Serotonin Strengthens a Developing Glutamatergic Synapse through a PI3K-Dependent Mechanism.

It is well established that, during neural circuit development, glutamatergic synapses become strengthened via NMDA receptor (NMDAR)-dependent upregulation of AMPA receptor (AMPAR)-mediated currents. In addition, however, it is known that the neuromodulator serotonin is present throughout most regions of the vertebrate brain while synapses are forming and being shaped by activity-dependent processes. This suggests that serotonin may modulate or contribute to these processes. Here, we investigate the role of serotonin in the developing retinotectal projection of the Xenopus tadpole. We altered endogenous serotonin transmission in stage 48/49 (∼10-21 days postfertilization) Xenopus tadpoles and then carried out a set of whole-cell electrophysiological recordings from tectal neurons to assess retinotectal synaptic transmission. Because tadpole sex is indeterminate at these early stages of development, experimental groups were composed of randomly chosen tadpoles. We found that pharmacologically enhancing and reducing serotonin transmission for 24 h up- and downregulates, respectively, AMPAR-mediated currents at individual retinotectal synapses. Inhibiting 5-HT2 receptors also significantly weakened AMPAR-mediated currents and abolished the synapse strengthening effect seen with enhanced serotonin transmission, indicating a 5-HT2 receptor-dependent effect. We also determine that the serotonin-dependent upregulation of synaptic AMPAR currents was mediated via an NMDAR-independent, PI3K-dependent mechanism. Altogether, these findings indicate that serotonin regulates AMPAR currents at developing synapses independent of NMDA transmission, which may explain its role as an enabler of activity-dependent plasticity.

Alpha-thiol deoxynucleotide triphosphates (S-dNTPs) as radioprotective agents: A novel approach.

In this study, the ability of a mixture of four different alpha-thiol deoxynucleotide triphosphates (S-dNTPs) each at a concentration of 10µM when incorporated into the genomic DNA of proliferating human HL-60 and Mono-Mac-6 (MM-6) cells in vitro to provide protection from 2, 5, and 10 Gy of gamma radiation was investigated. Incorporation of the four different S-dNTPs into nuclear DNA at 10 µM concentration for five days was validated by agarose gel electrophoretic band shift analysis. S-dNTP-treated genomic DNA reacted with BODIPY-iodoacetamide demonstrated a band shift to higher molecular weight to confirm the presence of sulfur moieties in the resultant phosphorothioate DNA backbones. No overt signs of toxicity or obvious morphologic cellular differentiation were noted in the presence of 10 µM S-dNTPs even after 8 days in culture. Significantly reduced radiation-induced persistent DNA damage measured at 24 and 48 h post-exposure by γ-H2AX histone phosphorylation using FACS analysis in S-dNTP incorporated HL-60 and MM6 cells indicated protection against radiation-induced direct and indirect DNA damage. Statistically significant protection by S-dNTPs was noted at the cellular level by CellEvent™ Caspase-3/7 assay, which assess the extent of apoptotic events, and by trypan blue dye exclusion to assed cell viability. The results appear to support an innocuous antioxidant thiol radioprotective effect built into genomic DNA backbones as the last line of defense against ionizing radiation and free radical-induced DNA damage.

Advancements in Rapid and Affordable Diagnostic Testing for Respiratory Infectious Diseases: Evaluation of Aptamer Beacon Technology for Rapid and Sensitive Detection of SAR-CoV-2 in Breath Condensate.

