Visualizing dynamic three-dimensional changes of human reticular dermal collagen under mechanical strain.

In clinical practice, plastic surgeons are often faced with large skin defects that are difficult to close primarily. Management of large skin wounds e.g. burns or traumatic lacerations requires knowledge of skin biomechanic properties. Research into skin microstructural adaptation to mechanical deformation has only been performed using static regimes due to technical limitations. Here, we combine uniaxial stretch tests with fast second harmonic generation imaging and we apply this for the first time to investigate dynamic collagen rearrangement in reticular human dermis.Ex vivohuman skin from the abdomen and upper thigh was simultaneously uniaxially stretched while either periodically visualizing 3D reorganization, or visualizing 2D changes in real time. We determined collagen alignment via orientation indices and found pronounced variability across samples. Comparing mean orientation indices at the different stages of the stress strain curves (toe, heel, linear) showed a significant increase in collagen alignment during the linear part of the mechanical response. We conclude that fast SHG imaging during uni-axial extension is a promising research tool for future studies on skin biomechanic properties.

Development of Three Molecular Diagnostic Tools for the Identification of the False Codling Moth (Lepidoptera: Tortricidae).

Three molecular protocols using qPCR TaqMan probe, SYBR Green, and loop-mediated isothermal amplification (LAMP) methods were set up for the identification of larvae and adults of an African invasive moth, Thaumatotibia leucotreta (Meyrick, 1913) (Lepidoptera: Tortricidae). The DNA extracts from larval and adult samples of T. leucotreta were perfectly amplified with an average Ct value of 19.47 ± 2.63. All assays were demonstrated to be inclusive for T. leucotreta and exclusive for the nontarget species tested; the absence of false positives for nontarget species showed a 100% of diagnostic specificity and diagnostic sensitivity for all assays. With the SYBR Green protocol, the Cq values were only considered for values less than 22 (cutoff value) to prevent false-positive results caused by the late amplification of nonspecific amplicons. The limit of detection (LoD) for the qPCR probe protocol was equal to 0.02 pg/µl while a value equal to 0.128 pg/µl for the qPCR SYBR Green assay and LAMP method were established, respectively. The intrarun variabilities of reproducibility and repeatability in all the assays evaluated as CV%, ranged between 0.21 and 6.14, and between 0.33 and 9.52, respectively; the LAMP values were slightly higher than other assays, indicating a very low interrun variability. In order for an operator to choose the most desirable method, several parameters were considered and discussed. For future development of these assays, it is possible to hypothesize the setup of a diagnostic kit including all the three methods combined, to empower the test reliability and robustness.

Power spectral analysis of two-channel EEG in very premature infants undergoing heat loss prevention.

OBJECTIVE: To evaluate whether wearing a wool cap, a routine practice used to prevent heat loss in premature infants, affects interpretation of electroencephalogram spectral analysis. METHODS: Eighteen premature infants (median gestational age 28 weeks, range 23-32) without neurological complications were randomized to two channel (C3, C4 referred to Cz) digital electroencephalogram recordings with (90 min) and without (90 min) wearing wool cap, at 4 days of life. Electroencephalogram was analyzed automatically by measurement of burst suppression ratio and asymmetry index and by Fast Fourier Transform to calculate total absolute spectral power; relative spectral power in the δ (0.5-3.5 Hz), θ (4-7.5 Hz), α (8-12.5 Hz), and ß (13-30 Hz) frequency bands; spectral edge frequency; and mean dominant frequency. RESULTS: The use of wool cap had no effect on all electroencephalogram parameters considered. Gestational age showed an effect on relative spectral power of all considered bands, spectral edge frequency and mean dominant frequency, while no effect was seen on burst suppression ratio and asymmetry index. Neonates born at gestational weeks lower than 28 had significantly higher relative power in the δ band and lower relative power in the α and ß bands. CONCLUSIONS: Heat loss prevention using wool cap does not affect interpretation of spectral electroencephalogram. Spectral values in our group of very premature infants without neurological complications correspond to normal data reported in the literature. Maturation changes consist of reduction of relative power of the δ band, spectral edge frequency and mean dominant frequency.

Norovirus gastroenteritis and seizures: an atypical case with neuroradiological abnormalities.

We describe an 8-year-old boy admitted because of prolonged seizures during norovirus gastroenteritis without any signs of encephalopathy. Blood tests were normal and cerebrospinal fluid examination resulted negative for both bacteria and viruses. A reverse transcriptase polymerase chain reaction revealed norovirus RNA in a stool sample. A cerebral computed tomography turned out to be normal whereas subsequent cerebral magnetic resonance imaging showed transitory signal abnormalities consistent with vasogenic edema. The post-ictal electroencephalogram revealed normal background activity with sporadic left posterior delta waves. The child was discharged after 10 days with an unremarkable physical examination. A cerebral magnetic resonance imaging and an electroencephalogram after 1 month were both negative. We report a new case of benign infantile convulsions due to norovirus gastroenteritis with neuroradiological abnormalities to the pertinent literature in order to improve knowledge about this disorder and increase the possibility of clarifying its pathogenesis.

