Survey of patient perception of pre-analytical requirements for blood testing in the UK and RoI.

BACKGROUND: A patient survey developed by the Pre-Analytical Phase Special Interest Group of the Association for Clinical Biochemistry and Laboratory Medicine (ACB-PA-SIG) was conducted during November and December 2019. The survey aimed to determine the quality of information provided to patients in preparation for their blood test(s). In addition, the ACB-PA-SIG provide a number of recommendations, which, if adopted, may yield higher quality test results and improve patient management. METHODS: The survey was distributed at phlebotomy suites in two Hospitals: Ipswich Hospital (United Kingdom [UK]), and Cork University Hospital (Republic of Ireland [RoI]). RESULTS: Overall, 235 survey responses were received from the two sites. A total of 103 respondents received no information about preparing for their blood test and 92 had been told they did not need to fast. None of the patients surveyed had been instructed to fast for 12 h. Twenty-two patients had been told to avoid certain foods, drinks or medication, 14 were told to avoid strenuous activity and 41 respondents had been informed of the need to avoid alcohol/smoking prior to their blood test. Overall, only approximately 78 felt well informed about the blood taking process. CONCLUSIONS: Based on the results of this survey, the ACB-PA-SIG conclude that: (1) clinicians should provide clear written information to patients regarding pre-analytical requirements; and (2) effective communication between laboratories and General Practitioners is required to disseminate information. In this paper, the ACB-PA-SIG provide a list of pre-analytical recommendations to standardize and improve practice across the UK and RoI.

No consistent evidence for association between mtDNA variants and Alzheimer disease.

OBJECTIVE: Although several studies have described an association between Alzheimer disease (AD) and genetic variation of mitochondrial DNA (mtDNA), each has implicated different mtDNA variants, so the role of mtDNA in the etiology of AD remains uncertain. METHODS: We tested 138 mtDNA variants for association with AD in a powerful sample of 4,133 AD case patients and 1,602 matched controls from 3 Caucasian populations. Of the total population, 3,250 case patients and 1,221 elderly controls met the quality control criteria and were included in the analysis. RESULTS: In the largest study to date, we failed to replicate the published findings. Meta-analysis of the available data showed no evidence of an association with AD. CONCLUSION: The current evidence linking common mtDNA variations with AD is not compelling.

Genome-wide association study of Alzheimer's disease with psychotic symptoms.

Psychotic symptoms occur in ~40% of subjects with Alzheimer's disease (AD) and are associated with more rapid cognitive decline and increased functional deficits. They show heritability up to 61% and have been proposed as a marker for a disease subtype suitable for gene mapping efforts. We undertook a combined analysis of three genome-wide association studies (GWASs) to identify loci that (1) increase susceptibility to an AD and subsequent psychotic symptoms; or (2) modify risk of psychotic symptoms in the presence of neurodegeneration caused by AD. In all, 1299 AD cases with psychosis (AD+P), 735 AD cases without psychosis (AD-P) and 5659 controls were drawn from Genetic and Environmental Risk in AD Consortium 1 (GERAD1), the National Institute on Aging Late-Onset Alzheimer's Disease (NIA-LOAD) family study and the University of Pittsburgh Alzheimer Disease Research Center (ADRC) GWASs. Unobserved genotypes were imputed to provide data on >1.8 million single-nucleotide polymorphisms (SNPs). Analyses in each data set were completed comparing (1) AD+P to AD-P cases, and (2) AD+P cases with controls (GERAD1, ADRC only). Aside from the apolipoprotein E (APOE) locus, the strongest evidence for association was observed in an intergenic region on chromosome 4 (rs753129; 'AD+PvAD-P' P=2.85 × 10(-7); 'AD+PvControls' P=1.11 × 10(-4)). SNPs upstream of SLC2A9 (rs6834555, P=3.0 × 10(-7)) and within VSNL1 (rs4038131, P=5.9 × 10(-7)) showed strongest evidence for association with AD+P when compared with controls. These findings warrant further investigation in larger, appropriately powered samples in which the presence of psychotic symptoms in AD has been well characterized.

The number of morphological synapses between neurons does not predict the strength of their physiological synaptic interactions: a study of dendrites in the nematode Ascaris suum.