The demand for rapid and efficient diagnostic point-of-care tests for respiratory infectious diseases has become increasingly critical in the current landscape. The emphasis on accessibility has been underscored over the past year, making it crucial to have biological components that exhibit fast and accurate kinetics. The foundation for precise, swift, and effective testing relies on the availability of highly responsive biological agents. Two published aptamer DNA sequences designated Song and MSA52 and their truncated internal stem-loop structures were studied for their potential to serve as aptamer beacons for rapid COVID detection. The candidate beacons were covalently labeled with Atto 633 dye attached to their 5' ends and Iowa Black quencher attached to their 3' ends. The whole aptamer structures exhibited the greatest fluorescence signal intensities and higher fluorescence background than their truncated internal stem-loop beacon structures suggesting that the distance between fluorophores and quenchers was greater for the whole aptamer beacon candidates versus the isolated stem-loop structures. Beacon candidates were tested against two heat- or gamma radiation-killed SARS-CoV-2 Washington 1/2020 virus samples and three different COVID spike (S) proteins to test their effectiveness. Despite the higher background fluorescence, the whole aptamer beacons showed better signal-to-noise ratios and were selected for further investigation. Limit of detection (LOD) studies revealed that both the whole Song and whole MSA52 aptamer beacon candidates had a LOD of 9.61 × 103 genome equivalents in phosphate-buffered saline using the red channel of a Promega Quantus™ fluorometer which correlated well with confirmatory spectrofluorometry. Cross-reactivity studies using numerous COVID variants, related coronaviruses, and other common respiratory pathogens suggested greater COVID selectivity for the whole MSA52 versus the whole Song aptamer beacon candidate, indicating promise for specific COVID detection. Importantly, both whole aptamer beacon candidates exhibited very rapid "bind and detect" fluorescence increases within the first 1-2 min of mixing the beacons with killed SARS-CoV-2 viruses in 100 µl samples. Overall, this work illustrates the strong potential for aptamer beacons for rapid, on-site detection and presumptive diagnosis of COVID in breath condensates or other small liquid samples. This research highlights the strong potential of aptamer beacons for addressing the need for fast and convenient diagnostic tools in global health contexts, especially in resource-limited settings.

DNA Aptamer-Based Staining and Fluorescence Microscopy for Rapid Detection of Cyclospora Cayetanensis Oocysts.

There are no commercial antibodies for detection of Cyclospora cayetanensis, only a relatively slow polymerase chain reaction (PCR) test developed by the U.S. Food and Drug Administration (FDA). However, DNA aptamers have recently been developed by our group against known proteins and whole oocysts of C. cayetanensis and shown to specifically detect the oocysts when attached on their 5' ends to red-emitting fluorophores and used as probes for fluorescence microscopy. Aptamers developed against recombinant wall protein 2 and TA4 antigen-like protein as well as whole oocysts specifically stained C. cayetanensis oocysts while exhibiting little, if any, staining of numerous other waterborne parasite species. Interestingly, the aptamers stained both exterior cell wall moieties and internal structures, suggesting that the aptamers penetrate the oocysts even without added detergents.

Remoteness does not enhance coral reef resilience.

Remote coral reefs are thought to be more resilient to climate change due to their isolation from local stressors like fishing and pollution. We tested this hypothesis by measuring the relationship between local human influence and coral community resilience. Surprisingly, we found no relationship between human influence and resistance to disturbance and some evidence that areas with greater human development may recover from disturbance faster than their more isolated counterparts. Our results suggest remote coral reefs are imperiled by climate change, like so many other geographically isolated ecosystems, and are unlikely to serve as effective biodiversity arks. Only drastic and rapid cuts in greenhouse gas emissions will ensure coral survival. Our results also indicate that some reefs close to large human populations were relatively resilient. Focusing research and conservation resources on these more accessible locations has the potential to provide new insights and maximize conservation outcomes.

Successes and Failures of Static Aptamer-Target 3D Docking Models.

While Molecular Dynamics simulation programs are probably superior for predicting the binding and affinity of aptamers and their cognate ligands, such molecular dynamics programs require more computing power and analysis time than static docking programs that are more widely accessible to the scientific community on the internet. Static docking programs can be used to investigate the geometric fit of rigid DNA or RNA aptamer 3D structures and their ligands to aid in predicting the relative affinities and cross-reactivity of various potential ligands. Herein, the author describes when such static 3D docking analysis has worked well to produce useful predictions or confirmation of high-affinity aptamer interactions or successful aptamer beacon behavior and when it has not worked well. The analysis of why failures may occur with static 3D computer models is also discussed.

