Near-infrared hyperspectral imaging (NIR-HSI) and normalized difference image (NDI) data processing: An advanced method to map collagen in archaeological bones.

In the present study, an innovative and highly efficient near-infrared hyperspectral imaging (NIR-HSI) method is proposed to provide spectral maps able to reveal collagen distribution in large-size bones, also offering semi-quantitative estimations. A recently introduced method for the construction of chemical maps, based on Normalized Difference Images (NDI), is declined in an innovative approach, through the exploitation of the NDI values computed for each pixel of the hyperspectral image to localize collagen and to extract information on its content by a direct comparison with known reference samples. The developed approach addresses an urgent issue of the analytical chemistry applied to bioarcheology researches, which rely on well-preserved collagen in bones to obtain key information on chronology, paleoecology and taxonomy. Indeed, the high demand for large-sample datasets and the consequent application of a wide variety of destructive analytical methods led to the considerable destruction of precious bone samples. NIR-HSI pre-screening allows researchers to properly select the sampling points for subsequent specific analyses, to minimize costs and time and to preserve integrity of archaeological bones (which are available in a very limited amount), providing further opportunities to understand our past.

Artificial nose, NIR and UV-visible spectroscopy for the characterisation of the PDO Chianti Classico olive oil.

An authentication study of the Italian PDO (Protected Designation of Origin) olive oil Chianti Classico, based on artificial nose, near-infrared and UV-visible spectroscopy, with a set of samples representative of the whole Chianti Classico production area and a considerable number of samples from other Italian PDO regions was performed. The signals provided by the three analytical techniques were used both individually and jointly, after fusion of the respective variables, in order to build a model for the Chianti Classico PDO olive oil. Different signal pre-treatments were performed in order to investigate their importance and their effects in enhancing and extracting information from experimental data, correcting backgrounds or removing baseline variations. Stepwise-Linear Discriminant Analysis (STEP-LDA) was used as a feature selection technique and, afterward, Linear Discriminant Analysis (LDA) and the class-modelling technique Quadratic Discriminant Analysis-UNEQual dispersed classes (QDA-UNEQ) were applied to sub-sets of selected variables, in order to obtain efficient models capable of characterising the extra virgin olive oils produced in the Chianti Classico PDO area.

Application of multi-way analysis to UV-visible spectroscopy, gas chromatography and electronic nose data for wine ageing evaluation.

In this study, a multi-way method (Tucker3) was applied to evaluate the performance of an electronic nose for following the ageing of red wines. The odour evaluation carried out with the electronic nose was combined with the quantitative analysis of volatile composition performed by GC-MS, and colour characterisation by UV-visible spectroscopy. Thanks to Tucker3, it was possible to understand connections among data obtained from these three different systems and to estimate the effect of different sources of variability on wine evaluation. In particular, the application of Tucker3 supplied a global visualisation of data structure, which was very informative to understand relationships between sensors responses and chemical composition of wines. The results obtained indicate that the analytical methods employed are useful tools to follow the wine ageing process, to differentiate wine samples according to ageing type (either in barrel or in stainless steel tanks with the addition of small oak wood pieces) and to the origin (French or American) of the oak wood. Finally, it was possible to designate the volatile compounds which play a major role in such a characterisation.

Characterisation of PDO olive oil Chianti Classico by non-selective (UV-visible, NIR and MIR spectroscopy) and selective (fatty acid composition) analytical techniques.

