Biomedical subjects

David J Marshall

Publications and source records attributed to David J Marshall.

11 recordsLinked to original sources

PubMed2006

Phenotypic plasticity of thermal tolerances in five oribatid mite species from sub-Antarctic Marion Island.

The extent to which phenotypic plasticity might mediate short-term responses to environmental change is controversial. Nonetheless, theoretical work has made the prediction that plasticity should be common, especially in predictably variable environments by comparison with those that are either stable or unpredictable. Here we examine these predictions by comparing the phenotypic plasticity of thermal tolerances (supercooling point (SCP), lower lethal temperature (LLT), upper lethal temperature (ULT)), following acclimation at either 0, 5, 10 or 15 degrees C, for seven days, of five, closely-related ameronothroid mite species. These species occupy marine and terrestrial habitats, which differ in their predictability, on sub-Antarctic Marion Island. All of the species showed some evidence of pre-freeze mortality (SCPs -9 to -23 degrees C; LLTs -3 to -15 degrees C), though methodological effects might have contributed to the difference between the SCPs and LLTs, and the species are therefore considered moderately chill tolerant. ULTs varied between 36 degrees C and 41 degrees C. Acclimation effects on SCP and LLT were typically stronger in the marine than in the terrestrial species, in keeping with the prediction of strong acclimation responses in species from predictably variable environments, but weaker responses in species from unpredictable environments. The converse was found for ULT. These findings demonstrate that acclimation responses vary among traits in the same species. Moreover, they suggest that there is merit in assessing the predictability of changes in high and low environmental temperatures separately.

Acclimatization↗

PubMed2006

A continuous assay for DNA cleavage using molecular break lights.

Exploring the properties of molecules that cleave DNA (i.e., enzymatic nucleases, chemical footprinting agents, and naturally occurring DNA cleaving antibiotics) has been an ongoing process with benefits extending toward both laboratory and clinical applications. Despite the progress that has been made toward understanding the mechanics of DNA cleavage, a simple and continuous assay for detecting DNA cleavage has been lacking. Herein, we describe the molecular break light assay, wherein a single oligo-nucleotide modified by a 5'-fluorophore-3'-quencher pair adopting a stem-loop structure with an appropriate DNA recognition site, provides for the rapid assaying of DNA cleavage with high sensitivity. Furthermore, the described methodology is highly convenient in that it is readily adaptable to common laboratory fluorometers and multi-well plate/ array systems, which may provide the basis for high-throughput screening of novel DNA cleaving agents. This assay may also be further extended to natural or "unnatural" transcription factor protection assay systems.

DNA↗

PubMed2004

Cold tolerance of Littorinidae from southern Africa: intertidal snails are not constrained to freeze tolerance.

All intertidal gastropods for which cold tolerance strategies have been assessed have been shown to be freeze tolerant. Thus, freeze tolerance is considered an adaptation to the intertidal environment. We investigated the cold tolerance strategies of three species of subtropical and temperate snails (Gastropoda: Littorinidae) to determine whether this group is phylogenetically constrained to freeze tolerance. We exposed 'dry' acclimated and 'wet' rehydrated snails to low temperatures to determine temperature of crystallisation (Tc), lower lethal temperature and LT(50) and to examine the relationship between ice formation and mortality. Tc was lowest in dry Afrolittorina knysnaensis (-13.6+/-0.4 degrees C), followed by dry Echinolittorina natalensis (-10.9+/-0.2 degrees C) and wet A. knysnaensis (-10.2+/-0.2 degrees C) . The Tc of both A. knysnaensis and E. natalensis increased with rehydration, whereas Tc of dry and wet Afrolittorina africana did not differ (-9.6+/-0.2 and -9.0+/-0.2 degrees C respectively). Wet snails of all species exhibited no or low survival of inoculative freezing, whereas dry individuals of A. knysnaensis could survive subzero temperatures above -8 degrees C when freezing was inoculated with ice . In the absence of external ice, Afrolittorina knysnaensis employs a freeze-avoidance strategy of cold tolerance, the first time this has been reported for an intertidal snail, indicating that there is no family-level phylogenetic constraint to freeze tolerance. Echinolittorina natalensis and A. africana both showed pre-freeze mortality and survival of some internal ice formation, but were not cold hardy in any strict sense.

