintain glucose homeostasis, and beta cell failure is a hallmark of type 2 diabetes. Glucose triggers insulin secretion in beta cells via oxidative mitochondrial pathways. However, it also feeds mitochondrial anaplerotic pathways, driving citrate export and cytosolic malonyl-CoA production by the acetyl-CoA carboxylase 1 (ACC1) enzyme. This pathway has been proposed as an alternative glucose-sensing mechanism, supported mainly by in vitro data. Here, we sought to address the role of the beta cell ACC1-coupled pathway in insulin secretion and glucose homeostasis in vivo. Methods Acaca, encoding ACC1 (the principal ACC isoform in islets), was deleted in beta cells of mice using the Cre/loxP system. Acaca floxed mice were crossed with Ins2cre mice (βACC1KO; life-long beta cell gene deletion) or Pdx1creER mice (tmx-βACC1KO; inducible gene deletion in adult beta cells). Beta cell function was assessed using in vivo metabolic physiology and ex vivo islet experiments. Beta cell mass was analysed using histological techniques. Results βACC1KO and tmx-βACC1KO mice were glucose intolerant and had defective insulin secretion in vivo. Isolated islet studies identified impaired insulin secretion from beta cells, independent of changes in the abundance of neutral lipids previously implicated as amplification signals. Pancreatic morphometry unexpectedly revealed reduced beta cell size in βACC1KO mice but not in tmx-βACC1KO mice, with decreased levels of proteins involved in the mechanistic target of rapamycin kinase (mTOR)-dependent protein translation pathway underpinning this effect. Conclusions/interpretation Our study demonstrates that the beta cell ACC1-coupled pathway is critical for insulin secretion in vivo and ex vivo and that it is indispensable for glucose homeostasis. We further reveal a role for ACC1 in controlling beta cell growth prior to adulthood

RNA with chemotherapeutic base analogues as a dual-functional anti-cancer drug

Nanoparticles of different sizes formulated with unmodified RNA and Protamine differentially engage Toll-like Receptors (TLRs) and activate innate immune responses in vitro. Here, we report that similar differential immunostimulation that depends on the nanoparticle sizes is induced in vivo in wild type as well as in humanized mice. In addition, we found that the schedule of injections strongly affects the magnitude of the immune response. Immunostimulating 130 nm nanoparticles composed of RNA and Protamine can promote lung metastasis clearance but provides no control of subcutaneous tumors in a CT26 tumor model. We further enhanced the therapeutic capacity of Protamine-RNA nanoparticles by incorporating chemotherapeutic base analogues in the RNA; we coined these immunochemotherapeutic RNAs (icRNAs). Protamine-icRNA nanoparticles were successful at controlling established subcutaneous CT26 and B16 tumors as well as orthotopic glioblastoma. These data indicate that icRNAs are promising cancer therapies, which warrants their further validation for use in the clinic. Keywords: 5FU; Chemotherapy; RNA; immunotherapy; toll like receptor; type I interferon

Seabed geomorphology: a two-part classification system

The BGS has developed a two- part classification system (‘Morphology’ and ‘Geomorphology’) to facilitate work on a new ‘S eabed Geomorphology’ mapping initia tive, and this classification system is the focus of this report. As stated in the Foreword, the rationale and the basic framework of the classification system were conceived and es tablished within BGS, but recent collaboration within the MAREANO -Norway, INFOMAR -Ireland, and MAREMAP -UK (MIM) partnership has led to significant improvement of the classifi cation system, and this report. To further support this effort, existing BGS GIS tools (SIGMA) ha ve been adapted to apply this two-part classification system for more efficient geom orphological mapping in the marine environment. This report: provides a brief background on seabed mapping and characterisation, as well as how this science has been addressed historically within BGS; describes the current motiva tion to conduct seabed geom orphological mapping, and the requirement for a new set of t ools to facilitate this work; describes the logical framework that underpins the classification system; outlines the attributes of the classification system, how it can be applied, and discusses the advantages and limitations of the approach. It is anticipated that through testing and usage, the classification syst em will be revised and improved over time, with updated versions released through MIM partnershi p. It is also planned that a further ‘user guide’ report will be produced for the classifi cation system and the GIS tools, including thematic details (e.g. background information on ‘coastal’ or ‘glacial’ features) and a feature glossary

Tracing differences in iron supply to the Mid-Atlantic Ridge valley between hydrothermal vent sites: implications for the addition of iron to the deep ocean