An authentication study of the Italian PDO (protected designation of origin) extra virgin olive oil Chianti Classico was performed; UV-visible (UV-vis), Near-Infrared (NIR) and Mid-Infrared (MIR) spectroscopies were applied to a set of samples representative of the whole Chianti Classico production area. The non-selective signals (fingerprints) provided by the three spectroscopic techniques were utilised both individually and jointly, after fusion of the respective profile vectors, in order to build a model for the Chianti Classico PDO olive oil. Moreover, these results were compared with those obtained by the gas chromatographic determination of the fatty acids composition. In order to characterise the olive oils produced in the Chianti Classico PDO area, UNEQ (unequal class models) and SIMCA (soft independent modelling of class analogy) were employed both on the MIR, NIR and UV-vis spectra, individually and jointly, and on the fatty acid composition. Finally, PLS (partial least square) regression was applied on the UV-vis, NIR and MIR spectra, in order to predict the content of oleic and linoleic acids in the extra virgin olive oils. UNEQ, SIMCA and PLS were performed after selection of the relevant predictors, in order to increase the efficiency of both classification and regression models. The non-selective information obtained from UV-vis, NIR and MIR spectroscopy allowed to build reliable models for checking the authenticity of the Italian PDO extra virgin olive oil Chianti Classico.

Computer-assisted modelling and optimisation of reversed-phase high-temperature liquid chromatographic (RP-HTLC) separations.

The use of high temperatures (above 100 °C) in reversed-phase liquid chromatography (RP-HTLC) has opened up novel and enhanced applications for this essential separation technique. Although the favourable effects of temperature on LC have been extensively studied both theoretically and practically, its potential application to method development has barely been investigated. These favourable effects include enhanced speed, efficiency, resolution and detectability, as well as changes in selectivity, especially for polar and ionisable compounds, and the emergence of new options such as temperature programming and the concomitant use of solvent and temperature gradients, green separations, and so on. The recent availability of silica-based columns that routinely support high temperatures in addition to more conventional temperature-resistant columns (based on graphitised carbon, polymers and zirconium oxide) and dedicated column ovens that allow accurate temperature control up to 200 °C makes it possible to conceive of RP-HTLC as a routine separation technique in the modern analytical laboratory. On the other hand, the addition of temperature as a new optimisable parameter to RPLC adds further complexity to method development. Thus, new computer-assisted optimisation tools that extend the capabilities of current computer-assisted tools are being specifically developed for this type of separation. A new specially developed computer-assisted method development (CAMD) tool is presented herein, and its efficiency is demonstrated. This CAMD is based on the development of a rugged retention model for peaks, allowing the simulation of any kind of RP-HTLC separation, including isocratic, linear, curved, multilinear and stepwise gradients of solvent composition concomitant with constant, linear and multilinear temperature gradients. Both the retention models and the unattended optimisation of separations are driven by evolutionary algorithms, thus providing negligible-cost, rapid, highly efficient, and user-friendly optimisation processes.

NIR and UV-vis spectroscopy, artificial nose and tongue: comparison of four fingerprinting techniques for the characterisation of Italian red wines.

Four rapid and low-cost vanguard analytical systems (NIR and UV-vis spectroscopy, a headspace-mass based artificial nose and a voltammetric artificial tongue), together with chemometric pattern recognition techniques, were applied and compared in addressing a food authentication problem: the distinction between wine samples from the same Italian oenological region, according to the grape variety. Specifically, 59 certified samples belonging to the Barbera d'Alba and Dolcetto d'Alba appellations and collected from the same vintage (2007) were analysed. The instrumental responses, after proper data pre-processing, were used as fingerprints of the characteristics of the samples: the results from principal component analysis and linear discriminant analysis were discussed, comparing the capability of the four analytical strategies in addressing the problem studied.

Multivariate range modeling, a new technique for multivariate class modeling: the uncertainty of the estimates of sensitivity and specificity.

MRM, multivariate range modeling, is based on models built as parallelepipeds in the space of the original variables and/or of discriminant variables as those of linear discriminant analysis. The ranges of these variables define the boundary of the model. The ranges are increased by a "tolerance" factor to take into account the uncertainty of their estimate. MRM is compared with UNEQ (the modeling technique based on the hypothesis of multivariate normal distribution) and with SIMCA (based on principal components) by means of the sensitivities and specificities of the models, the estimates of type I (sensitivity) and II error rates (specificity) evaluated both with the final model built with all the available objects and by means of cross validation. UNEQ and SIMCA models were obtained with the usual critical significance value of 5% and with the model forced to accept all the objects of the modeled category. The performance parameters of the class models are critically discussed focusing on their uncertainty.