Experimental evidence of signal-optical noise interferencelike effect in underwater amplitude-modulated laser optical radar systems.

We report experimental evidence that in an amplitude-modulated laser optical radar system for underwater 3D imaging the observed contrast oscillations as a function of the modulation frequency originate from an interference-like effect between target signal VT and water backscattered radiation VW. The demonstration relies on the ability to perform a direct measurement of VW in a 25 m long test tank. The proposed data processing method enables one to remove the contribution of water backscattering from the detected signal and drastically reduce signal fluctuations due to the medium. Experiments also confirm the possibility to improve the signal to optical noise ratio and contrast by increasing the modulation frequency.

Improvement in underwater phase measurement of an amplitude-modulated laser beam by polarimetric techniques.

The phase of the amplitude-modulated radiation reflected by a Lambertian target immersed in water was measured by using a linearly and circularly polarized sounding laser beam. Different values of the water extinction coefficient in the range of 0.06 - 2 m(-1) were realized by adding skim milk as a scattering element. It is shown that very efficient rejection of optical noise, resulting in reliable phase measurements, is accomplished with a cross-polarized and copolarized detection scheme for linear and circular polarization, respectively. The experiment demonstrates that phase measurements are very sensitive to optical noise suppression and that, as far as single scattering is the main involved mechanism, significant improvements can be achieved by adopting a polarization control on both the transmitter and the receiver stages of the apparatus.

Thrombophilic risk factors and unusual clinical features in three Italian CADASIL patients.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetically transmitted cerebrovascular disease. Typically, the first clinical manifestation is migraine and the full clinical spectrum of the disease with recurrent strokes of the subcortical type, cognitive, and mood disorders is seen during the fourth and fifth decades of life. Vascular risk factors are usually absent in CADASIL patients and the diagnosis of the disease is particularly suspected in young adults with cerebrovascular events of unknown cause, diffuse leukoencephalopathy on computed tomography or magnetic resonance imaging, and a history of cerebrovascular diseases or dementia in many family members. We describe three Italian CADASIL patients who presented to medical attention for cerebrovascular events occurred after the age of 55 and had, in addition to hypertension and hyperlipidemia, thrombophilic risk factors such as hyperhomocysteinemia, elevated levels of lipoprotein(a), and antiphospholipid antibodies. Symptoms possibly related to cortical involvement, such as dysphasia and visual field deficits, were reported by two of these patients. We conclude that a diagnosis of CADASIL should not be disregarded in patients with vascular risk factors and presenting with symptoms not immediately referable to subcortical damage at ages more advanced than commonly reported.

Longitudinal evaluation of leukoaraiosis with whole brain ADC histograms.

FLAIR and diffusion-weighted MRI were obtained twice (mean interval 20 +/- 4 months) in 10 patients with leukoaraiosis. At follow-up, visual extension of leukoaraiosis was unchanged, whereas the median of whole brain apparent diffusion coefficient (WB-ADC) histogram was increased (p= 0.008) and brain volume index (BVI) was decreased (p = 0.006). WB-ADC histogram and BVI are sensitive to leukoaraiosis and might be considered for monitoring progression of the disease.

Pulmonary function in non-insulin-dependent diabetes mellitus.

BACKGROUND: In type I diabetes mellitus, lung function has been investigated in several clinical studies, but there are few data concerning pulmonary function abnormalities in patients with non-insulin-dependent diabetes mellitus (NIDDM). OBJECTIVES: The aim of this study was to assess the presence of pulmonary function abnormalities in patients with NIDDM and to verify the possible associations between diabetic renal microangiopathy, retinopathy and diabetes control. METHOD AND PATIENTS: Thirty patients with NIDDM were collected and divided into two similar groups: subjects with retinopathy and/or diabetic glomerulopathy (group 1, n = 15) and patients without any complications (group 2, n = 15). 17 were males and 13 females, aged from 45 to 81 years. They had had diabetes for 3-23 years and were studied at the Division of Internal Medicine, with an outpatient service for diabetic patients. All patients were non-smokers. The presence of diabetic glomerulopathy was determined by measuring the 24-hour protein excretion rate using the nephelometric method. The presence of retinopathy was determined by using ophthalmoscopy. Glycosylated hemoglobin was measured as an indicator of glycemic control. We performed a global spirometry and measured pulmonary diffusion capacity by the single-breath method corrected by alveolar volume. RESULTS: We found a significant reduction in lung diffusion capacity for carbon monoxide (DL(CO)) in the group of patients with other signs of diabetic microangiopathy (p < 0.005) and a significative correlation between DL(CO )and the grade of albuminuria (r = -0.83, p < 0.001). CONCLUSIONS: Pulmonary function abnormalities, in particular a reduction in diffusion capacity, are common in patients with NIDDM and signs of diabetic microangiopathy. A possible explanation is related to an impaired pulmonary microvasculature and alveolar epithelial basal lamina.

