Body fluid and tissue analysis using filter paper sampling support prior to LC-MS/MS: application to fatal overdose with colchicine

Because of the various matrices available for forensic investigations, the development of versatile analytical approaches allowing the simultaneous determination of drugs is challenging. The aim of this work was to assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform allowing the rapid quantification of colchicine in body fluids and tissues collected in the context of a fatal overdose. For this purpose, filter paper was used as a sampling support and was associated with an automated 96-well plate extraction performed by the LC autosampler itself. The developed method features a 7-min total run time including automated filter paper extraction (2 min) and chromatographic separation (5 min). The sample preparation was reduced to a minimum regardless of the matrix analyzed. This platform was fully validated for dried blood spots (DBS) in the toxic concentration range of colchicine. The DBS calibration curve was applied successfully to quantification in all other matrices (body fluids and tissues) except for bile, where an excessive matrix effect was found. The distribution of colchicine for a fatal overdose case was reported as follows: peripheral blood, 29 ng/ml; urine, 94 ng/ml; vitreous humour and cerebrospinal fluid, < 5 ng/ml; pericardial fluid, 14 ng/ml; brain, < 5 pg/mg; heart, 121 pg/mg; kidney, 245 pg/mg; and liver, 143 pg/mg. Although filter paper is usually employed for DBS, we report here the extension of this alternative sampling support to the analysis of other body fluids and tissues. The developed platform represents a rapid and versatile approach for drug determination in multiple forensic media.

Spectral clustering independent component analysis for tissue classification from brain MRI

A spectral angle based feature extraction method, Spectral Clustering Independent Component Analysis (SC-ICA), is proposed in this work to improve the brain tissue classification from Magnetic Resonance Images (MRI). SC-ICA provides equal priority to global and local features; thereby it tries to resolve the inefficiency of conventional approaches in abnormal tissue extraction. First, input multispectral MRI is divided into different clusters by a spectral distance based clustering. Then, Independent Component Analysis (ICA) is applied on the clustered data, in conjunction with Support Vector Machines (SVM) for brain tissue analysis. Normal and abnormal datasets, consisting of real and synthetic T1-weighted, T2-weighted and proton density/fluid-attenuated inversion recovery images, were used to evaluate the performance of the new method. Comparative analysis with ICA based SVM and other conventional classifiers established the stability and efficiency of SC-ICA based classification, especially in reproduction of small abnormalities. Clinical abnormal case analysis demonstrated it through the highest Tanimoto Index/accuracy values, 0.75/98.8%, observed against ICA based SVM results, 0.17/96.1%, for reproduced lesions. Experimental results recommend the proposed method as a promising approach in clinical and pathological studies of brain diseases

Brain Tissue Classification from Multispectral MRI by Wavelet based Principal Component Analysis

In this paper, we propose a multispectral analysis system using wavelet based Principal Component Analysis (PCA), to improve the brain tissue classification from MRI images. Global transforms like PCA often neglects significant small abnormality details, while dealing with a massive amount of multispectral data. In order to resolve this issue, input dataset is expanded by detail coefficients from multisignal wavelet analysis. Then, PCA is applied on the new dataset to perform feature analysis. Finally, an unsupervised classification with Fuzzy C-Means clustering algorithm is used to measure the improvement in reproducibility and accuracy of the results. A detailed comparative analysis of classified tissues with those from conventional PCA is also carried out. Proposed method yielded good improvement in classification of small abnormalities with high sensitivity/accuracy values, 98.9/98.3, for clinical analysis. Experimental results from synthetic and clinical data recommend the new method as a promising approach in brain tissue analysis.

Animal Tissue Response To Mid-Term Electronic Modular Artificial Sphincter Implantation

