Temporal unpredictability increases error monitoring as revealed by EEG-EMG investigation.

Reacting in an unpredictable context increases error monitoring as evidenced by greater error-related negativity (ERN), an electrophysiological marker linked to an evaluation of response outcomes. We investigated whether ERN also increased when participants evaluated their responses to events that appeared in unpredictable versus predictable moments in time. We complemented electroencephalographic (EEG) analysis of cortical activity by measuring performance monitoring processes at the peripheral level using electromyography (EMG). Specifically, we used EMG data to quantify how temporal unpredictability would affect motor time (MT), the interval between the onset of muscle activity, and the mechanical response. MT increases following errors, indexing online error detection, and an attempt to stop incorrect actions. In our temporally cued version of the stop-signal task, symbolic cues predicted (temporally predictable condition) or not (temporally unpredictable condition) the onset of a target. In 25% of trials, an auditory signal occurred shortly after the target presentation, informing participants that they should inhibit their response completely. Response times were slower, and fewer inhibitory errors were made during temporally unpredictable than predictable trials, indicating enhanced control of unwanted actions when target onset time was unknown. Importantly, the ERN to inhibitory errors was greater in temporally unpredictable relative to temporally predictable conditions. Similarly, EMG data revealed prolonged MT when reactions to temporally unpredictable targets had not been stopped. Taken together, our results show that a temporally unpredictable environment increases the control of unwanted actions, both at cortical and peripheral levels, suggesting a higher subjective cost of maladaptive responses to temporally uncertain events.

Pre-hospital ECPR in an Australian metropolitan setting: a single-arm feasibility assessment-The CPR, pre-hospital ECPR and early reperfusion (CHEER3) study.

INTRODUCTION: Survival from refractory out of hospital cardiac arrest (OHCA) without timely return of spontaneous circulation (ROSC) utilising conventional advanced cardiac life support (ACLS) therapies is dismal. CHEER3 was a safety and feasibility study of pre-hospital deployed extracorporeal membrane oxygenation (ECMO) during cardiopulmonary resuscitation (ECPR) for refractory OHCA in metropolitan Australia. METHODS: This was a single jurisdiction, single-arm feasibility study. Physicians, with pre-existing ECMO expertise, responded to witnessed OHCA, age < 65 yrs, within 30 min driving-time, using an ECMO equipped rapid response vehicle. If pre-hospital ECPR was undertaken, patients were transported to hospital for investigations and therapies including emergent coronary catheterisation, and standard intensive care (ICU) therapy until either cardiac and neurological recovery or palliation occurred. Analyses were descriptive. RESULTS: From February 2020 to May 2023, over 117 days, the team responded to 709 "potential cardiac arrest" emergency calls. 358 were confirmed OHCA. Time from emergency call to scene arrival was 27 min (15-37 min). 10 patients fulfilled the pre-defined inclusion criteria and all were successfully cannulated on scene. Time from emergency call to ECMO initiation was 50 min (35-62 min). Time from decision to ECMO support was 16 min (11-26 min). CPR duration was 46 min (32-62 min). All 10 patients were transferred to hospital for investigations and therapy. 4 patients (40%) survived to hospital discharge neurologically intact (CPC 1/2). CONCLUSION: Pre-hospital ECPR was feasible, using an experienced ECMO team from a single-centre. Overall survival was promising in this highly selected group. Further prospective studies are now warranted.

Orienting attention to locations in perceptual versus mental representations.

Extensive clinical and imaging research has characterized the neural networks mediating the adaptive distribution of spatial attention. In everyday behavior, the distribution of attention is guided not only by extrapersonal targets but also by mental representations of their spatial layout. We used event-related functional magnetic resonance imaging to identify the neural system involved in directing attention to locations in arrays held as mental representations, and to compare it with the system for directing spatial attention to locations in the external world. We found that these two crucial aspects of spatial cognition are subserved by extensively overlapping networks. However, we also found that a region of right parietal cortex selectively participated in orienting attention to the extrapersonal space, whereas several frontal lobe regions selectively participated in orienting attention within on-line mental representations.

Brain activations during visual search: contributions of search efficiency versus feature binding.