Performance assessment of new multiplex probe assay for identification of mycobacteria.

A new DNA probe assay (INNO LiPA Mycobacteria; Innogenetics, Ghent, Belgium) for the simultaneous identification, by means of reverse hybridization and line-probe technology, of Mycobacterium tuberculosis complex, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium gordonae, the species of the Mycobacterium avium complex (MAC), Mycobacterium scrofulaceum, and Mycobacterium chelonae was evaluated on a panel of 238 strains including, besides representatives of all the taxa identifiable by the system, a number of other mycobacteria, some of which are known to be problematic with the only other commercial DNA probe system (AccuProbe; Gen-Probe, San Diego, Calif.), and two nocardiae. The new kit, which includes a control probe reacting with the whole genus Mycobacterium, correctly identified 99.6% of the strains tested; the one discrepancy, which remained unresolved, concerned an isolate identified as MAC intermediate by INNO LiPA Mycobacteria and as Mycobacterium intracellulare by AccuProbe. In five cases, because of an imperfect checking of hybridization temperature, a very slight, nonspecific, line was visible which was no longer evident when the test was repeated. Two strains whose DNA failed amplification at the first attempt were regularly identified when the test was repeated. Interestingly, the novel kit dodged all the pitfalls presented by the strains giving anomalous reactions with AccuProbe. A unique feature of INNO LiPA Mycobacteria is its ability to recognize different subgroups within the species M. kansasii and M. chelonae, while the declared overlapping reactivity of probe 4 with some M. kansasii and Mycobacterium gastri organisms and of probe 9 with MAC, Mycobacterium haemophilum, and Mycobacterium malmoense, may furnish a useful aid for their identification. The turnaround time of the method is approximately 6 h, including a preliminary PCR amplification.

Comparison of Mycobacterium tuberculosis susceptibility testing performed with BACTEC 460TB (Becton Dickinson) and MB/BacT (Organon Teknika) systems.

The recently introduced automated culture systems MB/BacT (Organon Teknika, Belgium) was compared with radiometric BACTEC 460TB (Becton Dickinson, USA) to test antimicrobial susceptibility of Mycobacterium tuberculosis to first line drugs. On 113 strains 97.5% agreement was obtained, with the difference being not significant. Concordance was practically complete for the most important drugs, isoniazid and rifampin. The two methods however significantly differed for the time needed to complete the test; in fact MB/BacT required on the average five days more than BACTEC 460TB. Despite the delay in the completion of the test, the excellent reliability along with the elimination of radioactivity and full automation make MB/BacT an attractive alternative for susceptibility testing of M. tuberculosis.

Effects of histamine H3 receptor agonists and antagonists on cognitive performance and scopolamine-induced amnesia.

In previous research we found that pre-training administration of histamine H3 receptor agonists such as (R)-alpha-methylhistamine and imetit impaired rat performance in object recognition and a passive avoidance response at the same doses at which they inhibited the release of cortical acetylcholine in vivo. Conversely, in the present study we report that the post-training administration of (R)-alpha-methylhistamine and imetit failed to affect rat performance in object recognition and a passive avoidance response, suggesting that H3 receptor influences the acquisition and not the recall processes. We also investigated the effects of two H3 receptor antagonists, thioperamide and clobenpropit, in the same behavioral tasks. Pre-training administration of thioperamide and clobenpropit failed to exhibit any procognitive effects in normal animals but prevented scopolamine-induced amnesia. However, also post-training administration of thioperamide prevented scopolamine-induced amnesia. Hence, the ameliorating effects of scopolamine-induced amnesia by H3 receptor antagonism are not only mediated by relieving the inhibitory action of cortical H3 receptors, but other mechanisms are also involved. Nevertheless, H3 receptor antagonists may have implications for the treatment of degenerative disorders associated with impaired cholinergic function.

Effect of subchronic treatment with metrifonate and tacrine on brain cholinergic function in aged F344 rats.