Previous electrophysiological and anatomical studies of Ascaris suum motor neurons demonstrated a strong correlation between functional interactions and the presence of anatomically defined synapses. However, one example of a physiologically robust synaptic connection was encountered for which no anatomical evidence of direct chemical synapses was found. This involved synaptic transmission from an identified excitatory motor neuron to its inhibitory partner. In this study, pressure injection of horseradish peroxidase or nickel lysine into inhibitory motor neurons revealed numerous spines projecting from the main neuronal process toward the neuromuscular surface that then branched and extended fine, longitudinal processes up to 130 microm in length. Subsequent examination of nickel lysine-injected spines by electron microscopy revealed numerous chemical synapses, including for the first time direct inputs from the excitatory neuron. However, the numbers of synapses from this motor neuron were very small relative to inputs from other identified cells. Thus, direct synapses are unlikely to explain the robust nature of this physiological interaction.

Structure-activity relationships of 18 endogenous neuropeptides on the motor nervous system of the nematode Ascaris suum.

Neuropeptides play an important role in all nervous systems and structure-activity studies of related peptides is one approach to understanding this role. This study of the motor nervous system of the parasitic nematode Ascaris suum describes the physiological effects of a family of 18 endogenous Ascaris FMRFamide-like peptides (AF peptides) on the membrane potential and input resistance of the dorsal excitatory type 2 (DE2) and dorsal inhibitory (DI) motor neurons. These motor neurons are part of the final common output pathway from the motor nervous system to the somatic muscle cells responsible for locomotion. AF peptide effects on the frequency of excitatory postsynaptic potentials (EPSPs) in DE2 motor neurons were also measured to infer peptide effects on central presynaptic spiking neurons. AF peptide injections into intact worms were made to assess their qualitative effects on behavior, providing a context for interpreting motor neuron data. One category of AF peptides, N-terminally extended -FIRFa peptides (AF5, AF7 and AF1), has pronounced behavioral effects and qualitatively similar, but quantitatively different effects on DE2 and DI motor neurons. A second category of AF peptides (AF2, AF9, and AF8) also produces dramatic behavioral effects and strong electrophysiological effects on DE2 and/or DI motor neurons. A third category of AF peptides, consisting of six members of the -PGVLRFa group (which are encoded by the same gene and have closely related sequences) and peptide AF11, have pronounced behavioral effects, but relatively weak or negligible effects on DE2 and DI motor neurons. A fourth category of AF peptides, also consisting of structurally unrelated members, has pronounced behavioral effects and, as individual peptides, similar effects on both DE2 and DI motor neurons; AF15 is excitatory, while AF17 and AF19 are inhibitory, on both motor neuron types. Finally, two AF peptides (AF6, AF16) are relatively weak or inactive in producing behavioral or motor neuronal effects. Based on comparisons of the effects of AF peptides on DE2 and DI motor neurons, a tentative list of 5 major response-types is proposed as a working hypothesis to guide the search for AF peptide receptors. The findings attest to the potential complexity of neurosignaling in this comparatively simple nervous system.

Changes in locomotory behavior and cAMP produced in Ascaris suum by neuropeptides from Ascaris suum or Caenorhabditis elegans.

Injection of Ascaris FMRFamide-like (AF) peptides and peptides encoded by genes in Caenorhabditis elegans were analyzed for effects on locomotion, body waveforms, and cAMP concentrations in adult female Ascaris suum. Injection of AF1 (KNEFIRFamide) or AF2 (KHEYLRFamide) inhibited the propagation of locomotory waves and reduced the number of waveforms, decreased the body length, and caused a large, long-lasting increase in cAMP. Muscle tissue was identified as a major source of the cAMP response induced by AF1. The AF1 analog AF1R6A did not affect cAMP levels by itself, but inhibited the cAMP response produced by AF1. AF8 (KSAYMRFamide) produced ventral coiling in the behavioral assay, and AF10 (GFGDEMSMPGVLRFamide) decreased the body length and increased the number of body waveforms. In dorsal muscle strips, AF10 produced a long-lasting contraction. Neither AF8 nor AF10 changed cAMP concentrations. AF17 (FDRDFMHFamide) increased body length and decreased cAMP. The neuropeptides encoded by C. elegans genes flp-4, flp-7, flp-9, and flp-13 produced paralysis and loss of waveforms, increased body length and, like AF17, decreased cAMP. Three new predicted peptides from C. elegans genome sequences were synthesized and tested. One produced ventral coiling but no change in cAMP; the other two gave no detectable responses. The fact that C. elegans neuropeptides produce behavioral and physiological effects in A. suum suggests that structurally related peptides may exist in A. suum. The profound changes in cAMP produced by some neuropeptides has important implications for understanding cAMP signaling and shows that neuropeptide-mediated signal transduction pathways are potential targets for anthelmintic drug development.