Introduction and objectives: The AMS 800TM is considered the gold standard for sphincter replacement. However, the one-ring design can erode the urethra and lead to severe complications. A mechanism that could alternatively compress successive segments of the urethra would limit such deleterious outcome. We report 12 weeks animal urethral tissue analysis following implantation of a new modular artificial sphincter. METHODS: The device is composed by three parts: the contractile unit, two rings and an integrated microprocessor. The contractile unit is made of Nitinol fibers. The rings are placed around the urethra to control the flow of urine by squeezing the urethra. They work in a sequential alternative mode and are controlled by a microprocessor connected to an external computer. The computer can reveal specific failure of device components. The device was impkanted in eight male sheep. The rings were positioned around the urethra and the control unit was placed 5cm away. The device was working twenty hours per day; it was open 10min. per hour to allow urination. The animals were sacrificed after 12 weeks. The urethra and the tissues surrounding the control unit were macroscopically and microscopically examined. Two transversal sections crossing the sphincter and two transversal sections crossing the urethra alone were obtained and stained with modified Paragon after resin embedding. Urethra was also embedded in paraffin. The first section was stained with safranin-hematoxylin-eosin, the second section was stained with Masson's Trichrome and the remaining eight sections were available for immunolabelling of the macrophages.Results: The chronic study went uneventful. No clinical infection or pain was observed. The computer registered no specific failure in ring function, Nitinol wires and tube connectors. At explantation, except for a slight grade of lymphocytes in two out of eight specimens, no urethral stricture or atrophy could be observed. Immunohistochemistry confirmed the absence of macrophages. Tissue structure and organization of the urethra with and without artificial sphincter were similar. No migration of the device was observed.Conclusions: The study clearly showed no tissue damage or inflammation of the urethra. Electronic design, preservation of urethral vascularisation and adjustability after implantation are the key ideas to improve the actual AUS. Further studies will be carried out to evaluate this potential.

Skeletal muscle fibre-type comparison of whole tissue and subcellular membrane phospholipids and fatty acids

Membranes are dynamic structures that affect cell structure and function. Compositional changes ofmembranes have been shown with the application of a perturbation; however these are limited to whole tissue analysis. The purpose of this thesis was to compare the phospholipid (PL) fatty acid (FA) composition of rat whole muscle (Wm) to 1) purified and non-purified subsarcolemmal (SS) mitochondria in soleus, plantaris, and red gastrocnemius, and 2) sarcolemma, transverse-tubules, SS and intermyofibrillar (IMF) mitochondria fix)m whole hindlimb. The major findings were that 1) contamination significantly altered the PL FA composition of the SS mitochondrial membrane fraction, 2) Wm and SS mitochondria compositions differed between muscle types, and 3) Wm did not accurately reflect the PL FA composition of any isolated subcellular membranes, with each being unique from each other. As such, the relevancy of the trends reported in the literature of the effects of perturbations on Wm may be limited.

Surgical management of gynecomastia--a 10-year analysis

BACKGROUND: Gynecomastia is defined as the benign enlargement of the male breast. Most studies on surgical treatment of gynecomastia show only small series and lack histopathology results. The aim of this study was to analyze the surgical approach in the treatment of gynecomastia and the related outcome over a 10-year period. PATIENTS AND METHODS: All patients undergoing surgical gynecomastia corrections in our department between 1996 and 2006 were included for retrospective evaluation. The data were analyzed for etiology, stage of gynecomastia, surgical technique, complications, risk factors, and histological results. RESULTS: A total of 100 patients with 160 operations were included. Techniques included subcutaneous mastectomy alone or with additional hand-assisted liposuction, isolated liposuction, and formal breast reduction. Atypical histological findings were found in 3% of the patients (spindle-cell hemangioendothelioma, papilloma). The surgical revision rate among all patients was 7%. Body mass index and a weight of the resected specimen higher than 40 g were identified as significant risk factors for complications (p < 0.05). CONCLUSIONS: The treatment of gynecomastia requires an individualized approach. Caution must be taken in performing large resections, which are associated with increased complication rates. Histological tissue analysis should be routinely performed in all true gynecomastia corrections, because histological results may reveal atypical cellular pathology.

Three-dimensional imaging methods for quantitative analysis of facial soft tissues and skeletal morphology in patients with orofacial clefts: a systematic review.

BACKGROUND Current guidelines for evaluating cleft palate treatments are mostly based on two-dimensional (2D) evaluation, but three-dimensional (3D) imaging methods to assess treatment outcome are steadily rising. OBJECTIVE To identify 3D imaging methods for quantitative assessment of soft tissue and skeletal morphology in patients with cleft lip and palate. DATA SOURCES Literature was searched using PubMed (1948-2012), EMBASE (1980-2012), Scopus (2004-2012), Web of Science (1945-2012), and the Cochrane Library. The last search was performed September 30, 2012. Reference lists were hand searched for potentially eligible studies. There was no language restriction. STUDY SELECTION We included publications using 3D imaging techniques to assess facial soft tissue or skeletal morphology in patients older than 5 years with a cleft lip with/or without cleft palate. We reviewed studies involving the facial region when at least 10 subjects in the sample size had at least one cleft type. Only primary publications were included. DATA EXTRACTION Independent extraction of data and quality assessments were performed by two observers. RESULTS Five hundred full text publications were retrieved, 144 met the inclusion criteria, with 63 high quality studies. There were differences in study designs, topics studied, patient characteristics, and success measurements; therefore, only a systematic review could be conducted. Main 3D-techniques that are used in cleft lip and palate patients are CT, CBCT, MRI, stereophotogrammetry, and laser surface scanning. These techniques are mainly used for soft tissue analysis, evaluation of bone grafting, and changes in the craniofacial skeleton. Digital dental casts are used to evaluate treatment and changes over time. CONCLUSION Available evidence implies that 3D imaging methods can be used for documentation of CLP patients. No data are available yet showing that 3D methods are more informative than conventional 2D methods. Further research is warranted to elucidate it.

