Superior Multiplexing Capacity of PlexPrimers Enables Sensitive and Specific Detection of SNPs and Clustered Mutations in qPCR.

BACKGROUND: Whilst qPCR provides an extremely powerful tool for genetic analysis, some applications such as multiplexing variant alleles (eg SNPs, point mutations or deletions), remain challenging using current primer/probe systems. The novel design features of PlexPrimers allow sensitive, multiplexed analysis of variant alleles even when these are tightly clustered. METHOD: PlexPrimers were combined with PlexZymes in qPCR assays for the detection of SNPs in human absorption, distribution, metabolism, and excretion (ADME) genes; clustered mutations in the 23S rRNA gene which confer antibiotic resistance to Mycoplasma genitalium; and deletions within the human epidermal growth factor receptor (EGFR) gene. RESULTS: The combination of PlexPrimers and PlexZymes allowed robust multiplexing of targets which resulted in 100% concordance with results obtained using hydrolysis probe kits for 14 SNPs in the ADME genes. A 7-plex qPCR assay targeting M. genitalium, 5 clustered mutations associated with macrolide resistance and an internal control, allowed efficient amplification of all targets, with all 5 mutations detected in a single channel. Finally, the strategy was employed to analyse common EGFR mutants with high sensitivity, detecting deletions present at only 0.01%. CONCLUSION: PlexPrime is a novel technology for the detection of genetic variants. Unlike previous strategies, the combination of PlexPrimers with PlexZymes enables both allele-specific detection and allele-specific amplification in qPCR. The study demonstrated highly sensitive and specific detection of mutations and SNPs, and superior multiplexing capacity. The ability to multiplex clustered genetic variants reduces the time to result providing more actionable information.

EzyAmp signal amplification cascade enables isothermal detection of nucleic acid and protein targets.

Advancements in molecular biology have improved the ability to characterize disease-related nucleic acids and proteins. Recently, there has been an increasing desire for tests that can be performed outside of centralised laboratories. This study describes a novel isothermal signal amplification cascade called EzyAmp (enzymatic signal amplification) that is being developed for detection of targets at the point of care. EzyAmp exploits the ability of some restriction endonucleases to cleave substrates containing nicks within their recognition sites. EzyAmp uses two oligonucleotide duplexes (partial complexes 1 and 2) which are initially cleavage-resistant as they lack a complete recognition site. The recognition site of partial complex 1 can be completed by hybridization of a triggering oligonucleotide (Driver Fragment 1) that is generated by a target-specific initiation event. Binding of Driver Fragment 1 generates a completed complex 1, which upon cleavage, releases Driver Fragment 2. In turn, binding of Driver Fragment 2 to partial complex 2 creates completed complex 2 which when cleaved releases additional Driver Fragment 1. Each cleavage event separates fluorophore quencher pairs resulting in an increase in fluorescence. At this stage a cascade of signal production becomes independent of further target-specific initiation events. This study demonstrated that the EzyAmp cascade can facilitate detection and quantification of nucleic acid targets with sensitivity down to aM concentration. Further, the same cascade detected VEGF protein with a sensitivity of 20nM showing that this universal method for amplifying signal may be linked to the detection of different types of analytes in an isothermal format.

Distribution and neurochemical characterization of neurons in the rat ventrolateral medulla activated by glucoprivation.

Hypoglycemia elicits physiological and behavioral responses which are mediated in part by neurons within the ventrolateral medulla (VLM). The present study describes the neurochemistry of neurons activated by glucoprivation (2-deoxy-D-glucose, 2DG), specifically those within regions containing the A1, caudal C1 (cC1) and rostral C1 (rC1) cell groups. 2DG induced c-Fos immunoreactivity throughout the VLM. Activated neurons expressing prepro-cocaine and amphetamine-regulated transcript (PPCART), neuropeptide Y (NPY), glutamic acid decarboxylase (GAD67) or prepro-enkephalin (PPE) mRNA and/or immunoreactivity (-ir) for tyrosine hydroxylase (TH) were identified. TH(+) neurons were recruited in a dose-dependent manner. At high doses of 2DG [400 mg/kg, (n = 6)], 76 ± 1.2 % of activated neurons were TH(+) representing 52 ± 1.3 % of the total TH population. Virtually all activated neurons in the A1 and cC1 regions but only 60 % in the rC1 region were TH(+). Within the A1 region, TH(+), TH(+)NPY(+) and TH(+)NPY(+)PPE(+) subpopulations were activated and likely regulate vasopressin, oxytocin, and corticotrophin releasing hormone (CRH) from the hypothalamus. Within the cC1 region, non-TH neurons, TH(+)NPY(+), TH(+)NPY(+)PPCART(+), and TH(+)NPY(+)PPE(+) subpopulations were activated, likely regulating autonomic hypothalamic neurons or CRH and thyrotropin releasing hormone secretion. Within the rC1 region, non-TH neurons (40 % of those activated) were predominantly PPE(+) and were recruited by higher 2DG doses. Of the TH(+) activated neurons in the rC1 region, many expressed PPCART and half expressed NPY. The activated spinally projecting population was almost entirely TH(+)PPCART(+) and is likely to regulate adrenaline and glucagon release. These data indicate that glucoprivation activates at least nine phenotypically distinct populations of neurons in the VLM.