afp-1: a gene encoding multiple transcripts of a new class of FMRFamide-like neuropeptides in the nematode Ascaris suum.

We have identified a gene, afp-1, that encodes a new subfamily of six FMRFamide-like neuropeptides in the nematode Ascaris suum. The predicted peptides share the C-terminal sequence PGVLRF-NH2 but have different N-terminal extensions. We discuss possible functional roles of these different peptides based upon experiments with Ascaris as well as results from other organisms. Three of the peptides were previously isolated from extracts of A. suum (4) and three other are novel sequences. The translated product of afp-1 is a precursor protein containing two main halves: a C-terminal region containing a series of putative peptides separated by characteristic processing sites and a relatively hydrophobic N-terminal region with no obvious peptides. Although the overall structure of the translated product of afp-1 is similar to flp-1 from C. elegans (18), there is little evidence for homology between the two nematode neuropeptide genes. At least four different transcripts of afp-1 have been identified. These transcripts differ in their 3' and 5' untranslated regions, and one of the transcripts predicts a truncated precursor protein which contains only the C-terminal peptide-containing region.

Heterogeneity of cholecystokinin/gastrin-like immunoreactivity in the nervous system of the nematode Ascaris suum.

A wholemount immunocytochemical method was used for the localization of cholecystokinin (CCK8)-like and gastrin-like immunoreactivity in Ascaris. The patterns of specific neuronal staining given by two antisera and four monoclonal antibodies made against CCK8, and one antiserum made against gastrin were investigated. Preabsorption of these antibodies with CCK8 or gastrin 17 resulted in complete loss of immunoreactivity in almost all of the neurons (two antisera also contained nonspecific antibodies), suggesting that all of the antibodies recognize epitopes, in Ascaris neurons, that include some or all of the C-terminal five amino acids that are identical in CCK8 and gastrin 17. However, the seven different antibodies showed immunoreactivity in different subpopulations of neurons, implying that there are at least seven different species of CCK-like molecules in Ascaris. Fractionation of Ascaris peptide extracts by high performance liquid chromatography (HPLC), monitoring fractions with a CCK8 radioimmunoassay (RIA), also shows heterogeneity of molecules immunologically related to CCK8.

Behavioral and cellular effects of serotonin on locomotion and male mating posture in Ascaris suum (Nematoda).

The site and mode of action of serotonin on locomotion were investigated in the parasitic nematode Ascaris suum. Injection of serotonin into Ascaris immediately caused paralysis in animals that were generating locomotory waveforms. Injected serotonin also increased body length and decreased the number of propagating body waves. Similar injections into the male tail produced a ventral tail curl. Injection of N-acetyl-serotonin had no effect on the generation of locomotory waveforms, but increased the body length and decreased the number of body waves in the waveform. Other biogenic amines were also tested but were much less potent. Serotonin decreased the amplitude of a submaximal acetylcholine-induced muscle contraction and increased the time to attain this contraction. The time course of this effect on the response to ACh was much slower than the action of injected serotonin on locomotory waveforms, suggesting that additional elements are involved in the action of serotonin on locomotory behavior. Serotonin abolished spontaneous slow potentials in VI motor neurons and decreased the frequency of EPSPs in DE2 motor neurons, probably by a pre-synaptic mechanism. In the male tail, serotonin depolarized the male-specific transverse ventral muscle cells, but did not affect either dorsal or ventral longitudinal muscle cells.

Neuronal localization of serotonin in the nematode Ascaris suum.