Homeobox genes and sea urchin development.

We describe the expression of three Paracentrotus lividus homeobox-containing genes of the dispersed class during sea urchin embryogenesis and discuss their possible roles in the mechanisms of cell specification and embryo morphogenesis. PlHbox12 represents the first regulator identified in sea urchin that belongs to the zygotic class of transcription factors. Its early and transient expression and the localization of transcripts suggests that PlHbox12 is involved in cell fate specification of the oral or aboral ectodermal territories at the early cleavage stages. PlHbox9 is expressed just after the completion of gastrulation in a narrow stripe of cells at the ectoderm-endoderm boundary. It probably organizes a novel spatial boundary which definitely separates the archenteron and the aboral ectoderm. Finally, the spatial and temporal expression of the PlOtp gene strongly indicate that this regulator is conditionally activated in few cells of the oral ectoderm and is involved in patterning of this territory at late stages. Furthermore, our data indicate that PlOtp acts upstream of signaling systems that lead to the activation of the primary mesenchyme cell gene expression program and skeletal morphogenesis.

Precambrian animal diversity: putative phosphatized embryos from the Doushantuo Formation of China.

Putative fossil embryos and larvae from the Precambrian phosphorite rocks of the Doushantuo Formation in Southwest China have been examined in thin section by bright field and polarized light microscopy. Although we cannot completely exclude a nonbiological or nonmetazoan origin, we identified what appear to be modern cnidarian developmental stages, including both anthozoan planula larvae and hydrozoan embryos. Most importantly, the sections contain a variety of small (

Spatially restricted expression of PlOtp, a Paracentrotus lividus orthopedia-related homeobox gene, is correlated with oral ectodermal patterning and skeletal morphogenesis in late-cleavage sea urchin embryos.

Several homeobox genes are expressed in the sea urchin embryo but their roles in development have yet to be elucidated. Of particular interest are homologues of homeobox genes that in mouse and Drosophila are involved in patterning the developing central nervous system (CNS). Here, we report the cloning of an orthopedia (Otp)-related gene from Paracentrotus lividus, PlOtp. Otp is a single copy zygotic gene that presents a unique and highly restricted expression pattern. Transcripts were first detected at the mid-gastrula stage in two pairs of oral ectoderm cells located in a ventrolateral position, overlying primary mesenchyme cell (PMC) clusters. Increases in both transcript abundance and the number of Otp-expressing cells were observed at prism and pluteus stages. Otp transcripts are symmetrically distributed in a few ectodermal cells of the oral field. Labelled cells were observed close to sites of active skeletal rod growth (tips of the budding oral and anal arms), and at the juxtaposition of stomodeum and foregut. Chemicals known to perturb PMC patterning along animal-vegetal and oral-aboral axes altered the pattern of Otp expression. Vegetalization by LiCl caused a shift in Otp-expressing cells toward the animal pole, adjacent to shifted PMC aggregates. Nickel treatment induced expression of the Otp gene in an increased number of ectodermal cells, which adopted a radialized pattern. Finally, ectopic expression of Otp mRNA affected patterning along the oral-aboral axis and caused skeletal abnormalities that resembled those exhibited by nickel-treated embryos. From these results, we conclude that the Otp homeodomain gene is involved in short-range cell signalling within the oral ectoderm for patterning the endoskeleton of the larva through epithelial-mesenchymal interactions.

Stable isotope techniques for the detection of Helicobacter pylori infection in clinical practice. 13C-Urea Breath Test in different experimental conditions.