Adaptation, Physiological↗

PubMed2004

Multiplexed genetic analysis using an expanded genetic alphabet.

BACKGROUND: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening. METHODS: MultiCode PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in approximately 3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine and isocytidine. We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns. RESULTS: In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls. CONCLUSIONS: The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting.

Autoanalysis↗

PubMed2004

Nucleic acid analysis using an expanded genetic alphabet to quench fluorescence.

Organic chemistry has made possible the synthesis of molecules that expand on Nature's genetic alphabet. Using the previously described nonstandard DNA base pair constructed from isoguanine and 5-methylisocytosine, we report a highly specific and sensitive method that allows for the fast and specific quantitation of genetic sequences in a closed tube format. During PCR amplification, enzymatic site-specific incorporation of a quencher covalently linked to isoguanine allows for the simultaneous detection and identification of multiple targets. The specificity of method is then established by analysis of thermal denaturation or melting of the amplicons. The appropriate functions of all reactions are further verified by incorporation of an independent target into the reaction mixture. We report that the method is sensitive down to the single copy level, and specificity is demonstrated by multiplexed end-point genotypic analysis of four targets simultaneously using four separate fluorescent reporters. The method is general enough for quantitative and qualitative analysis of both RNA and DNA using previously developed primer sets. Though the method described employs the commonly used PCR, the enzymatic incorporation of reporter groups into DNA site-specifically should find broad utility throughout molecular biology.

Adenosine↗

PubMed2004

A third base pair for the polymerase chain reaction: inserting isoC and isoG.

Two additional bases (isoguanosine and isocytosine), generating a third base pair, have been implemented in PCR. Enzyme fidelity for the third base pair is demonstrated using molecular thermodynamic melting, chemical cleavage and molecular beacons. When amplifying as few as 15 targets containing multiple non-natural base pairs with 40 cycles of amplification, our results confirm sequence conservation. The additional sequence space provided by three base pairs allows for the construction of molecular tools that achieve higher complexity and better discrimination than those possible with natural DNA alone.

Adenosine↗

PubMed2004

Regulated bradycardia in the pulmonate limpet Siphonaria (Gastropoda: Mollusca) during pollutant exposure: implication for biomarker studies.

Although the cardiac activity of invertebrates is now widely used as a biomarker of marine pollution, few studies consider the intrinsic factors causing the variability of heart rate (HR), including the physiological states associated with metabolic depression and behavioural isolation. We examined the cardiac responses to copper exposure of a pulmonate limpet (Siphonaria capensis Quoy and Gaimard), known to adaptively depress heart rate (and metabolic rate) under naturally stressful conditions (hyposalinity). Analysis of variance was used to assess the effects of copper concentration (0-1000 microg/L), exposure time (0-2 h), and individual difference (eight limpets per concentration) on heart rate parameters. Minute by minute heart rate (HR) and heart rate variability (HRV), and bradycardia (< or =10 beats/min) were determined from continuous recording traces. Most of the HR variance was attributable to the interaction of concentration and individuals (35.8%). Considerably lower mean HRs for the higher range of copper concentrations (250 to 1000 microg/L), compared to the lower range (0 to 50 microg/L), were attributable to the virtually exclusive induction of bradycardia in the higher concentration treatments. With increasing concentration, bradycardia was induced sooner and became less interspersed with near normal heart rates (i.e., less episodic). This regulated bradycardial response is apparently associated with isolation (avoidance) behaviour rather than with copper uptake. These findings have implications for biomarker exercises which use heart rate, in cases where invertebrates depress metabolism.

Animals↗

PubMed2004

Latitudinal variation in habitat specificity of ameronothrid mites (Oribatida).

