P2X7 receptor function in microglia in relation to glaucoma

Purpose: The purpose of this research was to investigate the role of P2X7 in microglial function, in relation to glaucoma. Specifically, a P2X7 knockout (K/O) microglial cell line was generated and used to explore the responses of purinergic stimulation, including [Ca2+]i signalling, cytotoxicity, and IL-1β production and release. Purinergic mediated consequences of oxygen glucose deprivation were also briefly explored. Methods: P2X7 K/O microglial cells were generated using CRISPR gene editing technology on BV-2 microglia, and tested for P2X7 expression using flow cytometry, gDNA PCR, qRT-PCR and Western blotting. [Ca2+] responses to purinergic agonist stimulation were measured in Fura-2 loaded BV-2 and P2X7 K/O cells. Viability and cytotoxicity of purinergic stimulated BV-2 cells were assessed with MTS, LDH and caspase-3/7 staining assays. IL-1β mRNA and protein expression was measured with qRT-PCR and ELISAs respectively. BV-2 cells were subject to OGD using a custom incubator Results: CRISPR modification generated a cell line, which upon analysis demonstrated knockout of P2X7 protein expression. Purinergic agonists demonstrated a multifaceted [Ca2+]i response mediated by multiple receptors including P2X7, P2X4, P2Y2 and P2Y6, with P2X7 giving a sustained rise in [Ca2+]i . Microglia were sensitive to ATP-induced cytotoxicity mediated by P2X7. An initial lower level of ATP-induced toxicity was P2X7-independent and mediated by ADP. ATP treatment was a sufficient priming stimulus for IL-1β mRNA and protein and P2X7 was responsible for IL-1β release upon subsequent, in a P2X7-dependent manner. Conclusions: The generation of a P2X7 K/O microglial cell line has provided a wealth of information that can contribute to the understanding of the role of microglia in the pathophysiological mechanisms in glaucoma

The water quality of the River Enborne, UK: observations from high-frequency Monitoring in a rural, lowland river system

This paper reports the results of a 2-year study of water quality in the River Enborne, a rural river in lowland England. Concentrations of nitrogen and phosphorus species and other chemical determinands were monitored both at high-frequency (hourly), using automated in situ instrumentation, and by manual weekly sampling and laboratory analysis. The catchment land use is largely agricultural, with a population density of 123 persons km−2. The river water is largely derived from calcareous groundwater, and there are high nitrogen and phosphorus concentrations. Agricultural fertiliser is the dominant source of annual loads of both nitrogen and phosphorus. However, the data show that sewage effluent discharges have a disproportionate effect on the river nitrogen and phosphorus dynamics. At least 38% of the catchment population use septic tank systems, but the effects are hard to quantify as only 6% are officially registered, and the characteristics of the others are unknown. Only 4% of the phosphorus input and 9% of the nitrogen input is exported from the catchment by the river, highlighting the importance of catchment process understanding in predicting nutrient concentrations. High-frequency monitoring will be a key to developing this vital process understanding

High-frequency water quality monitoring in an urban catchment: hydrochemical dynamics, primary production and implications for the Water Framework Directive

This paper describes the hydrochemistry of a lowland, urbanised river-system, The Cut in England, using in situ sub-daily sampling. The Cut receives effluent discharges from four major sewage treatment works serving around 190,000 people. These discharges consist largely of treated water, originally abstracted from the River Thames and returned via the water supply network, substantially increasing the natural flow. The hourly water quality data were supplemented by weekly manual sampling with laboratory analysis to check the hourly data and measure further determinands. Mean phosphorus and nitrate concentrations were very high, breaching standards set by EU legislation. Though 56% of the catchment area is agricultural, the hydrochemical dynamics were significantly impacted by effluent discharges which accounted for approximately 50% of the annual P catchment input loads and, on average, 59% of river flow at the monitoring point. Diurnal dissolved oxygen data demonstrated high in-stream productivity. From a comparison of high frequency and conventional monitoring data, it is inferred that much of the primary production was dominated by benthic algae, largely diatoms. Despite the high productivity and nutrient concentrations, the river water did not become anoxic and major phytoplankton blooms were not observed. The strong diurnal and annual variation observed showed that assessments of water quality made under the Water Framework Directive (WFD) are sensitive to the time and season of sampling. It is recommended that specific sampling time windows be specified for each determinand, and that WFD targets should be applied in combination to help identify periods of greatest ecological risk. This article is protected by copyright. All rights reserved