Three dimensional study to quantify the relationship between facial hard and soft tissue movement as a result of orthognathic surgery

Introduction Prediction of soft tissue changes following orthognathic surgery has been frequently attempted in the past decades. It has gradually progressed from the classic “cut and paste” of photographs to the computer assisted 2D surgical prediction planning; and finally, comprehensive 3D surgical planning was introduced to help surgeons and patients to decide on the magnitude and direction of surgical movements as well as the type of surgery to be considered for the correction of facial dysmorphology. A wealth of experience was gained and numerous published literature is available which has augmented the knowledge of facial soft tissue behaviour and helped to improve the ability to closely simulate facial changes following orthognathic surgery. This was particularly noticed following the introduction of the three dimensional imaging into the medical research and clinical applications. Several approaches have been considered to mathematically predict soft tissue changes in three dimensions, following orthognathic surgery. The most common are the Finite element model and Mass tensor Model. These were developed into software packages which are currently used in clinical practice. In general, these methods produce an acceptable level of prediction accuracy of soft tissue changes following orthognathic surgery. Studies, however, have shown a limited prediction accuracy at specific regions of the face, in particular the areas around the lips. Aims The aim of this project is to conduct a comprehensive assessment of hard and soft tissue changes following orthognathic surgery and introduce a new method for prediction of facial soft tissue changes.   Methodology The study was carried out on the pre- and post-operative CBCT images of 100 patients who received their orthognathic surgery treatment at Glasgow dental hospital and school, Glasgow, UK. Three groups of patients were included in the analysis; patients who underwent Le Fort I maxillary advancement surgery; bilateral sagittal split mandibular advancement surgery or bimaxillary advancement surgery. A generic facial mesh was used to standardise the information obtained from individual patient’s facial image and Principal component analysis (PCA) was applied to interpolate the correlations between the skeletal surgical displacement and the resultant soft tissue changes. The identified relationship between hard tissue and soft tissue was then applied on a new set of preoperative 3D facial images and the predicted results were compared to the actual surgical changes measured from their post-operative 3D facial images. A set of validation studies was conducted. To include: • Comparison between voxel based registration and surface registration to analyse changes following orthognathic surgery. The results showed there was no statistically significant difference between the two methods. Voxel based registration, however, showed more reliability as it preserved the link between the soft tissue and skeletal structures of the face during the image registration process. Accordingly, voxel based registration was the method of choice for superimposition of the pre- and post-operative images. The result of this study was published in a refereed journal. • Direct DICOM slice landmarking; a novel technique to quantify the direction and magnitude of skeletal surgical movements. This method represents a new approach to quantify maxillary and mandibular surgical displacement in three dimensions. The technique includes measuring the distance of corresponding landmarks digitized directly on DICOM image slices in relation to three dimensional reference planes. The accuracy of the measurements was assessed against a set of “gold standard” measurements extracted from simulated model surgery. The results confirmed the accuracy of the method within 0.34mm. Therefore, the method was applied in this study. The results of this validation were published in a peer refereed journal. • The use of a generic mesh to assess soft tissue changes using stereophotogrammetry. The generic facial mesh played a major role in the soft tissue dense correspondence analysis. The conformed generic mesh represented the geometrical information of the individual’s facial mesh on which it was conformed (elastically deformed). Therefore, the accuracy of generic mesh conformation is essential to guarantee an accurate replica of the individual facial characteristics. The results showed an acceptable overall mean error of the conformation of generic mesh 1 mm. The results of this study were accepted for publication in peer refereed scientific journal. Skeletal tissue analysis was performed using the validated “Direct DICOM slices landmarking method” while soft tissue analysis was performed using Dense correspondence analysis. The analysis of soft tissue was novel and produced a comprehensive description of facial changes in response to orthognathic surgery. The results were accepted for publication in a refereed scientific Journal. The main soft tissue changes associated with Le Fort I were advancement at the midface region combined with widening of the paranasal, upper lip and nostrils. Minor changes were noticed at the tip of the nose and oral commissures. The main soft tissue changes associated with mandibular advancement surgery were advancement and downward displacement of the chin and lower lip regions, limited widening of the lower lip and slight reversion of the lower lip vermilion combined with minimal backward displacement of the upper lip were recorded. Minimal changes were observed on the oral commissures. The main soft tissue changes associated with bimaxillary advancement surgery were generalized advancement of the middle and lower thirds of the face combined with widening of the paranasal, upper lip and nostrils regions. In Le Fort I cases, the correlation between the changes of the facial soft tissue and the skeletal surgical movements was assessed using PCA. A statistical method known as ’Leave one out cross validation’ was applied on the 30 cases which had Le Fort I osteotomy surgical procedure to effectively utilize the data for the prediction algorithm. The prediction accuracy of soft tissue changes showed a mean error ranging between (0.0006mm±0.582) at the nose region to (-0.0316mm±2.1996) at the various facial regions.