We investigated the involvement of the parietal cortex in binding features during visual search using functional magnetic resonance imaging. We tested 10 subjects in four visual search tasks across which we independently manipulated (1) the requirement to integrate different types of features in a stimulus (feature or conjunction search) and (2) the degree of search efficiency (efficient or inefficient). We identified brain areas that were common to all conditions of visual search and areas that were sensitive to the factors of efficiency and feature binding. Visual search engaged an extensive network of parietal, frontal, and occipital areas. The factor of efficiency exerted a strong influence on parietal activations along the intraparietal sulcus and in the superior parietal lobule. These regions showed a main effect of efficiency and showed a simple effect when inefficient conditions were compared directly with efficient pop-out conditions in the absence of feature binding. Furthermore, a correlation analysis supported a tight correspondence between posterior parietal activation and the slope of reaction-time search functions. Conversely, feature binding during efficient pop-out search was not sufficient to modulate the parietal cortex. The results confirm the important role of the parietal cortex in visual search, but suggest that feature binding is not a requirement to engage its contribution.

Direct detection and identification of African trypanosomes by fluorescence in situ hybridization with peptide nucleic acid probes.

We have developed a rapid and easy to perform fluorescence in situ hybridization test that allows specific identification of trypanosomes from the subgenus Trypanozoon, using peptide nucleic acid probes. Probes were designed to target subgenus-specific sequences on the multiple-copy 18S rRNA, greatly facilitating the detection of a single trypanosome.

Identification of indicator microorganisms using a standardized PNA FISH method.

A standardized fluorescent in situ hybridization (FISH) method using Peptide Nucleic Acid (PNA) probes for analysis of gram-negative and gram-positive bacteria, as well as yeast, has been developed. Fluorescently labeled PNA probes targeting specific rRNA sequences of Escherichia coli, Pseudomonas aeruginosa, Staphyloccocus aureus, Salmonella were designed, as well as PNA probes targeting eubacteria and eucarya. These PNA probes were evaluated by PNA FISH using 27 bacterial and 1 yeast species, representing both phylogenetically closely related species, as well as species important to both clinical and industrial settings. The S. aureus and P. aeruginosa PNA probes did not cross react with any of the organisms tested, whereas the E. coli PNA probe, as expected from sequence data, also detected Shigella species. The Salmonella PNA probe reacted with all of the 13 Salmonella strains, representing the 7 subspecies of Salmonella, however, it is also complementary to a few other bacterial species. The eubacteria- and eucarya-specific PNA probes detected all bacterial species and one yeast species, respectively. The general applicability of the PNA FISH method made simultaneous identification of multiple species, both gram-negative and gram-positive, in a mixed population an attractive possibility never accomplished using DNA probes. Four color images using differently labeled PNA probes showed simultaneous identification of E. coli, P. aeruginosa, S. aureus and Salmonella, thereby demonstrating the potential of multiplex FISH for various diagnostic applications within both clinical and industrial microbiology.

Region-specific developmental specialization of GABA-glycine cosynapses in laminas I-II of the rat spinal dorsal horn.

The spinal dorsal horn is the first level of the CNS in which nociceptive input from sensory afferents is integrated and transmitted. Although inhibitory control in this region has a crucial impact on pain transmission, the respective contribution of GABA and glycine to this inhibition remains elusive. We have previously documented co-release of GABA and glycine at the same inhibitory synapse in spinal laminas I-II of adult rats [older than postnatal day 30 (P30)]. However, despite this co-release, individual miniature inhibitory postsynaptic currents (mIPSCs) were mediated by either glycine receptors (GlyR) or GABA(A) receptors (GABA(A)R), yet never by the two together. In contrast, recent studies of ventral horn immature inhibitory synapses (

PNA beacons for duplex DNA.

We report here on the hybridization of peptide nucleic acid (PNA)-based molecular beacons (MB) directly to duplex DNA sites locally exposed by PNA openers. Two stemless PNA beacons were tested, both featuring the same recognition sequence and fluorophore-quencher pair (Fluorescein and DABCYL, respectively) but differing in arrangement of these groups and net electrostatic charge. It was found that one PNA beacon rapidly hybridized, with the aid of openers, to its complementary target within duplex DNA at ambient conditions via formation of a PD-like loop. In contrast, the other PNA beacon bound more slowly to preopened duplex DNA target and only at elevated temperatures, although it readily hybridized to single-stranded (ss) DNA target. Besides a higher selectivity of hybridization provided by site-specific PNA openers, we expect this approach to be very useful in those MB applications when denaturation of the duplex DNA analytes is unfavorable or undesirable. Furthermore, we show that PNA beacons are advantageous over DNA beacons for analyzing unpurified/nondeproteinized DNA samples. This feature of PNA beacons and our innovative hybridization strategy may find applications in emerging fluorescent DNA diagnostics.