A Combined Immunofluorescence and Fluorescent Viability Cocktail Staining Procedure for Rapid Microscopic Detection and Enumeration of Live Legionella pneumophila.

This report describes a combined immunofluorescence and fluorescence viability stain applied as one staining solution for rapid detection of live Legionella pneumophila in mixed bacterial populations. Instead of sequential viability staining with the Invitrogen BacLight LIVE/DEAD staining kit followed by antibody-Alexa Fluor (AF) 647 conjugate staining to identify live L. pneumophila, a combined single cocktail solution staining protocol was developed to simplify and accelerate the time to detection of viable L. pneumophila serogroup-1 (SG-1) in mixed species populations on a filter membrane. The stain cocktail will aid in accelerating fluorescence microscopic analysis of cooling tower, air conditioner and water fountain or other liquid samples for the presence of L. pneumophila and its viability status. Visibly red stained cells were identified as dead non-L. pneumophila SG-1 cells, while green fluorescing cells represented viable non-L. pneumophila SG-1 cells. Due to also staining red with antibody-AF 647, L. pneumophila SG-1 cells were pseudocolorized as blue to distinguish them from other dead cells. Fluorescence color emission mixing from the viability dyes (SYTO 9 and propidium iodide) with antibody-AF 647 stained L. pneumophila led to other fluorescent colors. For example, green plus pseudocolorized blue AF 647-antibody- labeled cells were identified as live cyan-colored L. pneumophila SG-1 cells. Magenta-colored cells resulted from dead L. pneumophila cells that combined red propidium iodide with blue pseudocolorized AF 647-antibody emissions. Analysis of measured RGB (red, green, blue) color values in microscopic images of mixed bacterial populations suggests the possibility of facile automated discrimination of subpopulations of live and dead L. pneumophila and non-L. pneumophila species by computers in 3-dimensional RGB color space after staining in the combined cocktail which will save time for more rapid microscopic detection of potential sources of Legionnaire's disease.

Prehospital end-tidal CO2 as an early marker for transfusion requirement in trauma patients.

OBJECTIVE: Below normal end-tidal carbon dioxide measurement (ETCO2) is associated with worse outcomes in sepsis and trauma patients as compared to patients with normal ETCO2. We sought to determine if ETCO2 can be used in the prehospital setting to predict transfusion requirement, operative hemorrhage control, or mortality in the first 24 h after admission for trauma. METHODS: This is a retrospective cohort study at a suburban, academic Level 1 Trauma Center. Patients were sequentially identified as prehospital trauma alerts from a single EMS system which requires, per policy, ETCO2 for all traumas. One year of prehospital data was collected and paired with hospital trauma registry data. Comparisons were made between ETCO2 values for patients who required transfusion, operative blood loss control, or who died, and those who did not. RESULTS: Two hundred thirty-five trauma patients were transported via the study EMS system, of which 105 (44.7%) had documented ETCO2 values. Patient mean age was 60 (SD24) years with 59 (56.2%) male. Three patients were intubated prehospital and seven were intubated in the trauma bay. Mean prehospital ETCO2 for those who needed transfusion, surgery, or died (n = 11) was 25.7 (9.1) compared to 30.6 (7.8) for those who did not (p = 0.049). Optimal cutoff for our population was EtCO2 ≤ 27 with a sensitivity of 72.7% (95% CI 32-93) and specificity of 72.2% (62-81). CONCLUSION: Below normal ETCO2 values were associated with increase need for transfusion, operative intervention, and death. Further study is warranted to determine if ETCO2 outperforms other predictors of severe trauma.

Geographical limits to species-range shifts are suggested by climate velocity.