Ginsenosides act as positive modulators of P2X4 receptors

We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by ∼twofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/ Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP 2 or ATP 1 ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain

The petrogenesis of Brazilian kimberlites and kamafugites intruded along the 125° lineament: improved geochemical and geochronological constraints on magmatism in Rondonia and the Alto Paranaiba Igneous Province

© 2014 Dr. Matthew Richard FelgateDespite a number of studies, the origin of ultrapotassic magmatism in Brazil remains contentious. In particular, problems relate to the timing of Cretaceous kimberlite and kamafugite magmatism within the Alto Paranaiba Igneous Province (APIP), the origin and relationship between the Cretaceous kimberlites and kamafugites, and the geochemical nature of the Permo-Triassic kimberlite magmatism. Many of these uncertainties arise as a consequence of either the implementation of now superseded analytical techniques or the use of highly altered samples. This study seeks to address these issues by performing state-of-the-art geochemical and geochronological analyses on a number of fresh kimberlite and kamafugite drill core samples situated along the 125° lineament. The first part of the thesis provides a comprehensive geochronological study of Brazilian kimberlites and kamafugites. This is built around a combination of the most recent U/Pb perovskite, Rb-Sr phlogopite, and Ar-Ar phlogopite dating techniques that together provide 33 age estimates, representing 22 separate intrusions, of which 11 are kimberlites and 11 kamafugites. The results are in broad agreement with previously published data, identifying a Permo-Triassic age for the Amazonian kimberlites, and kimberlites and kamafugites of Cretaceous age in the APIP. The new data from this study also support an age decrease from NW to SE within the APIP. However, data from this study suggest that this is not a continuous younging, instead indicating that two distinct magmatic episodes may exist. The data also reveal the presence of an “older” mid Cretaceous cluster (~88 Ma) encompassing Goias and the NW APIP (Southern Goiás and NW Minas Gerais) and a slightly younger Cretaceous cluster (~80 Ma) in the Central/Southern APIP. The importance of these new findings is discussed with reference to previously proposed petrogenetic models and casts doubt on the applicability of the plume-related hypothesis for ultrapossic magmatism in the region. In the second part of the thesis, an in-depth geochemical study of 15 kimberlites and 16 kamafugites from the APIP (27 samples) and Rondonia/Mato Grosso (4 samples) is presented. Bulk rock major and trace element data, together with Nd and Hf isotope determinations are combined with perovskite trace element, Sr and Nd isotope analyses in order to provide a comprehensive geochemical dataset. The elemental and isotopic similarity of the APIP kimberlite to South African transitional kimberlites, the APIP kamafugites to Ugandan and Chinese kamafugites and, for the first time, the Amazonian kimberlites to a mixture of South African Group I and transitional kimberlites is established. The overlapping range in Sr isotopic signatures identified between the APIP kimberlite and kamafugite magmas during previous bulk rock studies is confirmed by in situ perovskite analysis, with APIP kimberlites ranging between 70496 and 0.70596 and APIP kamafugites ranging between 0.70499 and 0.70574. Modelling suggests that this range can not result from contamination and must represent a heterogeneous source component, common in the formation of both rock types. It is argued that the combination of geochemical results is best explained by derivation of both rock types from the heterogeneous subcontinental lithospheric mantle. Kimberlites are believed to have been sourced from carbonated garnet lherzolite whereas the kamafugites were generated from phlogopite and clinopyroxene rich vein assemblages, with small volume MARID type metasomes indicated by the kamafugite Hf signatures. Finally, the geochronological and geochemical information is combined into a petrogenetic model: it is argued that the Cretaceous magmatism is likely not the result of plume heating but is instead a consequence of adiabatic decompression melting of the heterogeneous SCLM caused by extension. In parallel with the study of Brazilian ultrapotassic magmatism the thesis also investigates technical aspects of the U-Pb perovskite dating technique. Using a subset of Indian and Brazilian kimberlite samples, in situ perovskite dating was performed and combined with isotope dilution analysis of the co-magmatic, low uranium phase, titanomagnetite, in an attempt to improve precision of the Tera-Wasserburg regression. The inclusion of the titanomagnetite phase improved precision in all cases and enabled ages to be determined for the Indian samples without recourse to an assumed Stacey-Kramers common Pb value. The investigation also highlights some of the shortcomings associated with using an assumed Stacey-Kramers Pb value for young unradiogenic perovskite samples, whilst also highlighting the circularity of the commonly employed 207Pb correction method

