Non-invasive Investigations of Paintings by Portable Instrumentation: The MOLAB Experience.

The in situ non invasive methods have experienced a significant development in the last decade because they meet specific needs of analytical chemistry in the field of cultural heritage where  artworks are rarely moved from their locations, sampling is rarely permitted, and analytes are a wide range of inorganic, organic and organometallic substances in complex and precious matrices. MOLAB, a unique collection of integrated mobile instruments, has greatly contributed to demonstrate that it is now possible to obtain satisfactory results in the study of a variety of heritage objects without sampling or moving them to a laboratory. The current chapter describes an account of these results with particular attention to ancient, modern, and contemporary paintings. Several non-invasive methods by portable equipment, including XRF, mid- and near-FTIR, UV-Vis and Raman spectroscopy, as well as XRD, are discussed in detail along with their impact on our understanding of painting materials and execution techniques. Examples of successful applications are given, both for point analyses and hyperspectral imaging approaches. Lines for future perspectives are finally drawn.

Shear stress modulates endothelial KLF2 through activation of P2X4.

Vascular endothelial cells that are in direct contact with blood flow are exposed to fluid shear stress and regulate vascular homeostasis. Studies report endothelial cells to release ATP in response to shear stress that in turn modulates cellular functions via P2 receptors with P2X4 mediating shear stress-induced calcium signaling and vasodilation. A recent study shows that a loss-of-function polymorphism in the human P2X4 resulting in a Tyr315>Cys variant is associated with increased pulse pressure and impaired endothelial vasodilation. Although the importance of shear stress-induced Krüppel-like factor 2 (KLF2) expression in atheroprotection is well studied, whether ATP regulates KLF2 remains unanswered and is the objective of this study. Using an in vitro model, we show that in human umbilical vein endothelial cells (HUVECs), apyrase decreased shear stress-induced KLF2, KLF4, and NOS3 expression but not that of NFE2L2. Exposure of HUVECs either to shear stress or ATPγS under static conditions increased KLF2 in a P2X4-dependent manner as was evident with both the receptor antagonist and siRNA knockdown. Furthermore, transient transfection of static cultures of human endothelial cells with the Tyr315>Cys mutant P2X4 construct blocked ATP-induced KLF2 expression. Also, P2X4 mediated the shear stress-induced phosphorylation of extracellular regulated kinase-5, a known regulator of KLF2. This study demonstrates a major physiological finding that the shear-induced effects on endothelial KLF2 axis are in part dependent on ATP release and P2X4, a previously unidentified mechanism.

Trend analysis of plasma insulin level around parturition in relation to parity in Saanen goats.

The present study investigated the effect of parity on plasma insulin level around parturition in Saanen goats. On d -14, -7, 0, 3, 7, 10, and 14 from parturition, plasma glucose, NEFA, free AA, cortisol, and insulin concentrations were analyzed in 10 primiparous and 10 multiparous goats. At parturition, BW of primiparous goats was about 75% of that of multiparous ones (P < 0.001) and then their milk production was lower than that of multiparous ones (P < 0.001). At parturition, glucose increased (P < 0.01) in both primiparous and multiparous goats and then decreased (P < 0.01) on d 3 of lactation, remaining higher (P < 0.01) in primiparous than multiparous goats until the end of the study period. In both groups, free AA decreased (P < 0.01) at parturition, returning to prepartum levels (P < 0.01) on d 3 of lactation without difference between groups. Only in multiparous goats, plasma NEFA increased at parturition (P < 0.01), returning to prepartum levels on d 14 (P < 0.01). Changes in glucose and AA could have been caused by cortisol, which increased (P < 0.01) at parturition in both primiparous and multiparous goats, returning to prepartum levels (P < 0.01) on d 7 of lactation, without difference between the parity groups. In multiparous goats, insulin decreased soon after parturition (P < 0.05), remaining at low levels until the end of the study period, whereas in primiparous goats, insulin did not vary until d 14 of lactation, when it decreased (P < 0.05) also in this group. Therefore, between d 3 and 14 of lactation, insulin was higher in primiparous than multiparous goats (P < 0.05). Only in primiparous goats, at kidding, insulin was negatively correlated to BW (P < 0.01), and after parturition it was negatively correlated with milk yield (P < 0.05) and plasma NEFA (P < 0.05). We hypothesize that higher insulin levels in primiparous Saanen goats, which are still immature at their first breeding season, acted to limit both the mobilization of bodily reserves and the capture of nutrients by the lactating mammary gland, thus providing nutrients for their own physical and physiological development.

