bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracorporeal life support with an integrated left ventricular vent in children with a low cardiac output
Authors: Sandrio Stany , Springer Wolfgang , Karck Matthias , Gorenflo Matthias , Weymann Alexander , Ruhparwar Arjang , Loukanov Tsvetomir
Published: August 2013

Background: The aim of this study was to evaluate our experience in central extracorporeal life support with an integrated left ventricular vent in children with cardiac failure. Methods: Eight children acquired extracorporeal life support with a left ventricular vent, either after cardiac surgery (n = 4) or during an acute cardiac illness (n = 4). The ascending aorta and right atrium were cannulated. The left ventricular vent was inserted through the right superior pulmonary vein and connected to the venous line on the extracorporeal life support such that active left heart decompression was achieved. Results: No patient died while on support, seven patients were successfully weaned from it and one patient was transitioned to a biventricular assist device. The median length of support was 6 days (range 5–10 days). One patient died while in the hospital, despite successful weaning from extracorporeal life support. No intra-cardiac thrombus or embolic stroke was observed. No patient developed relevant intracranial bleeding resulting in neurological dysfunction during and after extracorporeal life support. Conclusions: In case of a low cardiac output and an insufficient inter-atrial shunt, additional left ventricular decompression via a vent could help avoid left heart distension and might promote myocardial recovery. In pulmonary dysfunction, separate blood gas analyses from the venous cannula and the left ventricular vent help detect possible coronary hypoxia when the left ventricle begins to recover. We recommend the use of central extracorporeal life support with an integrated left ventricular vent in children with intractable cardiac failure.
The use of dornase alpha for post-operative pulmonary atelectasis after congenital heart surgery
Authors: Ozturk Erkut , Tanidir Ibrahim C. , Haydin Sertac , Onan Ismihan S. , Odemis Ender , Bakir Ihsan
Published: August 2013

Objective:To investigate the efficacy of dornase alpha, a mucolytic agent, in children who developed pulmonary atelectasis after congenital heart surgery.Design:Retrospective case–control study.Setting: Paediatric cardiac intensive care unit at a tertiary care hospital.Patients: Between July, 2011 and July, 2012, 41 patients who underwent congenital cardiac operations and developed post-operative pulmonary atelectasis that was resistant to conventional treatment and chest physiotherapy.Interventions: In all, 26 patients received dornase alpha treatment. As a control group, 15 patients were treated with conventional medications and chest physiotherapy.Main results: The median age of patients was 25.5 (3–480) days in the study group and 50.0 (3–480) days in the control group. A total of 15 (57.6%) patients in the study group and 8 (53.3%) patients in the control group were male. The median weight was 4.2 (2.9–14.2) kg and 4.0 (3.5–13.6) kg in the study and control group, respectively. In the study group, pulmonary atelectasis was diagnosed at a median period of 5 (2–18) days after operations, whereas in the control group atelectasis was diagnosed at a median period of post-operative 6 (3–19) days. In the study group, the median atelectasis score decreased from 3.4 (1–6) to 0.8 (0–3) (p = 0.001). The median pO2level increased from 69 (17–142) mmHg to 89 (30–168) mmHg (p = 0.04). In addition, heart rate and respiratory rate per minute were significantly decreased (p < 0.05). There were no significant changes in these parameters in the control group.Conclusions: The use of dornase alpha can be effective for the management of pulmonary atelectasis that develops following congenital heart surgery.
Neural correlates of attentional bias in addiction
Authors: Hester Robert , Luijten Maartje
Published: August 2013

A small but growing neuroimaging literature has begun to examine the neural mechanisms underlying the difficulty that substance-use dependent (SUD) groups have with ignoring salient, drug-related stimuli. Drug-related attentional bias appears to implicate the countermanding forces of cognitive control and reward salience. Basic cognitive neuroscience research suggests that ignoring emotionally evocative stimuli in our environment requires both up-regulation of control networks and down-regulation of processing in emotion and reward regions. Research to date suggests that attentional biases for drug-related stimuli emerge from a failure to sufficiently increase control of attention over salient, but task-irrelevant stimuli. While SUD samples have typically shown increased activity in the cognitive control regions (ie, lateral prefrontal and dorsal anterior cingulate), during attentional bias such increases appear to have been insufficient for the concomitant increases in processing by the emotion/reward regions (ie, amygdala, insula, and striatum). Given the potential contribution of attentional biases to perpetuating drug use and the development of interventions (both pharmaceutical and cognitive-behavioral) to treat biases, understanding the neural basis of successfully reducing bias remains an important, but as yet unanswered, question for our field.
Breast-feeding in relation to weight retention up to 36 months postpartum in the Norwegian Mother and Child Cohort Study: modification by socio-economic status?
Authors: Brandhagen Martin , Lissner Lauren , Brantsaeter Anne Lise , Meltzer Helle Margrete , Häggkvist Anna-Pia , Haugen Margaretha , Winkvist Anna
Published: August 2013