The reorganization of patterns of species diversity driven by anthropogenic climate change, and the consequences for humans, are not yet fully understood or appreciated. Nevertheless, changes in climate conditions are useful for predicting shifts in species distributions at global and local scales. Here we use the velocity of climate change to derive spatial trajectories for climatic niches from 1960 to 2009 (ref. 7) and from 2006 to 2100, and use the properties of these trajectories to infer changes in species distributions. Coastlines act as barriers and locally cooler areas act as attractors for trajectories, creating source and sink areas for local climatic conditions. Climate source areas indicate where locally novel conditions are not connected to areas where similar climates previously occurred, and are thereby inaccessible to climate migrants tracking isotherms: 16% of global surface area for 1960 to 2009, and 34% of ocean for the 'business as usual' climate scenario (representative concentration pathway (RCP) 8.5) representing continued use of fossil fuels without mitigation. Climate sink areas are where climate conditions locally disappear, potentially blocking the movement of climate migrants. Sink areas comprise 1.0% of ocean area and 3.6% of land and are prevalent on coasts and high ground. Using this approach to infer shifts in species distributions gives global and regional maps of the expected direction and rate of shifts of climate migrants, and suggests areas of potential loss of species richness.

Integration of multiple computer modeling software programs for characterization of a brain natriuretic peptide sandwich DNA aptamer complex.

Several molecular modeling programs including Pep-Fold 3, Vienna RNA, RNA Composer, Avogadro, PatchDock, RasMol, and VMD were used to define the three-dimensional and basic binding characteristics of an extant sandwich DNA aptamer assay complex for human brain natriuretic peptide (BNP). In particular, the theoretical question of demonstrating likely binding of 72 base capture and reporter aptamers to at least two separate "epitopes" or binding sites on the small 32-amino acid BNP target was addressed, and the data support the existence of separate aptamer binding sites on BNP. The binding model was based on first docking BNP to the capture aptamer based on shape complementarity with PatchDock, followed by docking the capture aptamer-BNP complex with the reporter aptamer in PatchDock. Although, shape complementarity clearly dominated this binding model and aptamers are known to be somewhat flexible, the model demonstrates hydrogen bond stabilization within each of the two different aptamers and between the aptamers and the BNP target, thus suggesting a strong binding and high affinity sandwich assay that matches the author's former published assay results (Bruno et al., Microchem. J. 2014;115:32-38) with subpicogram per milliliter sensitivity and good specificity. Other aspects such as capture and reporter aptamer interactions in the absence of BNP are illustrated and suggest means for potentially improving the existing assay by truncating the capture and reporter aptamers where they overlap to further decrease background signal levels.

Beacons Contribute Valuable Empirical Information to Theoretical 3-D Aptamer-Peptide Binding.

DNA aptamers were developed against five different peptides from the known binding regions of anti-Cytomegalovirus and anti-Herpes Simplex Virus-2 antibodies and the aptamers were ranked by relative affinity based on an ELISA-like (ELASA) microplate assay. The secondary structures of the top five highest affinity aptamers were studied for stem-loop commonalities and the most probable peptide binding sites. Two of these stem-loop structures were converted into beacons by addition of TYE 665 dye on the 5' end and Iowa Black quencher on the 3' end. When competed against increasing concentrations of each of the five peptides, only three of the possible ten interactions demonstrated "lights on" fluorescence beacon responses. When modeled by generation of PDB files, after passage through PATCHDOCK and YASARA, two of the aptamer beacon-peptide interactions showed no theoretical evidence of separating the G-C stem-loop region, despite clear empirical evidence of separation of the fluorophore and quencher beyond the Förster distance leading to abundant fluorescence. And in the second beacon's case, YASARA modeling suggested that the beacon was always open despite clear empirical evidence that it was not (no fluorescence response) and only opened in the presence of one of the five peptides. These results are interpreted as a demonstration that 3-dimensional docking software such as PATCHDOCK and YASARA, which are based on rigid receptor-ligand shape complementarity may not reflect the "induced-fit" interactions between aptamers and their cognate targets. Therefore, for the most complete and accurate picture of aptamer-peptide binding, several theoretical and empirical (e.g., beacon fluorescence) analysis methods may be needed.