In-situ identification of copper-based green pigments on paintings and manuscripts by reflection FTIR.

In this work non-invasive reflection infrared spectroscopy was used to discriminate between different Cu-based green pigments (namely, malachite, verdigris, chrysocolla, emerald green and phthalo green). The pigments, chosen because of their historical widespread use in artworks, were investigated as pure powders and in situ on painted models by reflection FTIR spectroscopy. The distortion arising as a result of the specular and diffuse component of reflected radiation was evaluated as function of the optical and surface properties of the investigated mock-ups. Use of the Kramers-Kronig (KK) algorithm to correct for the distortion arising from the surface reflection gave k index maxima shifted by more than 20 cm(-1) relative to those obtained in conventional transmission mode. These findings stress the need to carefully manage use of the KK algorithm on reflection spectra to avoid erroneous assignment. On the other hand, combination and overtone bands (which are enhanced by the diffuse reflection) were proved to enable reliable and sensitive identification of most of the pigments studied. Knowledge acquired by study of pure compounds and painted models was subsequently applied to interpret the spectra acquired from paintings and manuscripts.

Analysis of weaning-induced stress in Saanen goat kids.

In young ruminants' life, weaning often coincides with a period of growth stasis and poor welfare. The present study aimed at evaluating the effect of coping with the new diet on behavioural and haematological stress indicators in goat kids subjected to a commonly adopted weaning practice. Immediately after birth, male Saanen goat kids were divided into two groups: MILK and WMIX. All were fed colostrum for the first 3 days and then goat milk to the age of 29 days. After that, MILK kids continued to receive milk, while the WMIX kids underwent weaning and were completely weaned by day 48. Animal behaviour was recorded daily. From day 23-50, blood samples were taken weekly and analysed for indicators of stress and immune function. No abnormal behaviour, such as injurious behaviours or stereotypies, was observed in either of the experimental groups throughout the experimental period. During the last week, fasting plasma cortisol level was significantly lower, whereas plasma activity of both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was significantly higher in WMIX kids, in relation to the MILK ones. Anyway, data were within the normal physiological range and no difference was observed neither in plasma haptoglobin, ceruloplasmin, albumin and antithrombin III, nor in plasma immunoglobulin A and G, at any time, signalling no stressful condition. Therefore, differences observed in cortisol, ALT and AST could be the consequence of the metabolic changes that occur during the transition from pre-ruminant to ruminant state. The gradual weaning at 48 days of age did not result in any stressful condition and had no negative effect on weight gain. Results suggest that parameters commonly adopted to provide information on animal stress, such as cortisol and aminotransferase activity, can vary in relation to the physiological status of the animals and may bias stress assessment.

Cocoa husks in diets of Italian heavy pigs.

The aim of the present study was to evaluate the effect of cocoa husks feeding on liver composition of the Italian heavy pig. Cocoa husks are by-products derived from chocolate production and have a high content of proteins, lipids, and NDF. Cocoa husks are also rich in antioxidants, polyphenols in particular. Eight finishing pigs were divided into 2 groups: control group fed a traditional diet, based on cereals, and treatment group fed a diet obtained by substitution of 10% of the control diet with coarsely ground cocoa husks. The trial was conducted during the hot season and lasted 6 wk, at the end of which all the pigs were slaughtered. Cocoa husks diet reduced dry matter intake (P < 0.01) and energy intake (P < 0.01) but neither body weight nor backfat thickness was affected by cocoa husks diet. Treatment did not influence carcass weight and hot dressing percentage but reduced liver weight (P < 0.05), liver dry matter percentage (P < 0.01), DNA (P = 0.01), and glycogen content (P = 0.01). By contrast, cocoa husks increased liver ether extract (P = 0.05) without affecting cholesterol content. Liver weight loss, reduction of protein synthesis, and a shift toward glycogen use instead of fat oxidation are considered metabolic strategies to reduce heat production under hot conditions. It is possible, therefore, that cocoa husks feeding promoted the process of acclimation because pigs needed less feeding to reach similar body and carcass weight as control pigs.