We have used immunocytochemical techniques to investigate the distribution of serotonin-like immunoreactivity in the nematode Ascaris suum. Antisera raised against serotonin (5-hydroxytryptamine, 5-HT) conjugated to bovine serum albumin (BSA) labelled a pair of neurons in the pharynx of both sexes and five cells in the ventral cord of the male tail. The labelling was blocked by 5-HT or by 5-HT conjugated to BSA. The 5-HT-immunoreactive cells in the pharynx resemble neurosecretory cells and are probably homologous to the neurosecretory motor neurons (NSM) in Caenorhabditis elegans; the cells in the male tail appear to be motor neurons that are homologous to CP neurons in C. elegans. Other cells that stain with 5-HT antisera have been observed in C. elegans but are not seen in Ascaris.

The motornervous system of Ascaris: electrophysiology and anatomy of the neurons and their control by neuromodulators.

Analysis of the electrical properties of neurons in the motornervous system of Ascaris sutom suggests that it is largely an analogue system. The motorneurons do not conduct action potentials and they release transmitter tonically at their normal resting potential; transmitter release is increased or decreased as a continuous function of membrane potential. Despite extensive physiological descriptions of the electrical properties of the neurons and their synapses, as well as morphological descriptions of the synaptic circuitry of the system, the predicted activities of the neurons in the circuit differ from those observed by direct recording in semi-intact behaving animals. We conclude that the description of the circuit is incomplete. There are several possibilities for the missing elements, including chemical, proprioceptive, and additional neuronal components. Recently, attention has been focussed most heavily on the intercellular chemical signalling systems; in addition to those mediated by classical neurotransmitters, a surprisingly complex array of neuropeptides has been identified. One family of these peptides, the AF peptides, has been analyzed in detail. It comprises at least 20 peptides, and they fall into sequence-related subfamilies. One of these subfamilies, containing 6 peptides, is encoded by a single transcript, suggesting that the AF peptides are under multiple genetic control. All AF peptides tested have potent activity on the motornervous system and/or on muscle. There are multiple physiological activities, and cellular localization studies show multiple patterns of cellular expression. Studies on Panagrellus and Caenorhabditis emphasize the diversity of this family and its genetic control.

Eight novel FMRFamide-like neuropeptides isolated from the nematode Ascaris suum.

Eight FMRFamide-like neuropeptides were isolated from an extract of heads and tails from the nematode Ascaris suum using seven steps of HPLC. The peptides ranged in size from 8 to 14 amino acid residues: AF3 (Ala-Val-Pro-Gly-Val-Leu-Arg-Phe-amide), AF4 (Gly-Asp-Val-Pro-Gly-Val-Leu-Arg-Phe-amide), AF5 (Ser-Gly-Lys-Pro-Thr-Phe-Ile-Arg-Phe-amide), AF7 (Ala-Gly-Pro-Arg-Phe-Ile-Arg-Phe-amide), AF9 (Gly-Leu-Gly-Pro-Arg-Pro-Leu-Arg-Phe-amide), AF10 (Gly-Phe-Gly-Asp-Glu-Met-Ser-Met-Pro-Gly-Val-Leu-Arg-Phe-amide), AF11 (Ser-Asp-Ile-Gly-Ile-Ser-Glu-Pro-Asn-Phe-Leu-Arg-Phe-amide), and AF12 (Phe-Gly-Asp-Glu-Met-Ser-Met-Pro-Gly-Val-Leu-Arg-Phe-amide). The effect of synthetic AF4 on muscle tension in a dorsal muscle strip preparation was a strong, long-lasting contraction. PF1, a peptide present in Panagrellus redivivus and Caenorhabditis elegans, relaxed the AF4-induced contraction.

Localization and differential expression of FMRFamide-like immunoreactivity in the nematode Ascaris suum.

By immunocytochemical and immunohistochemical methods, FMRFamide-like immunoreactivity (FLI) was localized to many neurons and processes in the Ascaris nervous system, including the head, tail, and lateral lines. Some of these cells were identified; they included sensory neurons, interneurons, and motor neurons. FLI was also present in the pharyngeal neurons and in their varicosities near the surface of the pharynx. By HPLC analysis of extracts, only a subset of the FMRFamide-like peptides (FLPs) expressed in Ascaris heads, and heads from which the pharynx had been removed, were expressed in the pharynx. Furthermore, FLPs appeared to be differentially expressed in female heads and tails and male heads and tails. Acetone and acid methanol differentially extracted subforms of FLI from Ascaris heads and from C. elegans.