New ultrasonic-based methods in proteomics

Dissertação apresentada para obtenção do Grau de Doutor em Bioquímica pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia.A presente dissertação foi preparada no âmbito do convénio bilateral existente entre a Universidade Nova de Lisboa e a Universidade de Vigo.

Complex networks and data mining: toward a new perspective for the understanding of complex systems

Complex systems, i.e. systems composed of a large set of elements interacting in a non-linear way, are constantly found all around us. In the last decades, different approaches have been proposed toward their understanding, one of the most interesting being the Complex Network perspective. This legacy of the 18th century mathematical concepts proposed by Leonhard Euler is still current, and more and more relevant in real-world problems. In recent years, it has been demonstrated that network-based representations can yield relevant knowledge about complex systems. In spite of that, several problems have been detected, mainly related to the degree of subjectivity involved in the creation and evaluation of such network structures. In this Thesis, we propose addressing these problems by means of different data mining techniques, thus obtaining a novel hybrid approximation intermingling complex networks and data mining. Results indicate that such techniques can be effectively used to i) enable the creation of novel network representations, ii) reduce the dimensionality of analyzed systems by pre-selecting the most important elements, iii) describe complex networks, and iv) assist in the analysis of different network topologies. The soundness of such approach is validated through different validation cases drawn from actual biomedical problems, e.g. the diagnosis of cancer from tissue analysis, or the study of the dynamics of the brain under different neurological disorders.

Non root feeding with two sources of zinc on Coffea arábica L. 'mundo novo'(B.Rodr.) Choussy

A trial was carried out on an eight old coffee plantation with visible zinc problems. The plantation was situated nearly the city of Jaú (22º30'S, 48º30'W). State of São Paulo, Brazil. The soil is classified as medium texture Oxisol of low base saturation (Latossol Vermelho Amarelo - fase arenosa). The pulverization program started in november 1977, followed in march and July 1978 (heavy harvest) and ended in march and July 1979 (light harvest). Is should be mentioned that a well reconized characteristic of arábica coffe is its habit of biennial bearing, a very heavy harvest is most often followed by a light load the next year. The following treatments and amounts of chemicals per cova hole (4 trees) were tested in accordance with a random block design: 1. 1 g of zinc (zinc sulphate, 0.5%) 2. 3 g of nitrogen (urea, 1.3%) 3. 1 g of zinc + 3 g of nitrogen (zinc sulphate 0.5% + urea 1.3%) 4. 0.25 g, 0.50 g, 1.00 g, 2.00 g of zinc plus 0.75 g, 1.50 g, 3.00 g and 6.00 of nitrogen (correspondent to NZN* 15-0-0-5 as 0.75%, 1-5%, 3.0% and 6.0% by v/v). Foliar absorption data were obtained by collecting the 3rd and 4th pairs of the coffee leaves and analysed them for N, P, K, Ca, Mg, S, B, Cu, Fe, Mn, and Zn. The main results may be summarized as follows: 1. The maximum calculated yields of clean coffee were obtained by the applications of 5.84 1 of NZN (1.13%) per hectare. 2. The applications of zinc sulphate (0.5%) and urea (1.3%) together or separate did not affected the coffee bean production. 3. The applications of 15.0 1 of NZN per hectare reduced the coffee yields. 4. Leaf damages and burning symptoms were observed by the applications of urea (1.3%) plus zinc sulphate (0.5%) and larger doses than 7.5 1 of NZN per hectare. 5. Leaf tissue analysis show that the concentrations of the elements were affecred by the age of the leaves and by the yields of the coffee trees. 6. The applications of increasing doses of NZN causes an increase in the concentration of zinc, manganese and boron in the leaves and decreased the concentration in calcium and potassium the leaves. 7. The concentration of zinc in the leaves associated with the heavy harvest, in July, was 70.0 ppm.

