Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples.

AIM: To evaluate commercial DNA extraction kits for their ability to isolate DNA from Yersinia pestis suspensions and spiked environmental samples. METHODS AND RESULTS: Five commercially available DNA extraction kits were evaluated: the ChargeSwitch gDNA Mini Bacteria Kit, the IT 1-2-3 Sample DNA Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit and the UltraClean Microbial DNA Isolation Kit. The extraction methods were performed upon six Y. pestis strains and spiked environmental specimens, including three swab types and one powder type. Taqman real-time PCR analysis revealed that the use of the MasterPure kit resulted in DNA with the most consistently positive results and the lowest limit of detection from Y. pestis suspensions and spiked environmental samples. CONCLUSION: Comparative evaluations of the five commercial DNA extraction methods indicated that the MasterPure kit was superior for the isolation of PCR-amplifiable DNA from Y. pestis suspensions and spiked environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can assist diagnostic laboratories with selecting the best extraction method for processing environmental specimens for subsequent detection of Y. pestis by real-time PCR.

Intracellular recordings of pontine medial gigantocellular tegmental field neurons in the naturally sleeping cat: behavioral state-related activity and soma size difference in order of recruitment.

Intracellular recordings and neurobiotin labeling of medial pontine gigantocellular tegmental field (m-PFTG) neurons in the undrugged, naturally sleeping cat were performed to establish the relationship between soma size and membrane potential (MP) activity before and during the onset of the rapid eye movement (REM) phase of sleep. Initial recordings without labeling revealed that recorded neurons in the m-PFTG had a tonic, sustained membrane depolarization in REM sleep as compared with more polarized MP levels in slow-wave sleep (S) and phasic depolarizations in wakefulness (W) on a more polarized MP level. In neurobiotin-labeled neurons, there was a strong correlation between the soma size of m-PFTG neurons and the 'lead time', the time of onset relative to the beginning of REM, of a sustained increase in membrane depolarization. Thirty-nine m-PFTG neurons with soma cross-sectional areas ranging from 2098 microm(2) to 5958 microm(2) (mean value 3833.8 microm(2)) were analyzed. A majority of these m-PFTG neurons showed an increase in membrane depolarization associated with depolarizing postsynaptic potentials (PSPs) and spike generation that occurred before electrographic signs of REM sleep onset, while the rest of the neurons depolarized at the beginning of or just after REM sleep onset. Our previous work had suggested that many of these m-PFTG neurons were output neurons to the spinal cord. Analysis of the onset time of sustained membrane depolarization (Leadtime(MP)) revealed that larger cells had a longer lead time, while analysis of the lead times for onset of sustained PSPs and action potentials (Leadtime(AP)) showed this measure not to be dependent on soma size, but to be rather uniform, occurring just before the onset of REM sleep. Hence recruitment time, defined as the difference between Leadtime(AP) and Leadtime(MP), was dependent on cell soma size, implying that larger neurons may take longer to depolarize to an MP level critical for generating sustained action potentials, while smaller neurons may require less time.

Extracellular histamine levels in the feline preoptic/anterior hypothalamic area during natural sleep-wakefulness and prolonged wakefulness: an in vivo microdialysis study.

Increased activity of the histaminergic neurons of the posterior hypothalamus has been implicated in the facilitation of behavioral wakefulness. Recent evidence of reciprocal projections between the sleep-active neurons of the preoptic/anterior hypothalamus and the histaminergic neurons of the tuberomammillary nucleus suggests that histaminergic innervation of the preoptic/anterior hypothalamic area may be of particular importance in the wakefulness-promoting properties of histamine. To test this possibility, we used microdialysis sample collection in the preoptic/anterior hypothalamic area of cats during natural sleep-wakefulness cycles, 6 h of sleep deprivation induced by gentle handling/playing, and recovery sleep. Samples were analyzed by a sensitive radioenzymatic assay. Mean basal levels of histamine in microdialysate during periods of wakefulness (1.155+/-0.225 pg/microl) did not vary during the 6 h of sleep deprivation. However, during the different sleep states, dramatic changes were observed in the extracellular histamine levels of preoptic/anterior hypothalamic area: wakefulness>non-rapid eye movement sleep>rapid eye movement sleep. Levels of histamine during rapid eye movement sleep were lowest (0.245+/-0.032 pg/microl), being significantly lower than levels during non-rapid eye movement sleep (0.395+/-0.081 pg/microl) and being only 21% of wakefulness levels. This pattern of preoptic/anterior hypothalamic area extracellular histamine levels across the sleep-wakefulness cycle closely resembles the reported single unit activity of histaminergic neurons. However, the invariance of histamine levels during sleep deprivation suggests that changes in histamine level do not relay information about sleep drive to the sleep-promoting neurons of the preoptic/anterior hypothalamic area.