AF2, an Ascaris neuropeptide: isolation, sequence, and bioactivity.

A FMRFamide-like neuropeptide, KHEYLRFamide (Lys-His-Glu-Tyr-Leu-Arg-Phe-amide; AF2) was isolated from a head extract of the nematode Ascaris suum by using a three-step HPLC separation. In a dorsal muscle strip preparation, synthetic AF2 produced multiple effects on muscle tension: a slow relaxation was followed by contraction and rhythmic activity. Sulfated AF2 was no more potent than AF2. The effects on muscle tension were correlated with electrical activity recorded intracellularly from muscle cells. AF2 markedly increased the tension change associated with change in muscle membrane potential.

Actions of cholinergic drugs in the nematode Ascaris suum. Complex pharmacology of muscle and motorneurons.

The cholinergic agonists acetylcholine (ACh), nicotine, and pilocarpine produced depolarizations and contractions of muscle of the nematode Ascaris suum. Dose-dependent depolarization and contraction by ACh were suppressed by about two orders of magnitude by 100 microM d-tubocurarine (dTC), a nicotinic antagonist, but only about fivefold by 100 microM N-methyl-scopolamine (NMS), a muscarinic antagonist. NMS itself depolarized both normal and synaptically isolated muscle cells. The muscle depolarizing action of pilocarpine was not consistently antagonized by either NMS or dTC. ACh receptors were detected on motorneuron classes DE1, DE2, DI, and VI as ACh-induced reductions in input resistance. These input resistance changes were reversed by washing in drug-free saline or by application of dTC. NMS applied alone lowered input resistance in DE1, but not in DE2, DI, or VI motorneurons. In contrast to the effect of ACh, the action of NMS in DE1 was not reversed by dTC, suggesting that NMS-sensitive sites may not respond to ACh. Excitatory synaptic responses in muscle evoked by depolarizing current injections into DE1 and DE2 motorneurons were antagonized by dTC; however, NMS antagonized the synaptic output of only the DE1 and DE3 classes of motorneurons, an effect that was more likely to have been produced by motorneuron conduction failure than by pharmacological blockade of receptor. The concentration of NMS required to produce these changes in muscle polarization and contraction, ACh antagonism, input resistance reduction, and synaptic antagonism was 100 microM, or more than five orders of magnitude higher than the binding affinity for [3H]NMS in larval Ascaris homogenates and adult Caenorhabditis elegans (Segerberg, M. A. 1989. Ph.D. thesis. University of Wisconsin-Madison, Madison, WI). These results describe a nicotinic-like pharmacology, but muscle and motorneurons also have unusual responses to muscarinic agents.

Extracellular recordings from the motor nervous system of the nematode, Ascaris suum.

1. The close association of muscle and neurons in Ascaris suum makes it difficult to determine whether spikes recorded from nerve cords originate in muscle or neurons. We have developed criteria that distinguish muscle and neuronal activity. There are two categories of extracellular spikes. 2. The first category consists of spikes with a wide range of amplitudes, marked by large spikes. These spikes, which can be recorded over lateral muscle and over the dorsal and ventral nerve cords, are abolished when muscle is disrupted or removed, or when curare is applied. Large spikes are relatively infrequent, are correlated with intracellularly recorded muscle events, and respond to polarizations of motor neurons, implying that they originate in muscle. 3. The second spike category, small amplitude spikes, is exclusive to the ventral nerve cord, occurs more frequently than large spikes and displays patterned firing. Small spikes are not affected by muscle removal or by curare, and are correlated with motor neuronal post-synaptic potentials, but not with intracellularly recorded muscle events. We infer that they originate in neurons. 4. Low level activity recorded extracellularly over nerve cords may represent muscle activity due to tonic motor neuronal synaptic transmission. It responds to motor neuronal polarization and is suppressed by curare or muscle removal.

Motor behavior and motor nervous system function in the nematode Ascaris suum.

Ascaris suum has a nervous system that is very simple both numerically and morphologically. It comprises only 298 neurons almost all of which are extremely simple in shape. Extensive anatomical descriptions of the morphology of neurons and of their synaptic connections, together with the study, by using intracellular recording techniques, of their physiological properties, have led to a prediction of how the motor nervous system controls behavior. Subsequent discovery of endogenous neuropeptides that have potent activity on subsets of the motor neurons suggests that the description of the motor circuitry is more complex than is apparent from its anatomy.