Objective

We investigated the association between full breast-feeding up to 6 months as well as partial breast-feeding after 6 months and maternal weight retention at 6, 18 and 36 months after delivery in the Norwegian Mother and Child Cohort Study (MoBa), conducted by the Norwegian Institute of Public Health.

Design

Cohort study. Information on exposure and outcome was collected by questionnaire.

Setting

Norway.

Subjects

Women at 6 months (n 49 676), 18 months (n 27 187) and 36 months (n 17 343) postpartum.

Results

Longer duration of full breast-feeding as well as partial breast-feeding was significantly related to lower weight retention at 6 months. At 18 months full breast-feeding (0–6 months) and partial breast-feeding for 12–18 months were significantly related to lower weight retention. At 36 months only full breast-feeding (0–6 months) was significantly related to lower weight retention. For each additional month of full breast-feeding, maternal weight was lowered by 0·50 kg/month at 6 months, 0·10 kg/month at 18 months and 0·14 kg/month at 36 months (adjusted for pre-pregnant BMI, pregnancy weight gain, age and parity). Partial breast-feeding resulted in 0·25 kg/month lower maternal weight at 6 months. Interactions were found between household income and full breast-feeding in relation to weight retention at 6, 18 and 36 months, indicating most benefit among women with low income.

Conclusions

The present study supports the hypothesis that full breast-feeding contributes to lower postpartum weight retention and shows that the effect is maintained for as long as 3 years postpartum.
Salicornia ramosissima ethanolic extract on mice: a light microscopy approach on liver and kidney
Authors: Ferreira D. , Pinto D. , Silva H. , Pereira M.L.
Published: August 2013

Salicornia ramosissima J. Woods (Chenopodiaceae), included in the species aggregate S. europaea agg., is an annual succulent halophyte and one of the most salt tolerant plants, broadly distributed in the salt marshes and salt pans of Ria de Aveiro and in many others of the Iberian Peninsula. Salicornia L. has been used not only as a seasoned vegetable, salad and fermented food in coastal areas of Europe and Asia, but also as folk medicine for disorders such as constipation, obesity and diabetes. To corroborate this, the literature reports immunomodulatory, antioxidative, anti-inflammatory, anti-hyperlipidemic and antidiabetic effects. Moreover, some bioactive compounds from its aerial parts were recently isolated and identified, exhibiting also antioxidant and cytotoxic activities.

Factors such as the proper botanical identification, season and harvest site, extraction and purification methods, characterisation of the active constituents and the extract effects in a dose-and time-dependent manner are crucial to assess the therapeutic potential of herbal extracts. The aim of this study was to investigate the possible hepatic and renal effects of an ethanolic extract of S. ramosissima on mice.

Aerial portions of S. ramosissima were collected from a salt pan in Ria de Aveiro (Portugal). The ethanolic extract (50 mg/kg b.w.) was orally administered, during 3 weeks, to male ICR-CD1 mice, purchased from Harlan (Spain). Control group was also considered. Liver and kidney were collected and prepared for histology. Animal procedures were followed according to guidelines for ethics and animal care.

Central and portal vein congestion and severe hydropic changes of the hepatocytes were noted in the liver of exposed group, compared with controls (Figs. 1A, 1B). Renal profile of control group evidenced normal features (Figs.1C, 1E). However, significant histological alterations were found in the exposed group: cortical interstitial haemorrhages (Fig. 1D), inflammatory cell infiltration and tubular cell degeneration within medulla and cortico-medullary junction (Fig. 1F).