Combination of ATP-bioluminescence and PNA probes allows rapid total counts and identification of specific microorganisms in mixed populations.

We have combined ATP-dependent bioluminescence with a novel chemiluminescent in situ hybridization (CISH) method using peroxidase-labeled peptide nucleic acid (PNA) probes targeting species-specific rRNA sequences to provide total counts and subsequent identification of specific microorganisms. Both methods are applied to the same membrane filter following a short incubation time and both methods provide results in the form of spots of light that are captured by the MicroStar detection system. Each spot of light represents individual micro-colonies detected by either ATP bioluminescence or PNA CISH. This new concept is particularly intended for in process and quality control of non-sterile products to rapidly provide total counts as well as presence/absence of specific indicators and/or pathogens in non-sterile, filterable samples.

A pilot study of the use of mycophenolate mofetil as a component of therapy for multidrug-resistant HIV-1 infection.

Mycophenolic acid (MPA) increases the activity of both abacavir (ABC) and didanosine (ddI) in vitro against wild-type and multinucleoside-resistant HIV. We treated 7 patients with diagnosed AIDS who did not respond to eight or more antiretroviral therapies in an open label pilot study with mycophenolate mofetil (MMF), ABC, ddI, amprenavir (APV), and ritonavir (RTV), with or without efavirenz (EFV). Therapy was well tolerated despite the patients' advanced disease states. No significant decline in lymphocyte or other blood counts was observed. Median HIV RNA was 5.26 log10 copies/ml at entry, 4.53 log10 copies/ml at 4 weeks, and 5.13 log10 copies/ml at 16 weeks. Median CD4+ count was 34 cells/microl at entry and 39 cells/microl at 16 weeks of therapy. CD4+ counts increased further in five study subjects on extended therapy to 25 weeks (median 27 cells/microl at entry, 66 cells/microl at close), despite loss of virologic suppression in 4 of 5 cases. MPA can induce apoptosis in lymphocytes in vitro. However despite viral rebound, cell surface markers of apoptosis and activation declined in total CD3+ cells and CD3+/CD4+ cells twofold to fourfold in 4 of 5 adherent study subjects at 16 weeks, reaching levels comparable with those found in seronegative donors. Although low-dose MMF appears safe in late-stage HIV disease, this study did not demonstrate virologic efficacy. Higher doses of MMF may be more effective. With careful monitoring of toxicities and pharmacokinetics, MMF deserves further testing in HIV therapy.

Self-reporting PNA/DNA primers for PCR analysis.

We report a new fluorogenic method for sealed-tube PCR analysis using a quencher-labeled peptide nucleic acid (Q-PNA) probe. The Q-PNA hybridizes to a complementary tag sequence located at the 5' end of a 5' fluorophore-labeled oligonucleotide primer, quenching the primer's fluorescence. Incorporation of the primer into a doublestranded amplicon causes displacement of the Q-PNA such that the fluorescence of the sample is a direct indication of the amplicon concentration. The Q-PNA is able to quench multiple primers bearing distinct 5' fluorophores in a single reaction. We show realtime quantitative detection of a single-copy gene, K-ras, from human genomic DNA, as well as an endpoint multiplex assay for Chlamydia trachomatis and Neisseria gonorrhoeae targets. Because the Q-PNA may be used to quench any primer that contains the 5' tag sequence, it is possible to inexpensively adapt an existing primer set for use in a self-reporting fluorescent assay by including the tag sequence in one of the primers.

Rapid detection, identification, and enumeration of Escherichia coli by fluorescence in situ hybridization using an array scanner.

A new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes and an array scanner for rapid detection, identification, and enumeration of Escherichia coli is described. The test utilizes Cy3-labeled peptide nucleic acid (PNA) probes complementary to a specific 16S rRNA sequence of E. coli. Samples were filtered and incubated for 5 h, the membrane filters were then analyzed by fluorescence in situ hybridization and results were visualized with an array scanner. Results were provided as fluorescent spots representing E. coli microcolonies on the membrane filter surface. The number of fluorescent spots correlated to standard colony counts up to 100 colony-forming units per membrane filter. Above this level, better accuracy was obtained with PNA FISH due to the ability of the scanner to resolve neighboring microcolonies, which were not distinguishable as individual colonies once they were visible by eye.