Ameronothroid mites, including Ameronothridae, Fortuyniidae and Selenoribatidae, are unique among the Oribatida through having a global distribution from the tropics to the poles, and occupying a diversity of habitats including terrestrial, marine and freshwater. Their ecological diversification is of considerable interest from both the perspective of evolution over geological timescales, and the detail of the underlying processes. Given their widespread global distribution, it seems likely that historical global events (tectonic and climatic) have played a fundamental role in their ecological diversification. Previous studies of sub-Antarctic island arthropods have generated considerable circumstantial evidence in support of glaciation being a primary factor influencing ecological patterns: lower habitat specificity and weaker interspecific interactions are associated with more recent (postglacial) vegetated terrestrial biotopes, as compared to the older epilithic and littoral biotopes (which are assumed to have been present, albeit reduced in extent, during Neogene glacial maxima). Here, we use ameronothrid mites as a case study to examine the extent to which the above island scenario generalizes globally across latitudes affected by glaciation. We show that, unlike congeners or even conspecifics at lower latitudes in each hemisphere which are restricted to marine environments, the species found at higher latitudes (especially Alaskozetes antarcticus, Ameronothrus dubinini, Ameronothrus lineatus, and Halozetes belgicae) show greater affinity for terrestrial environments. They show a transition or expansion of habitat use (from marine-influenced to terrestrial habitats) implicit with a lower degree of habitat specificity, in relation to increasing latitude. We contend that the terrestrial environment at higher latitudes in both hemispheres has been colonized by these ameronothrid mite species following the various glaciation events, facilitated by a lack of competition experienced in their low diversity communities, in a manner which represents a larger scale demonstration of the processes described on sub-Antarctic islands.

Animals↗

PubMed2003

Imposex in the indigenous Nassarius kraussianus (Mollusca: Neogastropoda) from South African harbours.

Nassarius kraussianus populations in the Durban and Richards Bay harbours (along the eastern seaboard of southern Africa) and in the Knysna lagoon (southern Cape coast) showed imposex development, confirming bioavailability of tributyltin (TBT) in these waterways. Incidence of imposex (which ranged from 29% to 100%) and relative penis length of females (RPL; which ranged from 1.3% to 55.2%) were markedly variable among populations from different sites in each waterway, indicative of localised effects of TBT contamination. Shell length of the snails was apparently not correlated with contamination level, as assessed from imposex measurements. While population imposex features are easy to determine and often provide a sensitive biomarker of TBT exposure, the discussion outlines reservations when extrapolating these to general ecological situations. Additionally, this study represents the first published report on the topic for sub-Saharan, African harbours, some of which seem remarkably less affected than suggested for other world regions.

Animals↗

PubMed2003

Respiratory responses of the mysid Gastrosaccus brevifissura (Peracarida: Mysidacea), in relation to body size, temperature and salinity.

The mysid Gastrosaccus brevifissura (Peracarida: Mysidacea) is widely distributed in southern Africa and is thought to be important in the functioning of estuarine systems. This mysid may experience highly variable physicochemical conditions, and its physiological responses to these are of interest considering its ecological role. This study presents data on the metabolic physiology in relation to body length, temperature (15-30 degrees C) and salinity (15-35 psu) of a G. brevifissura population on the sub-tropical eastern seaboard of South Africa. Oxygen consumption rate was linearly related to size (for body lengths ranging from 3 to 10 mm) and varied among individuals from 0.67 to 6.51 microgram h(-1), dependent on environmental conditions. Oxygen consumption rate was largely independent of salinity variation between 20 and 35 psu, although was significantly depressed at 15 psu. Aerobic rate generally increased with an acute increase in temperature (Q(10)=2.147), but was not affected by 7 days of acclimation at either 15 or 25 degrees C. The lack of a metabolic adjustment to meet the additional energetic demands associated with a decline in salinity may well be a factor limiting the estuarine distribution of G. brevifissura. Even though feeding behaviour substantially changes between summer and winter, this may best be explained by food availability or other ecological factors, rather than a metabolic adjustment, considering the apparent lack of metabolic acclimation.

Aerobiosis↗

PubMed2003

Exploiting the enzymatic recognition of an unnatural base pair to develop a universal genetic analysis system.

BACKGROUND: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target. METHODS: We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses. RESULTS: RNA quantification by reverse transcription-PCR was linear over four orders of magnitude for the simultaneous analysis of beta-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls). CONCLUSION: The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping.

Actins↗