Development of an oligonucleotide-based SNP detection method on lateral flow strips using hexapet tages

Detection of point mutations or single nucleotide polymorphisms (SNPs) is important in relation to disease susceptibility or detection in pathogens of mutations determining drug resistance or host range. There is an emergent need for rapid detection methods amenable to point-of-care applications. The purpose of this study was to reduce to practice a novel method for SNP detection and to demonstrate that this technology can be used downstream of nucleic acid amplification. The authors used a model system to develop an oligonucleotide-based SNP detection system on nitrocellulose lateral flow strips. To optimize the assay they used cloned sequences of the herpes simplex virus-1 (HSV-1) DNA polymerase gene into which they introduced a point mutation. The assay system uses chimeric polymerase chain reaction (PCR) primers that incorporate hexameric repeat tags ("hexapet tags"). The chimeric sequences allow capture of amplified products to predefined positions on a lateral flow strip. These "hexapet" sequences have minimal cross-reactivity and allow specific hybridization-based capture of the PCR products at room temperature onto lateral flow strips that have been striped with complementary hexapet tags. The allele-specific amplification was carried out with both mutant and wild-type primer sets present in the PCR mix ("competitive" format). The resulting PCR products carried a hexapet tag that corresponded with either a wild-type or mutant sequence. The lateral flow strips are dropped into the PCR reaction tube, and mutant sequence and wild-type sequences diffuse along the strip and are captured at the corresponding position on the strip. A red line indicative of a positive reaction is visible after 1 minute. Unlike other systems that require separate reactions and strips for each target sequence, this system allows multiplex PCR reactions and multiplex detection on a single strip or other suitable substrates. Unambiguous visual discrimination of a point mutation under room temperature hybridization conditions was achieved with this model system in 10 minutes after PCR. The authors have developed a capture-based hybridization method for the detection and discrimination of HSV-1 DNA polymerase genes that contain a single nucleotide change. It has been demonstrated that the hexapet oligonucleotides can be adapted for hybridization on the lateral flow strip platform for discrimination of SNPs. This is the first step in demonstrating SNP detection on lateral flow using the hexapet oligonucleotide capture system. It is anticipated that this novel system can be widely used in point-of-care settings.

Estimating percolation and lateral water flow on sloping land in rainfed lowland rice ecosystem

Quantifying water losses in paddy fields assists estimation of water availability in rainfed lowland rice ecosystem. Little information is available on water balance in different toposequence positions of sloped rainfed lowland. Therefore, the aim of this work was to quantify percolation and the lateral water flow with special reference to the toposequential variation. Data used for the analysis was collected in Laos and northeast Thailand. Percolation and water tables were measured on a daily basis using a steel cylindrical tube with a lid and perforated PVC tubes, respectively. Percolation rate was determined using linear regression analysis of cumulative percolation. Assuming that the total amount of evaporation and transpiration was equivalent to potential evapotranspiration, the lateral water flow was estimated using the water balance equation. Separate perched water and groundwater tables were observed in paddy fields on coarse-textured soils. The percolation rate varied between 0 and 3 mm/day across locations, and the maximum water loss by lateral movement was more than 20 mm/day. Our results are in agreement with the previously reported findings, and the methodology of estimating water balance components appears reasonably acceptable. With regard to the toposequential variation, the higher the position in the topoesquence, the greater potential for water loss because of higher percolation and lateral flow rates.

The water balance on sloping land in rainfed lowland rice ecosystem

Quantifying water losses in paddy fields assists estimation of water availability in rainfed lowland rice ecosystem. There is, however, no definite method for determining the water losses, and little information is available on water balance in different toposequence positions of a sloped rainfed lowland. Therefore, the aim of this work was to quantify percolation and the lateral water flow with special reference to the toposequential variation. Data used for the analysis was collected in Laos and northeast Thailand. Percolation and water tables were measured on a daily basis. The percolator is a steel cylindrical tube with a lid to prevent water loss from evapotranspiration. The water table meter is a short PVC tube for determination of perched water table and a long PVC tube for groundwater table, and the side is perforated with 5-mm diameter holes at 20-mm distance. Percolation rate was determined using linear regression analysis of cumulative percolation. Assuming that the total amount of evaporation and transpiration was equivalent to potential evapotranspiration, the lateral water flow was estimated using the water balance equation. Our results are in agreement with the previously reported findings, and the methodology of estimating water balance components appears reasonably acceptable. With regard to the toposequential variation, the higher position in the topoesquence, the greater potential of the water losses because of higher percolation and lateral flow rates.