Ghrelin, insulin and pancreatic activity in the peri-weaning period of goat kids.

Two groups of 3-day-old Saanen goat kids (MILK and WMIX) were studied in order to investigate the effect of weaning on plasma ghrelin and insulin and on pancreatic activity. MILK kids received goat milk to age 50 days; WMIX kids were initially fed milk but started weaning at 30 days and were completely weaned by 48 days. Dry matter intake and body weights were recorded, and plasma samples were analysed for metabolites, ghrelin, insulin, leptin and α-amylase. At 50 days, all the animals were slaughtered, pancreas samples were analysed for α-amylase, zymogen, DNA and ribosomal capacity (RNA/zymogen). Seven days after the beginning of the weaning program, dry matter intake in the WMIX group began to decrease in relation to the MILK group. Nonetheless, body weight did not differ throughout the study period. Weaning significantly decreased plasma levels of glucose, amino acids, urea and insulin, but increased creatinine and ghrelin. In weaning kids ghrelin secretion may help minimize the negative consequences of the new diet on dry matter intake. Pancreatic zymogen and ribosomal capacity did not differ between the groups, whereas pancreatic amylase activity was over three times higher in MILK than WMIX kids even though the former had no dietary starch. This finding could be a consequence of lower pancreatic secretion of amylase in the MILK group due to the lack of dietary starch, resulting in pancreatic accumulation, but could also be due to higher plasma insulin in the MILK group, suggesting a role for insulin in the functional development of the pancreas in weaning goats.

Non-invasive identification of organic materials in wall paintings by fiber optic reflectance infrared spectroscopy: a statistical multivariate approach.

The aim of this study is to develop a method for the non-invasive and in situ identification of organic binders in wall paintings by fiber optic mid-FTIR reflectance spectroscopy. The non-invasive point analysis methodology was set-up working on a wide set of wall painting replicas of known composition and using statistical multivariate methods, in particular principal component analysis (PCA), for the interpretation, understanding, and management of data acquired with reflectance mid-FTIR spectroscopy. Results show that PCA can be helpful in managing and preliminary sorting of the large amount of spectra typically collected during non-invasive measurement campaigns and highlight further avenues for research. The developed PCA model was finally applied to the case of a Renaissance wall painting by Perugino assessing it predictability as compared to the interpretation of the single spectrum.

Diet influences the content of bioactive peptides in goat milk.

Leptin, insulin, ghrelin, and IGF-I are circulating peptide hormones concerned with energy homeostasis and the regulation of GH axis. They are present in human milk, and are thought to promote neonatal development. The aim of the present study was to detect these substances in goat milk and determine whether their levels can be modified by changing the macronutrient content of the lactating animals' diet. Sixteen Saanen goats in mid-lactation were divided into two balanced groups, one given a diet containing 17% starch (LS) and the other a diet of 33% starch (HS). Eighty days later, leptin, insulin, ghrelin, and IGF-I were determined by human radioimmunoassay kits in plasma before and after feeding, and in sonicated milk centrifuged to remove fat from morning and evening milking. The HS diet was associated with higher plasma and milk insulin and IGF-I, and plasma ghrelin. Leptin, insulin, and ghrelin in milk were two-three times higher than in plasma; milk IGF-I was only 5-20% of plasma level. Plasma insulin correlated positively with plasma IGF-I; morning milk IGFI and insulin correlated positively with morning plasma levels. These findings demonstrate that human immuno-activities of bioactive peptides are present in goat milk, and also that levels of insulin and IGF-I in milk can be altered by changing the macronutrient content of the diet. Further research is required to determine whether these substances can be transferred from the milk to suckling animals and humans, and whether they have biological activity in such animals.

