Rapid detection of Legionella spp in water samples by ScanVIT method: comparison of acid vs heat treatment.

BACKGROUND: A rapid detection of Legionella bacteria in water samples is crucial to minimize the risk of acquiring infections, especially in health care facilities. Different detection methods and different decontamination procedures have been reported to affect the recovery of Legionella spp. Our goal was to test the recovery of Legionella pneumophila and Legionella non-pneumophila species using a kit based on non-specific and species-specific probes to treat water samples after two different decontamination procedures. METHODS: The study was conducted with samples collected in the teaching hospital "Le Scotte" of Siena (Italy). Waters samples were analyzed by: i) ScanVIT method after treatment with acids; ii) ScanVIT method after heating; and iii) cultural standard method after heating. The results of the decontamination procedures and the detection methods were evaluated by comparing the number of Legionella-positive and -negative samples, and the recovery rates (CFU l-1) obtained by ScanVIT and the standard method. RESULTS: We find that ScanVIT method is highly sensitive with both decontamination treatments, yielding a higher recovery of L. pneumophila compared to the standard method. Conversely, ScanVIT associated with the acid-treatment yielded the highest recovery of L. non-pneumophila. CONCLUSIONS: The acid-treatment combined to the ScanVIT method increases the recovery of L. non-pneumophila in water samples compared to both ScanVIT associated with heat-treatment and standard culture method. Thus, this method may represent the best choice to detect L. non-pneumophila in water samples and reduce the risk of infection due to underestimation of Legionella loads.

Effectiveness of ATP bioluminescence to assess hospital cleaning: a review.

INTRODUCTION: Contamination of hospital surfaces plays an important role in the transmission of several healthcare-associated microorganisms, therefore methods for evaluating hospital surfaces' cleaning gain particular importance. Among these, there are visual inspection, quantitative microbiology, fluorescent markers and adenosine triphosphate (ATP) bioluminescence. The latter seems to provide interesting features, detecting the presence of ATP on surface (as Relative Light Units, RLU), a proxy of organic matter and microbial contamination. Several studies have investigated the effectiveness of this technology; with this research, we aim to summarize the most significant results. METHODS: A systematic review was conducted. The keywords (namely, "ATP", "bioluminescence", "hospital" and "surfaces") were searched in PubMed/MEDLINE and Scopus databases, in order to find relevant data, from January 2000 to October 2014. After the selection, we globally considered 27 articles. RESULTS: Most of the studies were conducted in United Kingdom and in USA. Different threshold RLU benchmark values were identified by analyzed studies. Fourteen of these researches compared the ATP bioluminescence with microbiological methods, 11 identified a significant correlation between the two methods, although poor or not complete for 5. DISCUSSION: ATP bioluminescence is not a standardized methodology: each tool has different benchmark values, not always clearly defined. At the moment, we can say that the technique could be used to assess, in real time, hospital surfaces where cleanliness is required, but not sterility.

Single domain amnestic MCI: a multiple cognitive domains fMRI investigation.

Amnestic mild cognitive impairment (a-MCI) is associated with the highest annual incidence of conversion to Alzheimer's disease (AD) (10-15%). a-MCI patients may have only a memory deficit (single domain: sd-a-MCI) or additional dysfunctions affecting other cognitive domains (multiple domain: md-a-MCI). Using functional magnetic resonance imaging (fMRI), we investigated brain activation in 16 sd-a-MCI patients and 14 controls during four different tasks assessing language, memory, attention and empathy functions. We found greater activation in sd-a-MCI compared with controls in the left inferior temporal gyrus (language), the right superior temporal gyrus (memory) and the right dorsal precentral gyrus (attention). Moreover, patients' activation correlated significantly with neuropsychological scores obtained at tests exploring the corresponding function. These findings indicate that fMRI is sensitive to detect early changes occurring in AD pathology and that individuals with sd-a-MCI show increased activation in multiple task-related brain regions. We suggest that these functional changes relate to the development of early compensatory mechanisms that reduce cognitive deficits associated with the progressive accumulation of brain damage.

Intracortical excitability in patients with relapsing-remitting and secondary progressive multiple sclerosis.