In conclusion, this pilot study has demonstrated considerable effects in the mouse metabolism of S. ramosissima ethanolic extract, with significant hepatic and renal lesions. Further studies are needed to correlate these data with isolated active constituents for a more reliable evaluation of the potential of this species.

Acknowledgements

Grants were provided by CICECO, QOPNA and CESAM.
Ultrastructural study of a case of epidermolysis bullosa simplex superficialis (EBSS)
Authors: Alves de Matos A.P. , Gouveia C. , Sousa A.B. , Afonso A.
Published: August 2013

A very rare subset of Epidermolysis bullosa simplex (EBS), termed “EBS superficialis” (EBSS), has been described in two families. EBS superficialis is a rare autosomal dominant form of suprabasal EBS in which blistering arises just below the stratum corneum.

The original report of EBSS was subsequently questioned by genetic analysis of one of the families that identified a mutation in a collagen gene responsible for dystrophic epidermolysis bullosa (DEB), thus implying that this entity is in fact a variant of DEB. Nonetheless, the existence of some form of EBSS can not be ruled out.

We performed a transmission electron microscopy study of a skin biopsy from a 36 year old woman suspect of having EBSS. A skin biopsy was sequentially fixed in 3% glutaraldehyde in sodium cacodylate buffer 0.1M pH 7.3, osmium tetroxide in the same buffer and uranyl acetate in acetate-acetic acid buffer 0.1M pH 5. Dehydration was performed in ethanol and embedding in an Epon-Araldite mixture. Thin sections contrasted with uranyl acetate and lead cytrate were observed and photographed in a JEOL 100SX electron microscope.

She had a few intact acral bullae in the neonatal period and superficial erosions and crusts in friction areas since then. Her one year-old boy, her older sister and the latter’s adolescent son had the same clinical picture. The patient does not display any symptoms expected for DEB.

We found profound alterations of the structure of the outer layers of the epidermis. Degenerative changes were seen in the cells of the upper layers of the stratum spinosum. Nuclei are lost and flattened cells made mostly of compressed tonofilaments lay below a cleft that develops between the compressed cells and the granular cell layer (Fig. 1). The granular cell layer is very reduced and represented by a few damaged cells that stay attached to the stratum corneum (Fig. 2).

The alterations do not seem to be related to collagen defects, and in the reported case the cleft develops in the altered upper region of the prickle-cell layer, just below the granular layer.
Reservoirs of human pathogens: amoeba-associated microorganisms in the environment
Authors: Costa R. , Fugas M. , Caeiro M.F. , Vale F.F. , Amorim A. , Morgado F. , Alves de Matos A.P.
Published: August 2013

Microorganisms, that evolve to acquire resistance to environmental amoeba, are likely to become human pathogens due to the physiological similarities of free-living amoebae to human macrophages. In this sense free-living amoebae can be regarded as nurseries of pathogenic microorganisms. Due to the widespread distribution of amoebae in the environment and their resistance to current disinfection procedures, they may constitute important reservoirs of pathogenic microorganisms. Striking examples are Legionella pneumophila and several mycobacteria including Mycobacterium tuberculosis. This work represents the first attempt to detect and characterize amoebae and their associated microorganisms in Portugal.

Detection, isolation and in vitro culture of amoebae from several environmental sources, including lagoon and estuarine water and sediments, were performed according to previously developed methods. Identification of the isolated amoebae was done by optical microscopy based on morphological criteria and by DNA sequence analysis of PCR products amplified with one of the following two sets of primers: EUK and ITS. Transmission electron microscopy (TEM) studies and PCR/sequencing approaches were used to detect and identify amoeba-associated microorganisms (AAMs). Bacterial 16S region was amplified with the primers 616V/630R. TEM studies were performed according to standard procedures. In short, samples were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite. Thin sections contrasted with uranyl acetate and lead citrate were observed with a JEOL 100-SX electron microscope.