Effect of dietary conjugated linoleic acid supplementation in sows on performance and immunoglobulin concentration in piglets.

Conjugated linoleic acid isomers may affect the onset and severity of several diseases, including tumors, atherogenesis, and obesity. They may also modulate the immune response. However, little information regarding the most advantageous duration of CLA supplementation is available. The purpose of this study was to determine whether the length of dietary CLA supplementation of a sow affects growth, immune components, and metabolic and hormonal factors in lactating sows and piglets. Gestating sows were fed a control (0%) and a 0.5% CLA-supplemented diet beginning 7 d before parturition and ending 7 d postpartum (T1), or until weaning (T2; 7 sows per treatment). Colostrum and sow and piglet blood samples were collected for the determination of serum metabolite concentrations and immunoglobulin titer. Piglet BW at weaning were greater (P < 0.05) in the CLA groups compared with the control. Dietary CLA supplementation increased (P < 0.05) serum thyroxine concentration in sows, but serum insulin, glucose, NEFA, IGF-I, and leptin concentrations were not affected by CLA supplementation. Colostral IgG, IgA, and IgM titers were greater in sows fed CLA than in control sows (P < 0.05). At weaning (21 d), serum IgG titer of the piglets was greater (P < 0.05) in the T1 and T2 groups than the control group, but at 13 d postweaning, a difference (P < 0.05) was observed between the control and T2 group. The results from this study indicate potential beneficial effects of 0.5% dietary CLA supplementation from 7 d before parturition until 7 d postpartum in improving BW at weaning and immune components in piglets.

A 57-gene expression signature in B-cell chronic lymphocytic leukemia.

B-CLL is the most frequent type of leukemia in the Western countries. The disease, common among the elderly, follows a variable course in terms of survival time and symptoms. There is evidence that the accumulation of lymphocytes in peripheral blood and bone marrow is due to a cell resistance to apoptosis rather than to highly proliferative cells. Genetic mechanisms that lead to the development and progression of disease are mainly unknown, although a number of prognostically and diagnostically important genetic markers have been identified. The aim of this study is to investigate the gene expression profile, by a specific chip for microarray analysis, in B-CLL lymphocytes with regard to factors involved in apoptosis cascade, signal transduction, purine metabolism enzymes, interleukin expression, enzymes involved in the responses to oxidative stress. We found relevant results in a set of 19 of the 57 genes considered. IMP dehydrogenase, adenine phosphoribosyltransferase, adenylosuccinate lyase, adenylate kinase, ADORA1, G-protein-coupled receptor kinase 6, Bcl-2-like 1 isoform 2, caspase 6, and 8 were found underexpressed; while ADORA3, Gars-Airs-Gart, adenylate kinase 3, adenylate deaminase, NMN adenylyltransferase, CD26, CD38, interleukins 18 and 4 were found overexpressed. The microarray technique is a powerful method for identification of potential important diagnostic and prognostic markers, besides giving prominence to genes candidate for further studies.

[Preliminary results of the effects of electromagnetic fields ELF and TAMMEF on human lymphocytes: evaluation of some biological parameters]. / Studio preliminare sugli effetti di campi elettromagnetici, ELF e TAMMEF, nei linfociti umani: valutazione di alcuni parametri biologici.