Supply of iron (Fe) to the surface ocean supports primary productivity, and while hydrothermal input of Fe to the deep ocean is known to be extensive it remains poorly constrained. Global estimates of hydrothermal Fe supply rely on using dissolved Fe (dFe) to excess He (xs3He) ratios to upscale fluxes, but observational constraints on dFe/xs3He may be sensitive to assumptions linked to sampling and interpolation. We examined the variability in dFe/xs3He using two methods of estimation, for four vent sites with different geochemistry along the Mid-Atlantic Ridge. At both Rainbow and TAG, the plume was sampled repeatedly and the range of dFe/xs3He was 4 to 63 and 4 to 87 nmol:fmol, respectively, primarily due to differences in plume age. To account for background xs3He and shifting plume position, we calibrated He values using contemporaneous dissolved Mn (dMn). Applying this approach more widely, we found dFe/xs3He ratios of 12, 4–8, 4–44, and 4–86 nmol fmol−1 for the Menez Gwen, Lucky Strike, Rainbow, and TAG hydrothermal vent sites, respectively. Differences in plume dFe/xs3He across sites were not simply related to the vent endmember Fe and He fluxes. Within 40 km of the vents, the dFe/xs3He ratios decreased to 3–38 nmol fmol−1, due to the precipitation and subsequent settling of particulates. The ratio of colloidal Fe to dFe was consistently higher (0.67–0.97) than the deep N. Atlantic (0.5) throughout both the TAG and Rainbow plumes, indicative of Fe exchange between dissolved and particulate phases. Our comparison of TAG and Rainbow shows there is a limit to the amount of hydrothermal Fe released from vents that can form colloids in the rising plume. Higher particle loading will enhance the longevity of the Rainbow hydrothermal plume within the deep ocean assuming particles undergo continual dissolution/disaggregation. Future studies examining the length of plume pathways required to escape the ridge valley will be important in determining Fe supply from slow spreading mid-ocean ridges to the deep ocean, along with the frequency of ultramafic sites such as Rainbow. Resolving the ridge valley bathymetry and accounting for variability in vent sources in global biogeochemical models will be key to further constraining the hydrothermal Fe flux.</p

Tracing differences in iron supply to the Mid-Atlantic Ridge valley between hydrothermal vent sites: implications for the addition of iron to the deep ocean

Supply of iron (Fe) to the surface ocean supports primary productivity, and while hydrothermal input of Fe to the deep ocean is known to be extensive it remains poorly constrained. Global estimates of hydrothermal Fe supply rely on using dissolved Fe (dFe) to excess He (xs³He) ratios to upscale fluxes, but observational constraints on dFe/xs³He may be sensitive to assumptions linked to sampling and interpolation. We examined the variability in dFe/xs³He using two methods of estimation, for four vent sites with different geochemistry along the Mid-Atlantic Ridge. At both Rainbow and TAG, the plume was sampled repeatedly and the range of dFe/xs³He was 4 to 63 and 4 to 87 nmol:fmol, respectively, primarily due to differences in plume age. To account for background xs³He and shifting plume position, we calibrated He values using contemporaneous dissolved Mn (dMn). Applying this approach more widely, we found dFe/xs³He ratios of 12, 4–8, 4–44, and 4–86 nmol fmol−1 for the Menez Gwen, Lucky Strike, Rainbow, and TAG hydrothermal vent sites, respectively. Differences in plume dFe/xs³He across sites were not simply related to the vent endmember Fe and He fluxes. Within 40 km of the vents, the dFe/xs³He ratios decreased to 3–38 nmol fmol−1, due to the precipitation and subsequent settling of particulates. The ratio of colloidal Fe to dFe was consistently higher (0.67–0.97) than the deep N. Atlantic (0.5) throughout both the TAG and Rainbow plumes, indicative of Fe exchange between dissolved and particulate phases. Our comparison of TAG and Rainbow shows there is a limit to the amount of hydrothermal Fe released from vents that can form colloids in the rising plume. Higher particle loading will enhance the longevity of the Rainbow hydrothermal plume within the deep ocean assuming particles undergo continual dissolution/disaggregation. Future studies examining the length of plume pathways required to escape the ridge valley will be important in determining Fe supply from slow spreading mid-ocean ridges to the deep ocean, along with the frequency of ultramafic sites such as Rainbow. Resolving the ridge valley bathymetry and accounting for variability in vent sources in global biogeochemical models will be key to further constraining the hydrothermal Fe flux

Denudation of the continental shelf between Britain and France at the glacial-interglacial timescale