Preferential Disruption of Prefrontal GABAergic Function by Nanomolar Concentrations of the α7nACh Negative Modulator Kynurenic Acid.

Increased concentrations of kynurenic acid (KYNA) in the prefrontal cortex (PFC) are thought to contribute to the development of cognitive deficits observed in schizophrenia. Although this view is consistent with preclinical studies showing a negative impact of prefrontal KYNA elevation on executive function, the mechanism underlying such a disruption remains unclear. Here, we measured changes in local field potential (LFP) responses to ventral hippocampal stimulation in vivo and conducted whole-cell patch-clamp recordings in brain slices to reveal how nanomolar concentrations of KYNA alter synaptic transmission in the PFC of male adult rats. Our data show that prefrontal infusions of KYNA attenuated the inhibitory component of PFC LFP responses, a disruption that resulted from local blockade of α7-nicotinic acetylcholine receptors (α7nAChR). At the cellular level, we found that the inhibitory action exerted by KYNA in the PFC occurred primarily at local GABAergic synapses through an α7nAChR-dependent presynaptic mechanism. As a result, the excitatory-inhibitory ratio of synaptic transmission becomes imbalanced in a manner that correlates highly with the level of GABAergic suppression by KYNA. Finally, prefrontal infusion of a GABAAR positive allosteric modulator was sufficient to overcome the disrupting effect of KYNA and normalized the pattern of LFP inhibition in the PFC. Thus, the preferential inhibitory effect of KYNA on prefrontal GABAergic transmission could contribute to the onset of cognitive deficits observed in schizophrenia because proper GABAergic control of PFC output is one key mechanism for supporting such cortical functions.SIGNIFICANCE STATEMENT Brain kynurenic acid (KYNA) is an astrocyte-derived metabolite and its abnormal elevation in the prefrontal cortex (PFC) is thought to impair cognitive functions in individuals with schizophrenia. However, the mechanism underlying the disrupting effect of KYNA remains unclear. Here we found that KYNA biases the excitatory-inhibitory balance of prefrontal synaptic activity toward a state of disinhibition. Such disruption emerges as a result of a preferential suppression of local GABAergic transmission by KYNA via presynaptic inhibition of α7-nicotinic acetylcholine receptor signaling. Therefore, the degree of GABAergic dysregulation in the PFC could be a clinically relevant contributing factor for the onset of cognitive deficits resulting from abnormal increases of cortical KYNA.

Kynurenines in the mammalian brain: when physiology meets pathology.

The essential amino acid tryptophan is not only a precursor of serotonin but is also degraded to several other neuroactive compounds, including kynurenic acid, 3-hydroxykynurenine and quinolinic acid. The synthesis of these metabolites is regulated by an enzymatic cascade, known as the kynurenine pathway, that is tightly controlled by the immune system. Dysregulation of this pathway, resulting in hyper-or hypofunction of active metabolites, is associated with neurodegenerative and other neurological disorders, as well as with psychiatric diseases such as depression and schizophrenia. With recently developed pharmacological agents, it is now possible to restore metabolic equilibrium and envisage novel therapeutic interventions.

Long Shelf Life of a Lyophilized DNA Aptamer Beacon Assay.

An aptamer beacon previously developed to detect C-telopeptide (CTx) from human bone collagen breakdown was lyophilized and shown to give a "lights on" concentration-dependent spectral fluorescence response essentially identical to that of the fresh reagent despite storage in a dark dry environment for the past 5.5 years.

Living shorelines can enhance the nursery role of threatened estuarine habitats.