AIMS: The Authors have evaluated the effects of low frequency electromagnetic fields on human peripheral blood lymphocytes metabolism. MATERIALS AND METHODS: It has been assayed total proteins and the activities of some purine metabolism enzymes after exposure of the cells to sinusoidal ELF fields (100 Hz frequency) or to the new TAMMEF system fields, characterized by variable frequency, intensity and shape of wave. RESULTS: The protein lymphocytes content, increased after both treatments. Instead, the enzyme activities did not vary, with the exception of Myokinase activity, which, respect to the control, increased after ELF field treatment, and slightly decreased after TAMMEF treatment. This preliminary result was interpreted as a variation of the cellular electric charge stability, stimulated by ELF fields, which induce the Myokinase to increase its rate, to rearrange the correct equilibrium, while the TAMMEF field were useless to maintain the cellular electric charge at normal levels. CONCLUSIONS: We interpreted these preliminary results as follow: the ELF fields influence the cellular electric charge stability, so that the cells must increase MK activity to restore the correct equilibrium, while TAMMEF fields are useful to maintain and regulate cellular electric charge. The results obtained by this preliminary study opens interesting prospective to be further investigated.

Nitric oxide generation is associated with an unbalance of protein tyrosine phosphatases during liver transplantation.

Organ dysfunction secondary to ischemia-reperfusion (I/R) injury still represents a major problem in liver transplantation. Apoptosis has been observed in hepatocytes and sinusoidal endothelial cell, following I/R injury and it has been postulated as a contributing factor in ischemia-reperfusion graft dysfunction, involving a complex series of events, as changes of protein tyrosine-kinase phosphorylation. We evaluated hepatic purine metabolites, protein tyrosine phosphatases (PTPs), nitrate plus nitrite levels (NOx), caspase-3 (C-3) activity and DNA fragmentation in the time course of twelve pig orthotopic liver transplantation. Biopsies were taken before explantation (t0), after cold ischemic storage (t1) and 30 min from reperfusion (t2). During the ischemic period we observed a reduction of high energy phosphates and an increase of purine bases; PTP activity was largely increased. At t2 high energy phosphates showed a tendency to increase with respect to t1, with a partial restoration of phosphorylation potential, measured as ATP/ADT ratio. PTP activity was significantly reduced, with a concomitant increase of NOx production and C-3 activity; in a considerable number of cases we observed a sustained DNA fragmentation. We speculate that NOx production could be related to nitrosative stress, which in turn leads to dynamic alteration in PTP balance and cell signalling, regulating the activity of a number of proteins implicated in apoptotic cell death. These findings could be of interest in new potential strategy to prevent and treat I/R injury.

NT-proBNP changes, oxidative stress, and energy status of hypertrophic myocardium following ischemia/reperfusion injury.

N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a sensitive functional marker in heart disease, including left ventricular hypertrophy (LVH) secondary to valvular aortic stenosis (AS). We evaluated the association between NT-proBNP changes, oxidative stress, energy status and severity of LVH in patients with AS. Ten patients undergoing aortic valve replacement for AS were studied. Plasma NT-proBNP concentrations were performed by electroluminescence immunoassay 15min after the induction of anesthesia (t0), before aortic cross-clamping (t1), before clamp removal (t2), 15min after myocardial reperfusion (t3), and 24h after surgery (t4). Heart biopsies were obtained and high energy phosphates (ATP, ADP, AMP) were analyzed by capillary electrophoresis (CE). In plasma samples from the coronary sinus, nitrate plus nitrite (NOx) concentrations were also analyzed by CE. Echocardiographic measurements were acquired and correlations between biochemical markers and severity of AS were assessed. NT-proBNP peaked significantly at t4 (p<0.001). A linear correlation between NT-proBNP values measured at t0 and t4 was found (R(2)=0.89; p<0.001). A negative correlation between NT-proBNP production and phosphorylation potential (ATP/ADP ratio) was observed (R(2)=0.62; p<0.01). NOx values positively correlated with NT-proBNP levels (p<0.01). NT-proBNP inversely correlated with aortic valvular area (r=81, p<0.01), positively correlated with mean (r=0.82, p<0.01) and maximum left ventricle-to-aortic gradients (r=0.80, p<0.01), and with left ventricular mass (r=0.69, p<0.01). NT-proBNP is a useful marker of LVH and severity of AS. It may complement echocardiographic evaluation of patients with AS in identifying the optimum time for surgery.