Manual asymmetries in the preparation and control of goal-directed movements.

The primary purpose of this experiment was to determine if left hand reaction time advantages in manual aiming result from a right hemisphere attentional advantage or an early right hemisphere role in movement preparation. Right-handed participants were required to either make rapid goal-directed movements to small targets or simply lift their hand upon target illumination. The amount of advance information about the target for a particular trial was manipulated by precuing a subset of potential targets prior to the reaction time interval. When participants were required to make aiming movements to targets in left space, the left hand enjoyed a reaction advantage that was not present for aiming in right space or simple finger lifts. This advantage was independent of the amount or type of advance information provided by the precue. This finding supports the movement planning hypothesis. With respect to movement execution, participants completed their aiming movements more quickly when aiming with their right hand, particularly in right space. This right hand advantage in right space was due to the time required to decelerate the movement and to make feedback-based adjustments late in the movement trajectory.

Identification of Dekkera bruxellensis (Brettanomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes.

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.

Filter-based PNA in situ hybridization for rapid detection, identification and enumeration of specific micro-organisms.

AIMS: A method for rapid and simultaneous detection, identification and enumeration of specific micro-organisms using Peptide Nucleic Acid (PNA) probes is presented. METHODS AND RESULTS: The method is based on a membrane filtration technique. The membrane filter was incubated for a short period of time. The microcolonies were analysed by in situ hybridization, using peroxidase-labelled PNA probes targeting a species-specific rRNA sequence, and visualized by a chemiluminescent reaction. Microcolonies were observed as small spots of light on film, thereby providing simultaneous detection, identification and enumeration. The method showed 95-100% correlation to standard plate counts along with definitive identification due to the specificity of the probe. CONCLUSION: Using the same protocol, results were generated approximately three times faster than culture methods for Gram-positive and -negative bacterial species and yeast species. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is an improvement on the current membrane filtration technique, providing rapid determination of the level of specific pathogens, spoilage or indicator micro-organisms.

Rapid detection, identification, and enumeration of Escherichia coli cells in municipal water by chemiluminescent in situ hybridization.

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35 degrees C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.

Multicolor fluorescence in situ hybridization with peptide nucleic acid probes for enumeration of specific chromosomes in human cells.

In previous studies, we showed that peptide nucleic acid (PNA) probes have significant advantages over conventional synthetic RNA or DNA probes in FISH procedures for detecting telomeric and trinucleotide repeat sequences. Here, we report that directly labeled PNA probes recognizing chromosome-specific repeat sequences are also powerful tools for detecting and enumerating specific chromosomes in interphase and metaphase cells. This is illustrated by multicolor FISH experiments with cells from normal individuals and patients with numerical sex chromosome aberrations.

The noradrenergic alpha2 agonist clonidine modulates behavioural and neuroanatomical correlates of human attentional orienting and alerting.

We examined whether the known noradrenergic attenuation of the alerting effect (the beneficial effect of a warning cue) results from an underlying effect of noradrenaline on temporal orienting (orienting toward a particular moment in time). Following a within-subjects, counterbalanced design, 10 healthy human volunteers received placebo, 200 microg clonidine or 1 mg guanfacine (alpha2 agonists) in three separate testing sessions. Subjects were scanned by fMRI while performing attentional orienting tasks containing spatially informative, temporally informative, non-informative or no cues. The alerting effect primarily activated left-lateralized prefrontal, premotor and parietal regions. Clonidine, but not guanfacine, impaired behavioural measures of the alerting effect while attenuating activity in the left temporo-parietal junction. Replicating previous results, the temporal orienting task activated left parietal and frontal cortex, while parietal cortex was activated bilaterally during spatial orienting. Of these networks, clonidine, but not guanfacine, attenuated left prefrontal cortex and insula activity during temporal orienting and attenuated right superior parietal cortex activity during spatial orienting,. To complement these neuroanatomical changes, clonidine produced selective behavioural effects on both temporal and spatial orienting. The anatomical dissociation between the effects of clonidine during temporal orienting versus alerting suggests that noradrenergic modulation of the alerting effect does not result only from an underlying effect on temporal orienting. Furthermore, we have demonstrated lateralized neuroanatomical substrates for the noradrenergic modulation of human attentional orienting in the spatial and temporal domains.