The effects of 21-day treatment with the acetylcholinesterase inhibitors metrifonate (80 mg kg(-1) per os (p.o.)) and tacrine (3 mg kg(-1) p.o.), twice daily, on cortical and hippocampal cholinergic systems were investigated in aged rats (24-26 months). Extracellular acetylcholine levels were measured by transversal microdialysis in vivo; choline acetyltransferase and acetylcholinesterase activities were measured ex vivo by means of radiometric methods. Basal cortical and hippocampal extracellular acetylcholine levels, measured 18 h after the last metrifonate treatment, were about 15 and two folds higher, respectively, than in control and tacrine-treated rats. A challenge with metrifonate further increased cortical and hippocampal acetylcholine levels by about three and four times, respectively. Basal extracellular acetylcholine levels, measured 18 h after the last treatment with tacrine were not statistically different from those of the control rats. A challenge with tacrine increased cortical and hippocampal extracellular acetylcholine levels by about four and two times. A 75% inhibition of cholinesterase activity was found 18 h after the last metrifonate administration, while only a 15% inhibition was detectable 18 h after the last tacrine administration. The challenge with metrifonate or tacrine resulted in 90 and 80% cholinesterase inhibition, respectively. These results demonstrate that in aging rats a subchronic treatment with metrifonate results in a long-lasting, cholinesterase inhibition, and a persistent increase in acetylcholine extracellular levels which compensate for the age-associated cholinergic hypofunction. Metrifonate is therefore a potentially useful agent for the cholinergic deficit accompanying Alzheimer's disease.

Reticulocytes and reticulated platelets: simultaneous measurement in whole blood by flow cytometry.

Reticulated platelets are a fraction of newly released circulating elements characterized by a residual amount of RNA. It has been suggested that the reticulated platelet count, providing an estimate of thrombopoiesis in the same way as erythrocyte reticulocyte count is a measure of erythropoiesis, may be useful in the study of thrombocytopenic disorders. Reticulated red cells and platelets can be analyzed by flow cytometry using specific stains for nucleic acids such as Thiazole Orange and Auramine-O. The aim of our work was to perform the simultaneous evaluation of reticulated elements in whole blood using a standard flow cytometer and to correlate the results obtained with a dedicated cytometer. A group of 14 patients with abnormal absolute reticulocyte counts (range 1.1-11%) and a group of 41 patients showing a platelet discrimination error when analyzed with a dedicated flow cytometer (Sysmex R1000) were enrolled. Linear amplification of both scatter and fluorescence was used to perform reticulocyte count. A gate was set on platelet dimensions, and logarithmic amplification of scatter and fluorescence was used to count reticulated platelets. A good correlation was obtained both for results of reticulocyte count (r2 = 0.9825) and for reticulated platelets (r2 = 0.8717) between our method and those using dedicated instruments. These data show that reticulated platelet count may be easily introduced in clinical laboratories that routinely perform reticulocyte count by flow cytometry.

Acetylcholine release from the frontal cortex during exploratory activity.

The activation of the cortical cholinergic system was investigated in 3- and 25-month-old male Wistar rats, by measuring by transversal microdialysis the changes in cortical extracellular acetylcholine (ACh) levels during the performance of simple spontaneous tasks involving exploratory activity and working memory. Two days after implantation of the microdialysis probe in the frontal cortex, object recognition was investigated by either moving the rats from the home cage to the arena containing the objects or keeping the rats in the arena and introducing the objects. Spontaneous alternation was investigated in a Y runway. Young rats discriminated between familiar and novel objects and alternated in the Y runway, while aged rats were unable to discriminate. Whenever rats were moved from the home cage to the arena, ACh release increased (+70-80%) during the exploratory activity. Handling per se had no effect on extracellular ACh levels. When young rats were left in the arena, introduction of the objects caused some exploratory activity and object recognition but no increase in ACh release. ACh release increased by about 300% during spontaneous alternation. In aging rats basal extracellular ACh levels and their increase after placement in the arena were less than half that in young rats. Our work demonstrates that a novel environment activates the cortical cholinergic system, which presumably is associated with arousal mechanisms and selective attentional functions. It also demonstrates that in aging rats the cortical cholinergic hypofunction is associated with a loss of non-spatial working memory.

Effect of metrifonate on extracellular brain acetylcholine and object recognition in aged rats.

The effects of metrifonate were investigated in 4-6- and 22-24-month-old rats. Extracellular acetylcholine levels were measured by transversal microdialysis in vivo. Baseline extracellular acetylcholine levels in the cerebral cortex and hippocampus were 42% and 60% lower, respectively, in old than in young rats. Old rats did not discriminate between familiar and novel objects. In old rats, metrifonate (80 mg/kg p.o.) brought about 85% inhibition of cholinesterase activity in the cortex and hippocampus, a 4-fold increase in extracellular acetylcholine levels in the cortex only, and restored object recognition. In young rats, metrifonate caused 75% cholinesterase inhibition in the cerebral cortex and hippocampus, a 2-fold increase in cortical and hippocampal extracellular acetylcholine levels, and no effect on object recognition. The slight cholinesterase inhibition following metrifonate (30 mg/kg) in aged rats had no effect on cortical acetylcholine levels and object recognition. In conclusion, metrifonate may improve the age-associated cholinergic hypofunction and cognitive impairment.