The 13C-UBT has been demonstrated to be a reliable method for the evaluation of Helicobacter pylori infection. The aim of our work is to determine the cut-off point of the 13C-UBT for samples collected as gas or collected in a solution of triethanolamine. For this purpose, patients fasted for at least 6 hours were able to collect basal samples before the administration of 65 mg of 13C-urea solution. Breath samples were taken 10, 30 and 60 minutes after the administration of the labeled solution. All the samples were collected in gas collectors and in glass vials containing 1 ml of a 7% triethanolamine solution. The cut-off points for gas collected samples were established in 4.0/1000, 4.6/1000 and 4.4/1000 for 10, 30 and 60 minutes samples, respectively, while for the samples collected in triethanolamine solution, cut-off points were established in 5.0/1000, for the 10 minutes samples, in 3.5/1000 for the 30 minutes samples and 4.7/1000 for the 60 minutes samples. We found that this test has a sensitivity of 100% and a specificity of 100% for H. pylori detection in both experimental conditions, when multiple breath samples are taken. If we considered only the 30 minutes time, sensitivity and specificity diminish for the gas collected samples. We conclude that the collection of breath samples in triethanolamine solution allows a better differentiation between H. pylori infected and non infected patients than gas collected samples.

Stable isotope techniques for the detection of Helicobacter pylori infection in clinical practice. 13C-urea breath test in different experimental conditions

The (13)C-UBT has been demonstrated to be a reliable method for the evaluation of Helicobacter pylori infection. The aim of our work is to determine the cut-off point of the (13)C-UBT for samples collected as gas or collected in a solution of triethanolamine. For this purpose, patients fasted for at least 6 hours were able to collect basal samples before the administration of 65 mg of (13)C-urea solution. Breath samples were taken 10,30 and 60 minutes after the administration of the labeled solution. All the samples were collected in gas collectors and in glass vials containing 1 ml of a 7 per cent triethanolamine solution. The cut-off points for gas collected samples were established in 4.0 per cent and for 10, 30 and 60 minutes samples, respectively, while for the samples, collected in triethanolamine solution, cut-off points were established in 5.0 per cent, for the 10 minutes samples, in 3.5 per cent for the 30 minutes samples and 4.7 per cent for the 60 minutes samples. We found that this test has a sensitivity of 100 per cent and a specificity of 100 per cent for H. pylori detection in both experimental conditions, when multiple breath samples are taken. If we considered only the 30 minutes time, sensitivity and specificity diminish for the gas collected samples. We conclude that the collection of breath samples in triethanolamine solution allows a better differentiation between H. pylori infected and non infected patients than gas collected samples.

Stable isotope techniques for the detection of Helicobacter pylori infection in clinical practice. 13C-urea breath test in different experimental conditions

The (13)C-UBT has been demonstrated to be a reliable method for the evaluation of Helicobacter pylori infection. The aim of our work is to determine the cut-off point of the (13)C-UBT for samples collected as gas or collected in a solution of triethanolamine. For this purpose, patients fasted for at least 6 hours were able to collect basal samples before the administration of 65 mg of (13)C-urea solution. Breath samples were taken 10,30 and 60 minutes after the administration of the labeled solution. All the samples were collected in gas collectors and in glass vials containing 1 ml of a 7 per cent triethanolamine solution. The cut-off points for gas collected samples were established in 4.0 per cent and for 10, 30 and 60 minutes samples, respectively, while for the samples, collected in triethanolamine solution, cut-off points were established in 5.0 per cent, for the 10 minutes samples, in 3.5 per cent for the 30 minutes samples and 4.7 per cent for the 60 minutes samples. We found that this test has a sensitivity of 100 per cent and a specificity of 100 per cent for H. pylori detection in both experimental conditions, when multiple breath samples are taken. If we considered only the 30 minutes time, sensitivity and specificity diminish for the gas collected samples. We conclude that the collection of breath samples in triethanolamine solution allows a better differentiation between H. pylori infected and non infected patients than gas collected samples. (AU)

Stable isotope techniques for the detection of Helicobacter pylori infection in clinical practice. 13C-Urea Breath Test in different experimental conditions.