Neurochemical codes of sympathetic preganglionic neurons activated by glucoprivation.

Glucoprivation or hypoglycemia induces a range of counterregulatory responses, including glucose mobilization, reduced glucose utilization, and de novo glucose synthesis. These responses are mediated in part by the sympathetic nervous system. The aim of this study was to determine the chemical codes of sympathetic preganglionic neurons (SPN) activated by glucoprivation, induced by 2-deoxy-D-glucose (2DG). SPN controlling the adrenal glands and celiac ganglia, which ultimately can innervate the liver and pancreas, were targeted together with the superior cervical ganglia (control). 23.9% ± 1.3% of SPN in the T4-T11 region contained c-Fos immunoreactivity following 2DG; 70.3% ± 1.8% of SPN innervating the adrenal glands and 37.4% ± 3% of SPN innervating celiac ganglia were activated. 14.8% ± 3.5% of SPN (C8-T3) innervating superior cervical ganglia were activated. In the C8-T3 region 55% ± 10% of SPN activated contained PPCART, with only 12% ± 3% expressing PPE mRNA, whereas, in the T4-T11 region, 78% ± 4% contained PPE, with only 6.0% ± 0.6% expressing PPCART mRNA. Thus CART is not involved in glucose mobilization. Two chemically distinct populations of SPN (PPE⁺ 57.4% ± 5%, PPE⁻ ∼40%) were identified to regulate adrenaline release in response to glucoprivation. Multiple chemically distinct SPN populations innervating a specific target could suggest their graded recruitment. The two distinct populations of SPN (PPE⁺ 67.6% ± 9%, PPE⁻ ∼30%) projecting to celiac ganglia activated by glucoprivation could direct pancreatic and hepatic or other counterregulatory responses. Nearly all SPN that expressed PPE mRNA and projected to the adrenal glands or celiac ganglia were activated, suggesting a role for the inhibitory peptide enkephalin in responses evoked by glucoprivation.

Neuropeptide Y in the rostral ventrolateral medulla blocks somatosympathetic reflexes in anesthetized rats.

The rostral ventrolateral medulla (RVLM) is a major integrative center of cardiovascular reflexes that modulate vasomotor tone. The functions of Neuropeptide Y (NPY) in the RVLM on cardiorespiratory responses remain unknown. Arterial blood pressure (AP), heart rate (HR), splanchnic sympathetic (sSNA) and phrenic nerve activities, and responsiveness to baro-, somatosympathetic, and chemoreflex stimulation were recorded before and after bilateral NPY injection (100 pmol, 200 nl/side) in the RVLM of vagotomized urethane-anesthetized rats (n=7). Responses were characterized by an initial increase in AP followed by prolonged hypotension (P<0.01). A similar biphasic effect was exerted on HR (P<0.01). NPY caused a large increase of sSNA (P<0.01), that gradually recovered towards baseline. Somatosympathetic responses evoked by tibial nerve stimulation were largely abolished following NPY microinjection (P<0.01), but sympathoexcitatory responses evoked by acute hypoxia or sympathoinhibition evoked by aortic depressor nerve stimulation were unchanged following NPY. There was no effect of NPY on phrenic nerve amplitude or frequency. We conclude that NPY exerts excitatory effects on sympathetic tone, but inhibits responses evoked by somatic inputs. We speculate that this apparent contradiction may be due to differential expression of NPY receptor subtypes on the soma of sympathetic premotor neurons in the RVLM and on the presynaptic terminals of neurons that comprise excitatory afferent pathways.

Metabotropic neurotransmission and integration of sympathetic nerve activity by the rostral ventrolateral medulla in the rat.