Adenosinergic modulation of basal forebrain and preoptic/anterior hypothalamic neuronal activity in the control of behavioral state.

This review describes a series of animal experiments that investigate the role of endogenous adenosine (AD) in sleep. We propose that AD is a modulator of the sleepiness associated with prolonged wakefulness. More specifically, we suggest that, during prolonged wakefulness, extracellular AD accumulates selectively in the basal forebrain (BF) and cortex and promotes the transition from wakefulness to slow wave sleep (SWS) by inhibiting cholinergic and non-cholinergic wakefulness-promoting BF neurons at the AD A1 receptor. New in vitro data are also compatible with the hypothesis that, via presynaptic inhibition of GABAergic inhibitory input, AD may disinhibit neurons in the preoptic/anterior hypothalamus (POAH) that have SWS-selective activity and Fos expression. Our in vitro recordings initially showed that endogenous AD suppressed the discharge activity of neurons in the BF cholinergic zone via the AD A1 receptor. Moreover, in identified mesopontine cholinergic neurons, AD was shown to act post-synaptically by hyperpolarizng the membrane via an inwardly rectifying potassium current and inhibition of the hyperpolarization-activated current, I(h). In vivo microdialysis in the cat has shown that AD in the BF cholinergic zone accumulates during prolonged wakefulness, and declines slowly during subsequent sleep, findings confirmed in the rat. Moreover, increasing BF AD concentrations to approximately the level as during sleep deprivation by a nucleoside transport blocker mimicked the effect of sleep deprivation on both the EEG power spectrum and behavioral state distribution: wakefulness was decreased, and there were increases in SWS and REM sleep. As predicted, microdialyis application of the specific A1 receptor antagonist cyclopentyltheophylline (CPT) in the BF produced the opposite effects on behavioral state, increasing wakefulness and decreasing SWS and REM. Combined unit recording and microdialysis studies have shown neurons selectively active in wakefulness, compared with SWS, have discharge activity suppressed by both AD and the A1-specific agonist cyclohexyladenosine (CHA), while discharge activity is increased by the A1 receptor antagonist, CPT. We next addressed the question of whether AD exerts its effects locally or globally. Adenosine accumulation during prolonged wakefulness occurred in the BF and neocortex, although, unlike in the BF, cortical AD levels declined in the 6th h of sleep deprivation and declined further during subsequent recovery sleep. Somewhat to our surprise, AD concentrations did not increase during prolonged wakefulness (6 h) even in regions important in behavioral state control, such as the POAH, dorsal raphe nucleus, and pedunculopontine tegmental nucleus, nor did it increase in the ventrolateral/ventroanterior thalamic nucleii. These data suggest the presence of brain region-specific differences in AD transporters and/or degradation that become evident with prolonged wakefulness, even though AD concentrations are higher in all brain sites sampled during the naturally occurring (and shorter duration) episodes of wakefulness as compared to sleep episodes in the freely moving and behaving cat. Might AD also produce modulation of activity of neurons that have sleep selective transcriptional (Fos) and discharge activity in the preoptic/anterior hypothalamus zone? Whole cell patch clamp recordings in the in vitro horizontal slice showed fast and likely GABAergic inhibitory post-synaptic potentials and currents that were greatly decreased by bath application of AD. Adenosine may thus disinhibit and promote expression of sleep-related neuronal activity in the POAH. In summary, a growing body of evidence supports the role of AD as a mediator of the sleepiness following prolonged wakefulness, a role in which its inhibitory actions on the BF wakefulness-promoting neurons may be especially important.

Behavioral state-related changes of extracellular serotonin concentration in the pedunculopontine tegmental nucleus: a microdialysis study in freely moving animals.