We designed this study to investigate possible correlations between variables measuring primary motor cortex excitability detected by single and paired-pulse transcranial magnetic stimulation (TMS) and the severity of clinical manifestations in patients with multiple sclerosis (MS). Thirty patients with MS in remission, 16 with relapsing-remitting (RR), 14 with secondary progressive disease (SP) and 17 healthy subjects participated in the study. In each subject, the central motor conduction time (CMCT) was calculated, and single-pulse and paired-pulse TMS at 3 and 10 ms interstimulus intervals was delivered over the primary motor cortex of the dominant hemisphere to measure the amplitude of motor-evoked potentials (MEPs), motor threshold (MTh), intracortical inhibition (ICI) and facilitation (ICF). Correlations were determined between the patients' TMS findings and magnetic resonance imaging (MRI) (lesion load) and clinical features (expanded disability status scale, EDSS score). EDSS scores were significantly higher in SPMS than in RRMS patients. The MTh was significantly higher, and the MEP was significantly smaller in SPMS patients than in RRMS patients and control subjects. All patients had longer CMCTs than healthy subjects. In all patients, paired-pulse TMS elicited an inhibited test MEP at the 3-ms ISI and a facilitated test MEP at the 10 ms ISI. Post hoc analysis showed that ICI was significantly lower in SPMS patients than in those with RRMS and healthy subjects. EDSS scores correlated significantly with TMS measures (MEP, ICI, CMCT and MTh), but not with MRI lesion load. It was found that intracortical excitability as measured with TMS differs according to the clinical course of MS; it remains normal in patients with low EDSS scores and is altered in patients with high EDSS scores.

Neural basis of maternal communication and emotional expression processing during infant preverbal stage.

During the first year of life, exchanges and communication between a mother and her infant are exclusively preverbal and are based on the mother's ability to understand her infant's needs and feelings (i.e., empathy) and on imitation of the infant's facial expressions; this promotes a social dialog that influences the development of the infant self. Sixteen mothers underwent functional magnetic resonance imaging while observing and imitating faces of their own child and those of someone else's child. We found that the mirror neuron system, the insula and amygdala were more active during emotional expressions, that this circuit is engaged to a greater extent when interacting with one's own child, and that it is correlated with maternal reflective function (a measure of empathy). We also found, by comparing single emotions with each other, that joy expressions evoked a response mainly in right limbic and paralimbic areas; by contrast, ambiguous expressions elicited a response in left high order cognitive and motor areas, which might reflect cognitive effort.

Periodical in-situ re-calibration of force platforms: a new method for the robust estimation of the calibration matrix.

The paper provides a new technique based on a least-squares approach for the accurate estimation of a force platform calibration matrix using simple manual procedures, when the direction of the applied loads cannot be perfectly aligned with the axes of the platform. This new procedure can be applied to all force platforms and allows the combined application of vertical and horizontal forces, both static and time-varying. The robust calibration method includes the angular errors in the least-squares parameter vector, thus reducing the bias in the estimated calibration matrix parameters. The performance of the robust method was compared with the conventional one, using a numerical simulation approach starting from a known calibration matrix. With the conventional approach, in noiseless conditions, the maximum error due to load misalignment (SD = 3 degrees) was 6% for the direct terms and over 10% for the cross-talk terms. With the robust method, these errors reduced to zero and were always below 0.4%, even when realistic noise was superimposed on the measures. With perfectly aligned loads and realistic output noise, the confidence intervals of the calibration matrix parameters were very similar for the two methods, demonstrating that the increased number of parameters did not affect the reliability of the estimate.

Enhanced brain motor activity in patients with MS after a single dose of 3,4-diaminopyridine.

BACKGROUND: 3,4-diaminopyridine (3,4-DAP), a potassium (K+) channel blocker, improves fatigue and motor function in multiple sclerosis (MS). Although it was thought to do so by restoring conduction to demyelinated axons, recent experimental data show that aminopyridines administered at clinical doses potentiate synaptic transmission. OBJECTIVE: To investigate motor cerebral activity with fMRI and transcranial magnetic stimulation (TMS) after a single oral dose of 3,4-DAP in patients with MS. METHODS: Twelve right-handed women (mean +/- SD age 40.9 +/- 9.3 years) underwent fMRI on two separate occasions (under 3,4-DAP and under placebo) during a simple motor task with the right hand. FMRI data were analyzed with SPM99. After fMRI, patients underwent single-pulse TMS to test motor threshold, amplitude, and latency of motor evoked potentials, central conduction time, and the cortical silent period; paired-pulse TMS to investigate intracortical inhibition (ICI) and intracortical facilitation (ICF); and quantitative electromyography during maximal voluntary contraction. RESULTS: FMRI motor-evoked brain activation was greater under 3,4-DAP than under placebo in the ipsilateral sensorimotor cortex and supplementary motor area (p < 0.05). 3,4-DAP decreased ICI and increased ICF; central motor conduction time and muscular fatigability did not change. CONCLUSION: 3,4-DAP may modulate brain motor activity in patients with MS, probably by enhancing excitatory synaptic transmission.