Several species of amoeba (Acanthamoeba lenticulata, A. polyphaga, A. rhyzodes, Platyamoeba oblongata, Saccamoeba limax, Vannella simplex, Vannella sp. and other unidentified amoebae) were found and AAMs were detected both by TEM and PCR/sequencing. The bacteria species Variovorax paradoxus, previously found in association with Saccamoeba and Arcella, were isolated and identified by the PCR/sequencing approach, which also allowed the detection of an unidentified species with about 85% identity with Marivirga tractuosa, a species not yet associated with amoebae. These results point to the existence of AAMs in the environments subjected to this study and the need to evaluate their pathogenic potential. This is an important issue particularly in environments related to human recreational, nutritional and public health activities.
Histology and histochemistry of sea anemones in environmental contamination studies
Authors: Gadelha J.R. , Morgado F. , Soares A.M.V.M.
Published: August 2013

Since contaminants such as metals, POPs (Persistent Organic Pollutants) and PAHs (Polycyclic aromatic compounds represent a risk to human health and to the environment, it is therefore extremely necessary to study their biological effects. Man-made chemicals endocrine disruptors such as estrogens pose the potential to modulate endocrine function and thus adversely affect humans and other animal’s reproductive development. In this work, sublethal toxicity tests were carried out with the sea anemones Actinia equina and Anemonia sulcata exposed to 17-β-Estradiol. A. equina and A. sulcata are species that present a wide geographic distribution and might possibly be effective pollution indicators. Histological and histochemical techniques were used to detect morphological changes in sea anemones in order to find histological parameters that could be useful as early biomarkers of environmental contamination. The histological and histochemical procedures followed by standard methods to Hematoxylin and eosine (H & E), Periodic acid Shiff reagent (PAS) and Masson`s Tricrome (TMass) stained adaptated to Actiniidae conditions. Such as, the fixation (formalin and alcohol) time is high (96 hours), because this organisms are almost 98% of water body constitution. The slides obtained were observed by light microscopy means. The assemblage of methodologies described permitted the identification of several anomalies/pathologies in different parts of the sea anemones body, with special attention to reproductive structures. Results obtained for A. sulcata showed vitellogenic oocytes with anomalous dimensions, altered cytoplasm or without cellular membrane limits (Figure 1). It can also be observed lipid accumulations and cells membranes not always preserved. In certain areas oocytes presents small reactivity with atypical PAS low basophilic patterns. In the mesoglea the amoebocytes showed more eosinophilic cytoplasm or extracellular bodies suggesting necrosis or protein content.  The effects at 10 µg/L concentrations show a considerable number of oocytes with germinal vesicles membranes and indistinct cytoplasm boundaries (Figure 1). Results obtained for A. equina showed some morphological changes in the spermatocytes of male gonads and in the germinal vesicles the female gonads. The effects observed at higher concentrations shows oocytes and ovarian tissues disintegration. The morphologic alterations observed suggested a delay in spermatogenesis and although there have been no alterations in female vitellogenic granules, there are changes in their maturation. The whole effects lead to verify the role that the estradiol in the Anthozoan reproductive system.

Acknowledgments: This work was supported by the Portuguese Foundation for the Science and Technology – Portugal and FEDER funds, through the Projects: PTDC/MAR/464729/2006 and FCT/CNPq (Brazil), Project 6818, Programme 19/ 004.
Evaluation of biological agent association anti-TNF induced arthritis and laser therapy: an experimental study
Authors: Morsoleto M.J.M.S. , Amstalden E.M.I. , Morgado F.M.R. , Bértolo M.B.
Published: August 2013

Rheumatoid arthritis is a chronic, systemic inflammatory illness that predominantly affects the joints. Recent works tested inductor agents of arthritis models and their effects when associated to low intensity laser therapy. In this work the effects of the biological agent Infliximab and low intensity laser therapy were investigated in an animal experimental model of Zymosan induced arthritis. This model is widely used in pharmacological studies because it enhances the hypernociception of inflammation, detected throughout the observation of joint articular incapacitation, increase of vascular permeability, release of inflammatory mediators in the joint space and histological changes.

A laser instrument (GaAlAs) DMC brand, model Photon Laser III was used, and the radiation was applied at wavelength 808 nm, power density of 200 mW/cm2, energy density of 2000 mJ/cm2, dose of energy or fluence 2 J/cm2, spot area of 0.02 mm, length of 5 dots per second. Rats with 250g were separated in 5 groups; the control (GI) received intra-articular 50µl of 0.9% saline solution in the right knee. The experimental group GII received intra articular injections of Zymosan (AZy) in the right knee at day zero (1mg, in 50µl of saline solution 0.9%); group GIII received daily doses of the same solution in the medial face of the right knee and 10 applications of laser (904 nm); Group GIV received 3 doses of 1,6ml of endovenous Infliximab in the vein of tail; Group GV received 3 doses of infliximab and daily doses of laser (904 nm). In the 14th day of the experiment the group GII was not treated. The organisms were sacrificed 24 hours after each protocol was completed and submitted to histopathological analysis using standard histological procedures.