Neurons of the cholinergic mesopontine tegmentum preferentially discharge during REM sleep and are thought to promote this state. It has been hypothesized they are inhibited during wakefulness by serotonergic input. The present study used the microdialysis sampling procedure coupled to microbore HPLC to measure extracellular serotonin levels in the pedunculopontine tegmental nucleus (PPT) in naturally sleeping cats. Extracellular serotonin levels were found to be highest during periods of wakefulness, lower during slow wave sleep, and lowest during periods of REM sleep. During wakefulness serotonin levels (mean A+/-SEM) measured in 10 A microliter samples were 1.14 A+/- 0.13 fmol/sample, whereas during slow wave sleep levels declined significantly to 72% of the wakefulness baseline (0.85 A +/- 0.11 fmol/sample), and dropped further to 45% of the wakefulness baseline in REM samples (0.52 A +/- 0.10 fmol/sample; all p's<0.003). The decrease in PPT serotonin levels during sleep may be an important determinant in the timing of REM sleep cyclicity. The data support the hypothesis that, during slow wave sleep and REM sleep, the declining levels of serotonin release the PPT REM-promoting neurons from serotonergic inhibition, which, in turn, leads to increases in acetylcholine release in terminal areas, facilitating the emergence of REM sleep.

[Urinary infection in geriatric short stay: value of urinary strips]. / Infection urinaire en court séjour gériatrique: intérêt de la bandelette urinaire.

The blinded study of CBUE (cytobacteriological urine examination) test strips was carried out in 100 patients (65 women, 35 men), mean age 85.52 +/- 6.28 years, admitted for a short stay in a geriatrics department. Urinary infection was present in 19% of the cases. Typical clinical signs (pollakiuria, dysuria, hematuria, burning upon voiding) were rare, less than 10%. General signs, such as deterioration of general status (65% of the cases) and loss of autonomy (53% of the cases), on the other hand, were predominant and their relatively unspecific nature required a reliable screening test for urinary infection. Good results of sensitivity (89.5%), specificity (98.7%), and the leukocytes/nitrites values, confirmed the validity of the urine test strip in hospitalized elderly patients. In addition, the negative predictive value of the combination leukocytes and nitrites (90.8%) would have prevented 71 samples from being sent to the laboratory for CBUE, resulting in an obvious financial savings.

Electrical stimulation of the cholinergic laterodorsal tegmental nucleus elicits scopolamine-sensitive excitatory postsynaptic potentials in medial pontine reticular formation neurons.

A large and consistent body of data implicates mesopontine cholinergic neurons in the production of rapid eye movement sleep, and indicates that many rapid eye movement sleep events are mediated by activation of pontine reticular formation neurons. There is anatomical evidence for projections from the mesopontine cholinergic nuclei to the pontine reticular formation, but no study has shown that stimulation of this cholinergic zone produces excitatory postsynaptic potentials in pontine reticular formation neurons. In the present study, intracellular recording were made from 168 pontine reticular formation neurons, identified by antidromic activation from the bulbar reticular formation and by neurobiotin intracellular labeling, in acutely anesthetized cats. The effects of single-pulse electrical stimulation of the laterodorsal tegmental nucleus portion of the ipsilateral mesopontine cholinergic zone were evaluated in these neurons. Under urethane anesthesia this stimulation produced, in 21 of 22 recorded neurons, long-latency excitatory postsynaptic potentials (mean = 3 ms), consistent with the conduction velocity of unmyelinated cholinergic fibers (measured conduction velocity was 2 m/s). This excitatory postsynaptic potential was virtually abolished by intravenous administration of the muscarinic cholinergic receptor blocker scopolamine (n = 40 neurons), and by acute cuts separating the laterodorsal tegmental nucleus and the recorded neurons (n = 40). In contrast, a short-latency excitatory postsynaptic potential (0.7-1.5 ms) was not reduced in amplitude by scopolamine and could still be elicited following acute transverse cuts. Unlike the longer-latency excitatory postsynaptic potential, its amplitude was not reduced by barbiturate anesthesia. These data, suggesting the presence of an excitatory, cholinergic laterodorsal tegmental nucleus projection to the pontine reticular formation, provide further support to other lines of evidence implicating mesopontine cholinergic neurons in the production of rapid eye movement sleep, and are compatible with a model of rapid eye movement sleep generation in which a key element is mesopontine cholinergic input depolarizing and increasing the excitability of reticular core neurons.