Structure suggests function: the case for synaptic ribbons as exocytotic nanomachines.

Synaptic ribbons, the organelles identified in electron micrographs of the sensory synapses involved in vision, hearing, and balance, have long been hypothesized to play an important role in regulating presynaptic function because they associate with synaptic vesicles at the active zone. Their physiology and molecular composition have, however, remained largely unknown. Recently, a series of elegant studies spurred by technical innovation have finally begun to shed light on the ultrastructure and function of ribbon synapses. Electrical capacitance measurements have provided sub-millisecond resolution of exocytosis, evanescent-wave microscopy has filmed the fusion of single 30 nm synaptic vesicles, electron tomography has revealed the 3D architecture of the synapse, and molecular cloning has begun to identify the proteins that make up ribbons. These results are consistent with the ribbon serving as a vesicle "conveyor belt" to resupply the active zone, and with the suggestion that ribbon and conventional chemical synapses have much in common.

Stick to the rules.

A young patient under ACO (anticoagulant) therapy with acute embolic stroke is presented, and therapeutic rules are discussed.

An improved technique for the extraction of stochastic parameters from stabilograms.

An improved characterization of the dynamics of postural sway can provide a better understanding about the functional organization of the postural control system as well as a more robust tool for postural pattern recognition. To this aim, a novel parameterization was applied to the stabilogram diffusion analysis formerly proposed by Collins and De Luca [Collins JJ, De Luca CJ. Open-loop and closed-loop control of posture: a random-walk analysis of center-of-pressure trajectories. Exp Brain Res 1993;95:308-18] that considered the act of maintaining posture as a stochastic process. The main purpose of the present technique was to overcome some drawbacks of the model presented by Collins and De Luca that may restrain its potential application in clinical practice. The approach uses a unique non-linear model to describe the center of pressure (COP) dynamics that reduces the number of parameters and decreases their intra-subject variability; consequently, fewer trials are required to perform reliable estimates of stochastic parameters and this is of particular importance for subjects that cannot afford many repeated measurements because of age or pathology. Four new statistical mechanics parameters (NSMP) were computed on the log-log stabilogram diffusion plots and their estimates were compared in terms of reliability and sensitivity to the visual conditions with: (1) a minimal set of four summary statistic scores (SSS); and (2) the six statistical mechanics parameters (SMP) proposed by Collins and De Luca. All four NSMP showed at least a fair-to-good reliability (intraclass correlation coefficient, ICC>0.49) while SMP (ICC>0.20) showed some poor reliability. A better overall reliability was also observed with respect to SSS. Moreover, only NSMP had a similar score for eyes open and eyes closed conditions. Three out of four NSMP were also significantly sensitive to eyes open or closed conditions (P<0.001) while only three out of six SMP were sensitive to operating conditions (P<0.01).

Synaptic vesicle populations in saccular hair cells reconstructed by electron tomography.

We used electron tomography to map the three-dimensional architecture of the ribbon-class afferent synapses in frog saccular hair cells. The synaptic body (SB) at each synapse was nearly spherical (468 +/- 65 nm diameter; mean +/- SD) and was covered by a monolayer of synaptic vesicles (34.3 nm diameter; 8.8% coefficient of variation), many of them tethered to it by approximately 20-nm-long filaments, at an average density of 55% of close-packed (376 +/- 133 vesicles per SB). These vesicles could support approximately 900 msec of exocytosis at the reported maximal rate, which the cells can sustain for at least 2 sec, suggesting that replenishment of vesicles on the SB is not rate limiting. Consistent with this interpretation, prolonged K+ depolarization did not deplete vesicles on the SB. The monolayer of SB-associated vesicles remained after cell lysis in the presence of 4 mM Ca2+, indicating that the association is tight and Ca2+-resistant. The space between the SB and the plasma membrane contained numerous vesicles, many of which ( approximately 32 per synapse) were in contact with the plasma membrane. This number of docked vesicles could support maximal exocytosis for at most approximately 70 msec. Additional docked vesicles were seen within a few hundred nanometers of the synapse and occasionally at greater distances. The presence of omega profiles on the plasma membrane around active zones, in the same locations as coated pits and coated vesicles labeled with an extracellular marker, suggests that local membrane recycling may contribute to the three- to 14-fold greater abundance of vesicles in the cytoplasm (not associated with the SB) near synapses than in nonsynaptic regions.