The erosional morphology preserved at the sea bed in the eastern English Channel dominantly records denudation of the continental shelf by fluvial processes over multiple glacial-interglacial sea-level cycles rather than by catastrophic flooding through the Straits of Dover during the mid-Quaternary. Here, through the integration of multibeam bathymetry and shallow sub-bottom 2D seismic reflection profiles calibrated with vibrocore records, the first stratigraphic model of erosion and deposition on the eastern English Channel continental shelf is presented. Published Optical Stimulated Luminescence (OSL) and C ages were used to chronometrically constrain the stratigraphy and allow correlation of the continental shelf record with major climatic/sea-level periods. Five major erosion surfaces overlain by discrete sediment packages have been identified. The continental shelf in the eastern English Channel preserves a record of processes operating from Marine Isotope Stage (MIS) 6 to MIS 1. Planar and channelised erosion surfaces were formed by fluvial incision during lowstands or relative sea-level fall. The depth and lateral extent of incision was partly conditioned by underlying geology (rock type and tectonic structure), climatic conditions and changes in water and sediment discharge coupled to ice sheet dynamics and the drainage configuration of major rivers in Northwest Europe. Evidence for major erosion during or prior to MIS 6 is preserved. Fluvial sediments of MIS 2 age were identified within the Northern Palaeovalley, providing insights into the scale of erosion by normal fluvial regimes. Seismic and sedimentary facies indicate that deposition predominantly occurred during transgression when accommodation was created in palaeovalleys to allow discrete sediment bodies to form. Sediment reworking over multiple sea-level cycles (Saalian-Eemian-early Weichselian) by fluvial, coastal and marine processes created a multi-lateral, multi-storey succession of palaeovalley-fills that are preserved as a strath terrace. The data presented here reveal a composite erosional and depositional record that has undergone a high degree of reworking over multiple sea-level cycles leading to the preferential preservation of sediments associated with the most recent glacial-interglacial period

CARD14 Gain-of-Function Mutation Alone Is Sufficient to Drive IL-23/IL-17-Mediated Psoriasiform Skin Inflammation In Vivo.

Rare autosomal dominant mutations in the gene encoding the keratinocyte signaling molecule CARD14, have been associated with an increased susceptibility to psoriasis, but the physiological impact of CARD14 gain-of-function mutations remains to be fully determined in vivo. Here, we report that heterozygous mice harboring a CARD14 gain-of-function mutation (Card14ΔE138) spontaneously develop a chronic psoriatic phenotype with characteristic scaling skin lesions, epidermal thickening, keratinocyte hyperproliferation, hyperkeratosis, and immune cell infiltration. Affected skin of these mice is characterized by elevated expression of anti-microbial peptides, chemokines, and cytokines (including T helper type 17 cell-signature cytokines) and an immune infiltrate rich in neutrophils, myeloid cells, and T cells, reminiscent of human psoriatic skin. Disease pathogenesis was driven by the IL-23/IL-17 axis, and neutralization of IL-23p19, the key cytokine in maintaining T helper type 17 cell polarization, significantly reduced skin lesions and the expression of antimicrobial peptides and proinflammatory cytokines. Therefore, hyperactivation of CARD14 alone is sufficient to orchestrate the complex immunopathogenesis that drives T helper type 17-mediated psoriasis skin disease in vivo

Relationships between plasma lipids species, gender, risk factors and Alzheimer’s disease

Background: Lipid metabolism is altered in Alzheimer’s disease (AD); however, the relationship between AD risk factors (age, APOE ɛ4, and gender) and lipid metabolism is not well defined. Objective: We investigated whether altered lipid metabolism associated with increased age, gender, and APOE status may contribute to the development of AD by examining these risk factors in healthy controls and also clinically diagnosed AD individuals. Methods: We performed plasma lipidomic profiling (582 lipid species) of the Australian Imaging, Biomarkers and Lifestyle flagship study of aging cohort (AIBL) using liquid chromatography-mass spectrometry. Linear regression and interaction analysis were used to explore the relationship between risk factors and plasma lipid species. Results: We observed strong associations between plasma lipid species with gender and increasing age in cognitively normal individuals. However, APOE ɛ4 was relatively weakly associated with plasma lipid species. Interaction analysis identified differential associations of sphingolipids and polyunsaturated fatty acid esterified lipid species with AD based on age and gender, respectively. These data indicate that the risk associated with age, gender, and APOE ɛ4 may, in part, be mediated by changes in lipid metabolism. Conclusion: This study extends our existing knowledge of the relationship between the lipidome and AD and highlights the complexity of the relationships between lipid metabolism and AD at different ages and between men and women. This has important implications for how we assess AD risk and also for potential therapeutic strategies involving modulation of lipid metabolic pathways