Estimating parent population of pottery vessels from a sample of fragments: a case study from inter-tidal surface collections, Roviana Lagoon, Solomon Islands

Archaeologists frequently interpret the duration and type of prehistoric occupation at a location/site from the relative abundance of prehistoric materials (accumulations research). Taphonomic factors have the potential to alter substantially the archaeological record leading to inaccurate assumptions about the nature of prehistoric settlement. Where vessel families of potsherds can be established, we provide a general method to estimate the parent population from which the sample of sherds derives, through analysis of vessel completeness of the sample. This provides a basis for archaeological interpretation of the scale of discard represented at a location. The approach used has broad applicability to many archaeological settings where behavioural inferences are made from poorly preserved ceramic samples. Statistical analysis of a ceramic assemblage from Roviana Lagoon stilt villages indicates that most of the ceramic assemblage has been destroyed over time. An unexpectedly severe taphonomic regime is inferred for this relatively sheltered landlocked lagoon setting. Rather than marking an ephemeral occupation, estimating a parent ceramic population indicates instead that early stilt village sites in the Solomon Islands region were probably permanently occupied for several centuries. Results also suggest that the relative absence of recorded Lapita sites in the main Solomon Islands, and possibly elsewhere in Near Oceania may be a result of a harsh taphonomic regime for the remains of stilt village occupation

Human history and palaeoenvironmental change at Site 17, Freshwater Beach, Lizard Island, northeast Queensland, Australia

Late Holocene patterns of change in occupation and use of islands along the eastern coast of Queensland have long been debated in terms of various drivers, though much of this discussion relates to regions south of Cairns, with comparatively little study of the far northern Great Barrier Reef islands. The numerous middens, stone arrangements and art sites on Lizard Island suggest long-term use by Indigenous people, but recent discoveries of pottery give tantalising glimpses of a prehistoric past that may have included a prehistoric economy involving pottery. Here we review previous archaeological surveys and studies on Lizard Island and report on new archaeological and palaeoenvironmental studies from the Site 17 midden at Freshwater Beach, with an oldest date of 3815–3571 cal BP. We identify two major changes in the archaeological and palaeoenvironmental records, one associated with more recent European influences and the other at c.2000 cal BP. Pottery from the intertidal zone is as yet undated. When dates become available the relationship between the Site 17 results reported here and the use of pottery on the island may be clarified

A Solomon Sea interaction sphere?

The Solomon Sea is the body of water bounded by New Britain to the north, New Guinea to the west, the Louisiade Archipelago to the South, and the Solomon Island chain down to Makira on the east. Our thinking and writing on the culture history of the broader region has focused primarily on a terrestrial model of movement with arrows showing migration or influence moving down the Solomons chain or along the eastern coast of New Guinea, rarely have we considered arrows moving across the Solomon Sea. This is despite the steady increase of evidence for relatively sophisticated maritime transport from the Late Pleistocene in the Bismarck Archipelago. It is commonplace now to consider the sea as more of a highway than a barrier. Within the relatively dispersed islands of the Massim region, history and archaeology have documented extensive long-distance interaction using sophisticated sailing craft, yet we rarely consider the ease with which such patterns of interaction might extend east across the Solomon Sea to the Solomon Islands. It is certainly true that historically there is no evidence for such an interaction sphere; however increasingly there is evidence for increased interaction in the past. In this paper we will review the evidence for such a pattern

Phosphorus dynamics and productivity in sewage-impacted lowland chalk stream

Hourly in situ phosphorus, conductivity, turbidity, dissolved oxygen, pH, and chlorophyll measurements were collected from January 2004 to November 2006 for the River Kennet, 2 km downstream of a sewage effluent inlet. Excess carbon dioxide pressure (EpCO2) was calculated from continuous pH and spot alkalinity measurements. EpCO2 and dissolved oxygen were used to estimate rates of photosynthesis and respiration. These parameters were examined alongside flow, water temperature and solar radiation to explore controls on phosphorus dynamics and in-stream productivity. Diurnal, event and seasonal patterns were observed in phosphorus concentrations. The diurnal and seasonal variations appeared to be related to the upstream sewage treatment works. The event patterns coincided with periods of high flow, and were attributed to diffuse sources. Chlorophyll behaved independently of phosphorus concentration and returned to baseline levels before photosynthesis rate. This indicated that, during the period of study, in-stream productivity was primarily controlled by aquatic plants other than phytoplankton