The 13C-UBT has been demonstrated to be a reliable method for the evaluation of Helicobacter pylori infection. The aim of our work is to determine the cut-off point of the 13C-UBT for samples collected as gas or collected in a solution of triethanolamine. For this purpose, patients fasted for at least 6 hours were able to collect basal samples before the administration of 65 mg of 13C-urea solution. Breath samples were taken 10, 30 and 60 minutes after the administration of the labeled solution. All the samples were collected in gas collectors and in glass vials containing 1 ml of a 7

triethanolamine solution. The cut-off points for gas collected samples were established in 4.0/1000, 4.6/1000 and 4.4/1000 for 10, 30 and 60 minutes samples, respectively, while for the samples collected in triethanolamine solution, cut-off points were established in 5.0/1000, for the 10 minutes samples, in 3.5/1000 for the 30 minutes samples and 4.7/1000 for the 60 minutes samples. We found that this test has a sensitivity of 100

and a specificity of 100

for H. pylori detection in both experimental conditions, when multiple breath samples are taken. If we considered only the 30 minutes time, sensitivity and specificity diminish for the gas collected samples. We conclude that the collection of breath samples in triethanolamine solution allows a better differentiation between H. pylori infected and non infected patients than gas collected samples.

MIN 14C UBT: a combination of gastric basal transit and 14C-urea breath test for the detection of Helicobacter pylori infection in human beings.

The purpose of this work is to demonstrate that the 14C-urea breath test (UBT) performed at different times combined with the study of the gastric basal transit, which evaluates the intragastric displacement of a labeled solution under fasting conditions, has the advantage of being representative of the whole stomach surface and constitutes a non-aggressive test for the detection of H. pylori. This test, which has been called MIN 14C UBT, is a modification of the conventional 14C UBT in which low volumes of a solution of 14C-urea together with 99mTc-sulfur colloid are administered. The 99mTc-sulfur colloid is not absorbed in the gastrointestinal tract and has the great advantage of allowing the "visualization" of the transit of the 14C-urea within the gastrointestinal tract. This modification allows the simultaneous determination of the production of the 14CO2 and the place where this process occurs. The results show that there is a good correlation between the images obtained and the breath samples collected. We found that this test has a sensitivity of 98% and a specificity of 96% for H. pylori detection.

Homeobox-containing gene transiently expressed in a spatially restricted pattern in the early sea urchin embryo.

In the sea urchin embryo, the lineage founder cells whose polyclonal progenies will give rise to five different territories are segregated at the sixth division. To investigate the mechanisms by which the fates of embryonic cells are first established, we looked for temporal and spatial expression of homeobox genes in the very early cleavage embryos. We report evidence that PlHbox12, a paired homeobox-containing gene, is expressed in the embryo from the 4-cell stage. The abundance of the transcripts reaches its maximum when the embryo has been divided into the five polyclonal territories--namely at the 64-cell stage--and it abruptly declines at later stages of development. Blastomere dissociation experiments indicate that maximal expression of PlHbox12 is dependent on intercellular interactions, thus suggesting that signal transduction mechanisms are responsible for its transcriptional activation in the early cleavage embryo. Spatial expression of PlHbox12 was determined by whole-mount in situ hybridization. PlHbox12 transcripts in embryos at the fourth, fifth, and sixth divisions seem to be restricted to the conditionally specified ectodermal lineages. These results suggest a possible role of the PlHbox12 gene in the early events of cell specification of the presumptive ectodermal territories.

Expression of homeobox-containing genes in the sea urchin (Parancentrotus lividus) embryo.

Two homeobox-containing genes that belong to different homeodomain classes have been isolated from a sea urchin genomic library. One, PlHbox11, is the sea urchin homologue of the human and mouse Hox B3 gene, the other, PlHbox12, shows about 55% identity with paired class genes. Expression profile analysis of the two sea urchin Hbox genes suggests that they play different roles during embryogenesis. In fact, PlHbox11 transcripts are rare and are detected only in the pluteus larva and in the Aristotle's lantern and intestine of the adult. The PlHbox12 gene is, on the contrary, transiently expressed in the very early embryo already at the four cell stage; it accumulates at the 64 cell stage and disappears at later stages of development. In situ hybridization experiments to 16 and 32 cell stage whole mount embryos showed localization of the PlHbox12 mRNA to part of the mesomere-macromere region of the early cleavage embryo. These observations suggest a possible role of this gene in early events of cell specification.