Coastal ecosystems provide numerous services, such as nutrient cycling, climate change amelioration, and habitat provision for commercially valuable organisms. Ecosystem functions and processes are modified by human activities locally and globally, with degradation of coastal ecosystems by development and climate change occurring at unprecedented rates. The demand for coastal defense strategies against storms and sea-level rise has increased with human population growth and development along coastlines world-wide, even while that population growth has reduced natural buffering of shorelines. Shoreline hardening, a common coastal defense strategy that includes the use of seawalls and bulkheads (vertical walls constructed of concrete, wood, vinyl, or steel), is resulting in a "coastal squeeze" on estuarine habitats. In contrast to hardening, living shorelines, which range from vegetation plantings to a combination of hard structures and plantings, can be deployed to restore or enhance multiple ecosystem services normally delivered by naturally vegetated shores. Although hundreds of living shoreline projects have been implemented in the United States alone, few studies have evaluated their effectiveness in sustaining or enhancing ecosystem services relative to naturally vegetated shorelines and hardened shorelines. We quantified the effectiveness of (1) sills with landward marsh (a type of living shoreline that combines marsh plantings with an offshore low-profile breakwater), (2) natural salt marsh shorelines (control marshes), and (3) unvegetated bulkheaded shores in providing habitat for fish and crustaceans (nekton). Sills supported higher abundances and species diversity of fishes than unvegetated habitat adjacent to bulkheads, and even control marshes. Sills also supported higher cover of filter-feeding bivalves (a food resource and refuge habitat for nekton) than bulkheads or control marshes. These ecosystem-service enhancements were detected on shores with sills three or more years after construction, but not before. Sills provide added structure and may provide better refuges from predation and greater opportunity to use available food resources for nekton than unvegetated bulkheaded shores or control marshes. Our study shows that unlike shoreline hardening, living shorelines can enhance some ecosystem services provided by marshes, such as provision of nursery habitat.

Studies of DNA Aptamer OliGreen and PicoGreen Fluorescence Interactions in Buffer and Serum.

Spectrofluorometric and emission peak titration and timed studies of OliGreen (OG) and PicoGreen (PG) were conducted in Tris EDTA (TE) buffer, pooled rat and fetal bovine serum with two different aptamers of 72 and 192 bases in length to determine if OG or PG were suitable for aptamer pharmacokinetic (PK) studies in sera. Results indicated that OG and PG detected the single-stranded (ss) and double-stranded (ds) stem-loop structures of the two aptamers quite well in TE with reliable standard curves having exponential character (or several linear detection regions) up to 1 µg/ml of aptamer DNA with detection limits of ~1 ng/ml. The intensity of OG and PG staining appeared to correlate with the number and percentage of ss and ds bases in each aptamer. OG and PG fluorescence in pooled rat serum or fetal bovine serum (FBS) did not titer as a function of DNA aptamer concentration from 1 µg/ml to 1 ng/ml. This lack of OG or PG aptamer assays in serum is contrary to most published reports of OG or PG assays for ss antisense oligonucleotides, ds PCR amplicons or other types of DNA in serum or plasma. Further studies suggested that the lack of OG and PG assay titration in serum might not be entirely due to aptamer degradation from nucleases in serum since the fluorescence signals in serum appeared relatively stable over time from 30 min to 4 hours. A hypothesis is presented which attributes the inability of OG or PG to assay aptamers in serum to a combination of high blue-green autofluorescence in serum with possible serum nuclease degradation of aptamers over time and the changing aptamer to serum protein ratio coupled to nonspecific binding of serum proteins to aptamers thereby possibly changing aptamer conformations as a function of aptamer concentration during titration experiments.

Exploring the role of temperature in the ocean through metabolic scaling.