Fiber-optic fourier transform mid-infrared reflectance spectroscopy: a suitable technique for in situ studies of mural paintings.

A prototypical in situ noninvasive study of ancient mural painting materials has been carried out using an easily manageable fiber-optic Fourier transform mid-infrared (mid-FT-IR) reflectance spectrophotometer. The reported object of the study is the Renaissance fresco by Pietro Vannucci, called il Perugino, located in the church of Santa Maria delle Lacrime (1521, Trevi, Perugia Italy). For the first classification and interpretation of infrared spectra, principal components analysis was used. Spectral artifacts due to lacunas, restoration materials, or alteration products have been identified, as well as two different secco refinements bound in a tempera medium. For the characterization of inorganic pigments, mid-FT-IR spectra have been integrated with other data obtained through in situ X-ray fluorescence (XRF) elemental analysis. This complementary noninvasive approach led to the characterization of Perugino's pigments, even in the presence of complex mixtures. The mid-FT-IR noninvasive technique, in combination with XRF, is thus recommended as a valuable first approach for the examination of mural paintings, permitting the assessment of the execution technique as well as contributing to the evaluation of the conservation state.

Purine metabolism in B-cell lymphocytic leukemia: a microarray approach.

B-cell chronic lymphocytic leukemia (B-CLL) is an adult-onset highly heterogeneous malignancy characterized by a cells resistance to apoptosis rather than to highly proliferative cells. In previous research, we evidenced an imbalance of purine metabolism in B-CLL cells. Since the extracellular adenosine has been proved to induce apoptosis via A2b receptor, enzymes involved in adenosine metabolism could play an important role in apoptosis resistance of B-CLL cells. We prepared a microarray chip for the analysis of 50 selected genes that could be of interest in B-CLL: enzymes of purine de-novo, salvage and catabolic pathway, oxidative stress enzymes, and apoptotis-related proteins. Preliminary results identify many genes of purine metabolism that exhibit low or high expression, while genes involved in signal transduction and apoptosis exhibit lower alterations even if of remarkable interest. This application of microarray technique seems promising and at least a subset of these genes will be valid candidates for further studies.

Leptin in bovine colostrum and milk.

We studied leptin content in bovine colostrum, milk and plasma during the first month of lactation, and investigated relationships between selected milk components and milk leptin in five multiparous dairy cows. Colostrum/milk yield and composition were measured on days 0, 10, 20, and 30 of lactation. Leptin was assayed using a multi-species leptin RIA kit. Leptin concentration was 56 % lower in mature milk (day 10) than colostrum (13.90 vs. 6.14 microg/l; p < 0.001), but remained steady over the twenty days afterwards. Daily secretion of leptin into mature milk was 28 % lower than into colostrum (173.2 microg/d vs. 220.0 microg/d; p = 0.09) notwithstanding an 80 % increase in production. Colostrum and milk leptin levels correlated with fat (0.90; p < 0.001) and choline phospholipid (0.76; p < 0.05). Plasma and milk leptin decreased during the first month, but remained higher in milk, and highest in colostrum. Thus, leptin is present in large quantities in colostrum, less so and more variably in untreated milk, and is likely to be decreased in skimmed milk. These findings have implications for the use of untreated milk and colostrum-based (functional) food products.

Evaluation of ADA gene expression and transduction efficiency in ADA/SCID patients undergoing gene therapy.

A capillary electrophoresis (CE) method was developed for ADA/SCID diagnosis and monitoring of enzyme replacement therapy, as well as for exploring the transfection efficiency for different retroviral vectors in gene therapy.

Liver transplant: adenosine metabolism and apoptosis.

Apoptosis and necrosis coexist in ischemia-reperfusion (I/R) injury following organ transplant. During experimental liver transplant we evidenced a deep alteration in energy and antioxidant status. The activity of purine catabolic enzymes was also altered. Caspase-3 (C-3), protein tyrosine phosphatase (PTP) showed significative alterations that lead to DNA fragmentation. These findings could be of interest in new potential strategy to prevent and treat I/R injury.