Urinary sphincter based on electronics and artificial muscles

Objectives: The AMS 800TM is the current artificial urinary sphincter (AUS) for incontinence due to intrinsic sphincter deficiency. Despite good clinical results, technical failures inherent to the hydraulic mechanism or urethral ischemic injury contribute to revisions up to 60%. We are developing an electronic AUS, called ARTUS to overcome the rigors of AMS. The objective of this study was to evaluate the technical efficacy and tissue tolerance of the ARTUS system in an animal model.Methods: The ARTUS is composed by three parts: the contractile unit, a series of rings and an integrated microprocessor. The contractile unit is made of Nitinol fibers. The rings are placed around the urethra to control the flow of urine by squeezing the urethra. They work in a sequential alternative mode and are controlled by a microprocessor. In the first phase a three-rings device was used while in the second phase a two-rings ARTUS was used. The device was implanted in 14 sheep divided in two groups of six and eight animals for study purpose. The first group aimed at bladder leak point pressure (BLPP) measurement and validation of the animal model; the second group aimed at verifying mid-term tissue tolerance by explants at twelve weeks. General animal tolerance was also evaluated.Results: The ARTUS system implantation was uneventful. When the system was activated, the BLPP was measured at 1.038±0.044 bar (mean±SD). Urethral tissue analysis did not show significant morphological changes. No infection and no sign of discomfort were noted in animals at 12 weeks.Conclusions: The ARTUS proved to be effective in continence achievement in this study. Histological results support our idea that a sequential alternative mode can avoid urethral atrophy and ischemia. Further technical developments are needed to verify long-term outcome and permit human use.

Urinary Sphincter Based on Electronics and Artificial Muscles

Objectives: The AMS 800 is the current artifi cial urinary sphincter (AUS) forincontinence due to intrinsic sphincter defi ciency. Despite good clinical results,technical failures inherent to the hydraulic mechanism or urethral ischemicinjury contribute to revisions up to 60%. We are developing an electronic AUS,called ARTUS to overcome the rigors of AMS. The objective of this study wasto evaluate the technical effi cacy and tissue tolerance of the ARTUS systemin an animal model.Methods: The ARTUS is composed by three parts: thecontractile unit, a series of rings and an integrated microprocessor. The contractileunit is made of Nitinol fi bers. The rings are placed around the urethrato control the fl ow of urine by squeezing the urethra. They work in a sequentialalternative mode and are controlled by a microprocessor. In the fi rst phase athree-rings device was used while in the second phase a two-rings ARTUS wasused. The device was implanted in 14 sheep divided in two groups of six andeight animals for study purpose. The fi rst group aimed at bladder leak pointpressure (BLPP) measurement and validation of the animal model; the secondgroup aimed at verifying midterm tissue tolerance by explants at twelve weeks.General animal tolerance was also evaluated.Results: The ARTUS systemimplantation was uneventful. When the system was activated, the BLPP wasmeasured at 1.038 ± 0.044 bar (mean ± SD). Urethral tissue analysis did notshow signifi cant morphological changes. No infection and no sign of discomfortwere noted in animals at 12 weeks.Conclusions: The ARTUS proved to beeffective in continence achievement in this study. Histological results supportour idea that a sequential alternative mode can avoid urethral atrophy andischemia. Further technical developments are needed to verify long-termoutcome and permit human use.

DRIS norms for 'Valencia' sweet orange on three rootstocks

Diagnosis and Recommendation Integrated System (DRIS) applies nutrient ratios instead of the isolated concentration values of each nutrient in interpretation of tissue analysis. The objectives of this research were to establish adequate DRIS norms for 'Valencia' sweet orange irrigated commercial groves budded on three rootstocks and correlate indexes of nutrition balance with yield. Experiments were conducted in São Paulo State, Brazil. Rootstocks Rangpur lime, Caipira sweet orange, and Poncirus trifoliata, with more than six years old and yield above 40 ton ha-1 were utilized. Data referred to yield, tree spacing, rootstock and foliar concentrations of N, P, K, Ca, Mg, S, Fe, Mn, Cu, Zn, and B in non fruiting terminals for each grove were processed for the years 1994 through 1998. DRIS indexes were calculated by Nick criterion for choosing the ratio order of the nutrients and Jones calculation method of the ratio functions. Indexes of nutritional balance calculated from DRIS norms presented high correlation with yield for the three scion/rootstock combinations. DRIS norms defined in this research are valid, since leaf sampling is done on non fruiting terminals and the grove is irrigated.