1. Cardiovascular sympathetic nerve activity at rest is grouped into waves, or bursts, that are generally, although not exclusively, related to the heart rate and to respiration. In addition, activity is also generated in response to central commands and to environmental stimuli. 2. Responsibility for the integration of all these different elements of sympathetic activity rests with pre-motoneurons in the rostral ventrolateral medulla oblongata. These pre-motoneurons are glutamatergic and spinally projecting where they form synapses with sympathetic preganglionic neurons. 3. Pre-motoneurons also contain and presumably release, neurotransmitters other than glutamate, including amines and neuropeptides that act on metabotropic receptors with long-term effects on cell function. 4. Similarly, in the rostral ventrolateral medulla oblongata the pre-motoneurons are mainly regulated by excitatory influences from glutamate and inhibitory influences from gamma-aminobutyric acid (GABA). Major focuses of recent studies are the interactions between non-glutamatergic and GABAergic systems and reflexes that regulate the activity of the sympathetic nervous system. 5. The results indicate that neurotransmitters acting at metabotropic receptors selectively affect different reflexes in the rostral ventrolateral medulla. It is suggested that this differential activation or attenuation of reflexes by different neurotransmitters is a mechanism by which the organism can fine-tune its responses to different homeostatic requirements.

Significance of multiple neurochemicals that regulate respiration.

Current efforts to characterize the neuronal mechanisms that underlie automatic breathing generally adopt a 'minimalist' approach. In this review, we survey three of the many neurochemicals that are known to be present in raphe neurons and may be involved in respiration. Specifically, we ask the question, 'Is the minimalist approach consistent with the large number of neuronal types and neurochemicals found in respiratory centres'?

Targeting brain stem centers of cardiovascular control using adenoviral vectors: impact of promoters on transgene expression.

Adenoviral vectors (AVV) are widely used as tools for exploring gene function in studies of the central autonomic control, but the cellular specificity of the promoters commonly used in these vectors has not been studied. We evaluated AVV with four "wide-spectrum" promoters, human cytomegalovirus promoter (HCMV), synapsin-1 promoter (Syn1), tubulin-alpha1 promoter (Talpha1), and neuron-specific enolase (NSE) for their ability to express enhanced green fluorescent protein (EGFP) within the dorsal vagal complex and the adjacent brain stem. They were compared with the PRSx8 promoter, specifically designed to target catecholaminergic neurons. AdHCMVEGFP, AdSyn1EGFP-WHE (woodchuck hepatitis enhancer element), AdTalpha1EGFP, and AdNSEEGFP were unable to drive expression of EGFP in dopamine beta-hydroxylase-immunoreactive neurons of the A2 cell group, although the adjacent dorsal vagal motonucleus and especially hypoglossal motoneurons did express high levels of EGFP. AdPRSx8EGFP efficiently drove EGFP expression in the A2 cell group but also in choline acetyltransferase-positive vagal motoneurons. However, catecholaminergic neurons could be selectively and efficiently transduced via a retrograde route by injecting the vector into their target areas. Thus AVV with "wide-spectrum" promoters have strikingly different activity in the diverse cellular populations within brain stem cardiovascular control centers. The PRSx8 promoter is a valuable tool for the study of the role of catecholaminergic neurons.

Mu opioid receptors in rat ventral medulla: effects of endomorphin-1 on phrenic nerve activity.

Anatomical and in vitro studies suggest that mu opioid receptors (MOR) on pre-Bötzinger complex neurons are responsible for opioid induced respiratory depression (Grey et al., Science 286 (1999) 1566). However, mu opioid agonists injected in vivo, in other regions of the ventral respiratory group (VRG), produce respiratory depression, suggesting that opioids are widely distributed in the VRG. We therefore re-examined the distribution of the MOR in the ventral medulla and found MOR-immunoreactive neurons and terminals in all subdivisions of the VRG. Furthermore, we determined, in rats, the effects of a MOR agonist (endomorphin-1, 10 mM, 60 nl, unilateral), microinjected into different subdivisions of the VRG, on phrenic nerve activity. Endomorphin-1 produced changes in phrenic nerve frequency and amplitude, throughout the VRG. Unexpectedly, endomorphin-1 microinjected into the Bötzinger and pre-Bötzinger complexes consistently increased phrenic nerve frequency. These results support the widespread distribution of MOR in the VRG and also indicate that endomorphin-1, a postulated endogenous ligand, may differentially regulate respiration.