Calcium-triggered exocytosis and endocytosis in an isolated presynaptic cell: capacitance measurements in saccular hair cells.

Depolarization of isolated frog saccular hair cells caused Ca(2+)-dependent increases in membrane capacitance that we interpret as the fusion of synaptic vesicles with the plasma membrane. During a maintained depolarization to -10 mV, the capacitance increased at a rate corresponding to the fusion of approximately 500 vesicles per second at each active zone. Release continued at this high rate for up to 2 s, long enough to exhaust > 5 times the number of vesicles initially in close apposition to the plasma membrane at active zones. We therefore propose that hair cells are specialized for rapid replenishment of vesicles at release sites. Upon repolarization to -70 mV, the capacitance returned exponentially (time constant, approximately 14 s) to near the prestimulus level in perforated-patch recordings, but not in whole-cell recordings, suggesting that a mobile intracellular factor is required for membrane retrieval.

Calcium signalling in hair cells: multiple roles in a compact cell.

Ca2+ is critical for mechanosensory adaptation, frequency tuning, afferent synaptic transmission, and efferent modulation in hair cells. These four processes involve cytoplasmic Ca2+ in three independent signalling pathways. Recent work suggests that Ca2+ regulates a myosin adaptation motor, and that a mobile Ca2+ buffer is highly concentrated in hair cells. Focal Ca2+ entry and the cytoplasmic Ca2+ buffer help to separate these pathways by limiting the spread of Ca2+ signals.

Symmetrical segregation of potassium channels at cytokinesis.

To determine how voltage-gated ion channels segregate between sibling cells at cytokinesis, we used a whole-cell patch clamp to measure the electrophysiological phenotypes of siblings within 45 min of division. Recently born siblings in an immortalized line of embryonic retinal cells were identified as pairs of spherical cells adhering to one another. All siblings were electrically coupled when cells were simultaneously voltage clamped, whereas nonsiblings were not coupled. Twelve pairs of siblings were electrically isolated by mechanical separation so that their phenotypes could be measured independently. Cells expressed two principal membrane conductances, delayed rectifier-like (IK) and inward rectifier (IK(IR)) potassium currents. Despite qualitative and quantitative variability in IK and IK(IR) expression within the population, each cell of a given pair expressed similar steady-state current densities between -110 and +50 mV. We estimated IK(IR) slope conductance by blocking the current specifically with 5 mM Cs and calculated IK(IR) ratios in siblings and nonsiblings. Three pairs of siblings expressed IK(IR) ratios of approximately 1.2, while ratios in three pairs of adhered nonsiblings varied between 1.6 and 5.4. When currents were sampled continuously through cytokinesis by using the perforated-patch recording mode, current amplitude showed no net change within 30 min of division. Because channel number did not appear to change in siblings during this interval, parental channels were inherited by each daughter in proportion to the area of membrane received. Heterogeneity therefore arises after siblings reenter interphase and is not due to the asymmetrical segregation of channels at cytokinesis.

Clonal cells from embryonic retinal cell lines express qualitative electrophysiological differences.

Cells from the embryonic quail retina were immortalized with the v-mil oncogene and cloned by limiting dilution. Their phenotype was examined using the whole-cell patch clamp method. Three membrane currents, IK(IR), INa and IK, were found at different frequencies within a sample of 170 cells drawn from a large clone. Nearly all combinations of these three markers were found and the frequency of combinations showed that the markers assorted independently. Examination of clones of less than 10 cells showed that heterogeneity originates with a high probability within clones, arguing that chromosomal mutation, for example, is unlikely to account for phenotypic diversity. A possible explanation is that phenotypic differences between cells might reflect the local exchange of instructive signals. If so, then the genes for the three phenotypic markers are controlled independently.