Effect of pindolol and propranolol on sinus node recovery time and atrioventricular conduction intervals.

In a randomized, observer-blind study, the effect of incremental doses of pindolol 0.001, 0.002, 0.003, and 0.004 mg/kg IV and propranolol 0.01, 0.02, 0.03, and 0.04 mg/kg IV on SA nodal recovery time (SNRT) and atrioventricular conduction interval (AH) was assessed in 20 patients (15 men and 5 women age range thirty to seventy-two, mean age fifty-three). AH and His bundle-to-ventricle (HV) intervals and SNRT were measured at spontaneous heart rate and at incremental atrial pacing rates (80, 100, 120, 140 bpm). Both drugs caused significant beta blockade as estimated by the percentage suppression of heart rate increment induced by 3 mcg isoproterenol administered intravenously (pindolol 67.6 +/- 5.3%, P less than 0.007; propranolol 38.6 +/- 10.6%, P less than 0.001). Propranolol significantly prolonged SNRT (P less than 0.05) and AH interval (P less than 0.05). Pindolol did not significantly affect either SNRT (P = 0.25) or AH intervals (P = 0.78). Significant effects on HV interval were not seen. Thus, in the doses tested that resulted in significant beta blockade, propranolol prolonged SA nodal recovery times and depressed AV nodal conduction while pindolol did not affect these variables.

Sympathetic nervous system mediates urinary kallikrein excretion in conscious rats.

1. The influence of the sympathetic nervous system on urinary kallikrein excretion (UKal) was investigated in conscious rats during stress produced by inserting a silastic catheter through the urethra into the bladder. The effect of this stress on blood glucose (BG), mean arterial pressure (MAP), urinary volume (Uv), urinary sodium (UVNa), and glomerular filtration rate (GFR) was also studied. 2. In intact animals stress of 120 min duration produced a non-significant increase of MAP, a significant increase of BG and a decrease of UNa (P less than 0.001 t-test, 9 d.f.), but it did not affect Uv and GFR. In stressed rats UKal was considerably lower (52 milli Amidasic Units [mAU] per 100 g bodyweight, s.e.m. = 5, n = 6) than in control rats (170 mAU per 100 g, s.e.m. = 21, n = 5). The inhibitory effect on UKal was also observed when kallikrein was measured by the kininogenase method. 3. Adrenal medullectomy, performed 1 week before the experiment, suppressed the stress hyperglycaemia but did not affect the reduction of urinary kallikrein or the anti-natriuresis. 4. Intracerebroventricular (i.c.v.) injection of saline also had no effect in the control or in the stressed rats, while i.c.v. d-l-propranolol decreased MAP, suppressed the stress hyperglycaemia and the anti-natriuresis and stimulated UKal, without changes in Uv and GFR. Non-stressed control rats i.c.v. injected with saline excreted considerably less kallikrein than rats i.c.v. injected with d-l-propranolol (control saline: 162, s.e.m. = 14, n = 6; vs control d-l-propranolol: 559, s.e.m. = 20, n = 6). Even in stressed rats this difference was registered (stressed saline: 56, s.e.m. = 8, n = 6; vs stressed d-l-propranolol: 554 mAU per 100 g, s.e.m. = 33, n = 6). 5. Peripheral sympathectomy with 6-hydroxydopamine (6-OHDA) did not suppress the hyperglycaemic response to stress, but it stimulated UKal. Kallikrein excretion was similar in 6-OHDA stressed (534 mAU per 100 g, s.e.m. = 30, n = 6) than in 6-OHDA control rats (491 mAU per 100 g, s.e.m. = 34, n = 6). No differences were observed on UNa and GFR between control 6-OHDA treated rats and stressed 6-OHDA treated rats. 6. The present results suggest strongly that urinary kallikrein excretion is modulated by sympathetic activity. Results after central beta-adrenergic blockade and peripheral sympathectomy led to the hypothesis that normal sympathetic tone in the kidney inhibits the release of kallikrein into the urine.