Timing and chemistry of fluid-flow events in the Lawn Hill Platform, Northern Australia

Mudrocks and carbonates of the Isa superbasin in the Lawn Hill platform in northern Australia host major base metal sulfide mineralization, including the giant strata-bound Century Zn-Pb deposit. Mineral paragenesis, stable isotope, and K-Ar dating studies demonstrate that long-lived structures such as the Termite Range fault acted as hot fluid conduits several times during the Paleoproterozoic and Mesoproterozoic in response to major tectonic events. Illite and chlorite crystallinity studies suggest the southern part of the platform has experienced higher temperatures (up to 300 degrees C) than similar stratigraphic horizons in the north. The irregular downhole variation of illite crystallinity values provides further information oil the thermal regime in the basin and shows that clay formation was controlled not only by temperature increase with depth but also by high water/rock ratios along relatively permeable zones. K-Ar dating of illite, in combination with other data, may indicate three major thermal events in the central and northern Lawn Hill platform Lit 1500, 1440 to 1400, and 1250 to 1150 Ma. This study did not detect the earlier Century base metal mineralizing event at 1575 Ma. 1500 Ma ages are recorded only in the south and correspond to the age of the Late Isan orogeny and deposition of the Lower Roper superbasin. They may reflect exhumation of a provenance region. The 1440 to 1300 Ma ages are related to fault reactivation and a thermal pulse at similar to 1440 to 1400 Ma possibly accompanied by fluid flow, with subsequent enhanced cooling possibly due to thermal relaxation or further crustal exhumation. The youngest thermal and/or fluid-flow event at 1250 to 1150 Ma is recorded mainly to the cast of the Tern-lite Range fault and may be related to the assembly of the Rodinian supercontinent. Fluids in equilibrium with illite that formed over a range of temperatures, at different times in different parts of the platform. have relatively uniform oxygen isotope compositions and more variable hydrogen isotope compositions (delta O-18 = 3.5-9.7 parts per thousand V-SMOW; delta D = -94 to -36 parts per thousand V-SMOW). The extent of the 180 enrichment and the variably depleted hydrogen isotope compositions suggest the illite interacted with deep-basin hypersaline brines that were composed of evaporated seawater and/or highly evolved meteoric water. Siderite is the most abundant iron-rich gangue phase in the Century Zn-Pb deposit, which is surrounded by all extensive ferroan carbonate alteration halo. Modeling suggests that the ore siderite formed at temperatures of 120 degrees to 150 degrees C, whereas siderite and ankerite in the alteration halo formed at temperatures of 150 degrees to 180 degrees C. The calculated isotopic compositions of the fluids are consistent with O-18-rich basinal brines and mixed inorganic and organic carbon Sources (6180 = 3-10 parts per thousand V-SMOW, delta C-13 = -7 to -3 parts per thousand V-PDB). in the northeast Lawn Hill platform carbonate-rich rocks preserve marine to early diagenetic carbon and oxygen isotope compositions, whereas ferroan carbonate cements in siltstones and shales in the Desert Creek borehole are O-18 and C-13 depleted relative to the sedimentary carbonates. The good agreement between temperature estimates from illite crystallinity and organic reflectance (160 degrees-270 degrees C) and inverse correlation with carbonate delta O-18 values indicates that organic maturation and carbonate precipitation in the northeast Lawn Hill platform resulted from interaction with the 1250 to 1150 Ma fluids. The calculated isotopic compositions of the fluid are consistent with evolved basinal brine (delta O-18 = 5.1-9.4 parts per thousand V-SMOW; delta C-13 = -13.2 to -3.7 parts per thousand V-PDB) that contained a variable organic carbon component from the oxidation and/or hydrolysis of organic matter in the host sequence. The occurrence of extensive O-18- and C-13-depleted ankerite and siderite alteration in Desert Creek is related to the high temperature of the 1250 to 1150 Ma fluid-flow event in the northeast Lawn Hill platform, in contrast to the lower temperature fluids associated with the earlier Century Zn-Pb deposit in the central Lawn Hill platform.

Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay

Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUN(TM) beads and the whole blood Lyse/No-Wash protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 mul of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 It after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained. (C) 2003 Elsevier B.V. All rights reserved.

Wave-generated flow on coral reefs - an analysis for two-dimensional horizontal reef-tops with steep faces

Waves breaking on the seaward rim of a coral reef generate a flow of water from the exposed side of the reef to the sheltered side and/or to either channels through the reef-rim or lower sections of the latter. This wave-generated flow is driven by the water surface gradient resulting from the wave set-up created by the breaking waves. This paper reviews previous approaches to modelling wave-generated flows across coral reefs and discusses the influence of reef morphology and roughness upon these flows. Laboratory measurements upon a two-dimensional horizontal reef platform with a steep reef face provide the basis for extending a previous theoretical analysis for wave set-up on a reef in the absence of a flow [Gourlay, M.R., 1996b. Wave set-up on coral reefs. 2. Set-up on reefs with various profiles. Coastal Engineering 28, 1755] to include the interaction between a unidirectional flow and the wave set-up. The laboratory model results are then used to demonstrate that there are two basic reef-top flow regimes-reef-top control and reef-rim control. Using open channel flow theory, analytical relationships are derived for the reef-top current velocity in terms of the offreef wave conditions, the reef-top water depth and the physical characteristics of the reef-top topography. The wave set-up and wave-generated flow relationships are found to predict experimental values with reasonable accuracy in most cases. The analytical relationships are used to investigate wave-generated flows into a boat harbour channel on Heron Reef in the southern Great Barrier Reef. (c) 2005 Elsevier B.V. All rights reserved.

Thermal evolution of the ore-hosting Isa Superbasin: Central and Northern Lawn Hill Platform

Hydrocarbon migration pathways and organic mineral matter associations were used to identify brine pathways in Paleoproterozic to early Mesoproterozoic rocks from the Lawn Hill platform, Mount Isa. Several types of organic matter are identified, and their thermal imprints are used to reconstruct the thermal history of the northern to central parts of the Isa superbasin. Three major thermal hydrothermal episodes are recognized from the organic maturation studies. Isotherm plots on a 175-km-long structural-sedimentological north-south section of the Isa superbasin highlight specific fault systems that acted as hot fluid conduits during the geologic history of the basin. Some of these systems indicate continuing activity into the south Nicholson basin, supported by the presence of low reflectance (type B) bitumen. This bitumen has not been overprinted by later hydrothermal episodes and therefore represents the latest thermal event. Along the north-south profile a general southward increase in temperature is evident. The lowest temperatures are recorded in proximity to the basin margin on the southern flank of the Murphy inlier. Thermal processes and their sequence of events in the basin are recorded by organic maturation, subsequent hydrocarbon generation, its migration and destruction coincident with transport and precipitation of minerals. As some timing and trapping mechanisms for minerals may have analogues with hydrocarbon entrapment, relative timing of processes leading to organic maturation, hydrocarbon generation and migration are utilized in this study to enhance understanding of ore-grade mineralization. In the Proterozoic successions of the Mount Isa basin multiple hydrocarbon generation events are recognized. These events record the transient passage of potential metal-bearing fluids rather than background conductive heat flow from the basement. Such hydrothermal fluids are responsible for inverse maturation profiles in the vicinity of the Termite Range fault and extreme maturation (reflectance values) up to 6 percent Ro at the Grevillea prospect. At Century, intermediate Ro values of