A versatile dot-ELISA method with femtomole sensitivity for detecting small peptides.

Several protocols for conjugating peptides in situ to a protein carrier on paper, nitrocellulose, or nylon membranes were explored for their usefulness in dot-ELISA detection of the peptides. The most sensitive method in which peptide diluted in bovine serum albumin is applied to nitrocellulose, then fixed with glutaraldehyde, can detect several peptides, ranging from 4 to 38 amino acids in length, at the level of 2-10 fmol. Both immunohistochemical grade antisera and monoclonal antibodies have been used successfully. The method may be a useful alternative to radioimmunoassay since there is no requirement for radiolabelled peptide, or (for quantitation) for known quantities of unlabelled peptide. The method has been used to monitor, semiquantitatively, the fractionation of FMRFamide-like or CCK-like peptides from the nematode Ascaris, and to detect peptide-like immunoreactivities in tissue extracts.

GABA-immunoreactive neurons in the nematode Ascaris.

gamma-Aminobutyric acid (GABA) immunoreactive neurons in the cephalic, somatic, and caudal regions of the Ascaris nervous system were visualized with serial section and whole-mount GABA immunocytochemistry. In the ventral and dorsal nerve cords, GABA-like immunoreactivity (GLIR) is localized to the neurites and cell bodies of identified inhibitory motor neurons and to two fibers, one in each cord, that arise from neurons in the nerve ring. GLIR is absent from identified excitatory motor neurons and from ventral cord interneurons. In neurons containing GLIR, immunoreactivity was present throughout the cell, which argues against an exclusive localization of GABA at conventional synapses. In whole mounts, ten GABA-immunoreactive neurons were present in the cephalic region. These include four nerve ring-associated cells (the RME-like cells), two bilaterally symmetrical pairs of lateral ganglia neurons (the amphid-GABA and deirid-GABA cells) and one bilaterally symmetrical pair of ventral ganglion cells (the VG-GABA cells). In sections, the RME-like cells and the VG-GABA cells were consistently stained through the cephalic region. However, anti-GABA staining of the lateral ganglia cells in sections was light, thus suggesting that they contain less GLIR than the other more intensely stained GABA-immunoreactive neurons. In the caudal region, a single GABA-immunoreactive neuron was present in the dorsal rectal ganglion. Our data suggest that these ten cephalic neurons, and a single dorsal rectal ganglion neuron, use GABA as a neurotransmitter.

Distribution of 3H-GABA uptake sites in the nematode Ascaris.

The distribution of uptake sites for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the nematode Ascaris suum was examined by autoradiography of 3H-GABA uptake. Single neural processes in both the ventral and dorsal nerve cords were labeled with 3H-GABA. Serial section analysis identified the cells of origin of these processes as the RMEV-like and RMED-like neurons. These cells belong to a set of four neurons in the nerve ring, all of which are labeled by 3H-GABA. 3H-GABA labeling of at least two other sets of cephalic neurons was seen. One of these pairs consists of medium-sized lateral ganglia neurons, located at the level of the amphid commissure bundle. A second pair is located in the lateral ganglia at the level of the deirid commissure bundle. The position and size of these lateral ganglia cells suggest that they are the GABA-immunoreactive lateral ganglia cells frequently seen in whole-mount immunocytochemical preparations (Guastella et al., J Comp Neurol 307:584-597, 1991). Four neuronal cell bodies located in the retrovesicular ganglion were also labeled with 3H-GABA. These cells, which are probably cholinergic excitatory motor neurons, do not contain detectable GABA-like immunoreactivity. Heavy labeling of muscle cells was also observed. The ventral and dorsal nerve cord inhibitory motor neurons, which are known to contain GABA-like immunoreactivity, were not labeled above background with 3H-GABA. Together with the experiments reported previously (Guastella et al., J Comp Neurol 307:584-597, 1991), these results define three classes of GABA-associated neurons in Ascaris: 1) neurons that contain endogenous GABA and possess a GABA uptake system; 2) neurons that contain endogenous GABA, but that either lack a GABA uptake system or possess a GABA uptake system of low activity; 3) neurons that possess a GABA uptake system, but that lack endogenous GABA.