Temperature imposes a constraint on the rates and outcomes of ecological processes that determine community- and ecosystem-level patterns. The application of metabolic scaling theory has advanced our understanding of the influence of temperature on pattern and process in marine communities. Metabolic scaling theory uses the fundamental and ubiquitous patterns of temperature-dependent metabolism to predict how environmental temperature influences patterns and processes at higher levels of biological organization. Here, we outline some of these predictions to review recent advances and illustrate how scaling theory might be applied to new challenges. For example, warming can alter species interactions and food-web structure and can also reduce total animal biomass supportable by a given amount of primary production by increasing animal metabolism and energetic demand. Additionally, within a species, larval development is faster in warmer water, potentially influencing dispersal and other demographic processes like population connectivity and gene flow. These predictions can be extended further to address major questions in marine ecology, and present an opportunity for conceptual unification of marine ecological research across levels of biological organization. Drawing on work by ecologists and oceanographers over the last century, a metabolic scaling approach represents a promising way forward for applying ecological understanding to basic questions as well as conservation challenges.

Stochastic dynamics of a warmer Great Barrier Reef.

Pressure on natural communities from human activities continues to increase. Even unique ecosystems like the Great Barrier Reef (GBR), that until recently were considered near-pristine and well-protected, are showing signs of rapid degradation. We collated recent (1996-2006) spatiotemporal relationships between benthic community composition on the GBR and environmental variables (ocean temperature and local threats resulting from human activity). We built multivariate models of the effects of these variables on short-term dynamics, and developed an analytical approach to study their long-term consequences. We used this approach to study the effects of ocean warming under different levels of local threat. Observed short-term changes in benthic community structure (e.g., declining coral cover) were associated with ocean temperature (warming) and local threats. Our model projected that, in the long-term, coral cover of less than 10% was not implausible. With increasing temperature and/or local threats, corals were initially replaced by sponges, gorgonians, and other taxa, with an eventual moderately high probability of domination (> 50%) by macroalgae when temperature increase was greatest (e.g., 3.5 degrees C of warming). Our approach to modeling community dynamics, based on multivariate statistical models, enabled us to project how environmental change (and thus local and international policy decisions) will influence the future state of coral reefs. The same approach could be applied to other systems for which time series of ecological and environmental variables are available.

An acridine orange spore germination fluorescence microscopy versus spectral paradox.

The metachromatic fluorophore acridine orange (AO) has demonstrated green fluorescent staining of dormant Bacillus spores and orange to red staining of transcriptionally active vegetative cells when used in the mid-micoMolar range. Despite the microscopic observation of numerous bright orange to red fluorescent vegetative cells following germination induction, no clear spectral emission peaks > 590 nm have ever been reported for spectrofluorometric analysis involving AO in conjunction with spore germination. This microscopy versus spectrofluorometry paradox is documented in the present report and hypotheses are put forth to explain the very weak spectral changes in the red region which do not appear to correlate with the abundant orange-red fluorescence of nascent vegetative cells seen through the fluorescence microscope.

Development of a fluorescent enzyme-linked DNA aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid listeria detection.

A fluorescent DNA aptamer-magnetic bead sandwich assay was developed to detect listeriolysin O (LLO) protein from pathogenic Listeria bacteria using a peroxidase-linked system, Amplex Ultra Red (AUR; derivatized resazurin) substrate, and a custom-designed handheld fluorometer. The assay is highly sensitive with demonstrated limits of detection (LODs) in the range of 4 to 61 L. monocytogenes cells or the equivalent LLO produced by 4 to 61 cells on average in separate titration trials. Total assay processing and analysis time was approximately 30 mins. The assay has demonstrated the ability to detect 6 species of Listeria as desired by the USDA's Food Safety Inspection Service (FSIS). The portable system was designed to be used primarily with surface swab samples from fomites, but it can also be used to assess enrichment cultures. The minimal time to detect a positive enrichment culture in our hands from an initial 10 cell inoculum in 200 ml of broth has been 8 h post-incubation at 37 °C in shaker flask cultures. An optional automated magnetic bead assay processing and wash device capable of simultaneously processing 6 samples with low and consistent fluorescence background for higher volume central laboratories is also described.