Results showed that, with 14 days of AZy, the groups presented a moderate to intense inflammation, presence of giant cell granulomas and synovial hyperplasia. When treatment was associated with laser therapy the inflammation was substantially reduced. The results suggest the efficiency of laser therapy associated or not with the biological agent application, to the treatment of induced chronical synovitis.
A novel formed classifier for analyzing cellular contents in cells images
Authors: López M.A.G. , Posada N.G. , Gadelha J.R. , Morgado F.
Published: August 2013

Several approaches for cell image segmentation have been proposed and applied with success in the biomedical sciences field, but cell content estimation is still a not solved problem. This paper presents a novel formed classifier that combine appropriately “intelligent scissors” (livewire), object’s pixel information (RGB values of colors) and probabilistic artificial neural networks (ANN) techniques for segmenting and estimating precisely the cytoplasm composition in relation to protein, lipid, and carbohydrates. Based on this it is possible to estimate the gonad’s state of development and the maturation stage of oocytes in adult ovigerous females gonads.

The ovigerous mature females (identifiable by their brood pouch – empty, with eggs, with eyeless or eyed embryos) were selected under a dissecting microscope in the laboratory. A total of 170 gonadic masses from females containing oocytes in different developmental stages were analyzed. A total of 100 sections were observed by light microscopy in order to record the arrangement of the ovaries and determine the maturity stage of the various germ cells.

With the ANN network (Fig. 1) trained is possible to carry out the classification step. Classification aims to associate each cytoplasm pixel to one of the selected classes. In this process each cytoplasm pixel is simulated (Figure 2 A). As result a new image containing the classified cytoplasm is produced, where each class is represented by a different colour level (Figure 2 B). Figure 2 C show from left to right the full “classification flow process” including ROI selection, cytoplasm and nucleus contour extraction, cytoplasm segmentation and cytoplasm content classification. The proposed classifier was tested successfully in a representative image dataset, demonstrating the effectiveness and robustness of this new method facilitating the histochemical analysis of cell contents, based on a gonads model.
Automatic detection of polyomavirus particles with local texture indicators
Authors: Proença M.C. , Moura Nunes J.F. , Alves de Matos A.P.
Published: August 2013

A fully automatic approach to locate polyomavirus particles in images of transmission electronic microscopy is presented, that can localize intact particles, many damaged capsids and an acceptable percentage of superposed ones; the performance is quantified in 25 electron micrographs containing nearly 390 particles, compared to the interpretation of two independent electron microscopy experts. As these particles often does not have a well-defined edge, in highly textured backgrounds (Figure 1) the initial guess of candidate points from local entropy proportion measures retains a large set of points, in order to avoid false negatives.

Based in human criteria - focused on the pattern generated by the capsomeres typical of polyomavirus surface, this approach uses indicators calculated from the local co-occurrence matrix of grey levels to assess the textured pattern and prune the initial set of candidates; in some more complicated backgrounds about 2-10% of the elements will survive. A restricted set of the points accepted (Figure 2) is used to evaluate typical average and variance and reduce the set of survivors accordingly. These intermediate points are locally evaluated using i) a statistical index concerning the radiometric distribution of a circular neighborhood around the centroid of each candidate, and ii) a structural index resuming the expected morphological characteristics of eight radial intensity profiles encompassing the area of the possible particle. All the parameterization is based on the particle expected dimensions.

This hierarchical approach attains 95% efficiency in the detection of intact virus particles, tolerating a certain lack of differentiation in the borders and a certain amount of shape alterations. Only 5 to 7% (according to the reference used) of the intact particles are missed by the algorithm – cases of particles with abnormal radial structures, particles with different degrees of agglutination or particles showing empty (or with different degrees of permeation) capsids. Superposed particles are detected with a success rate of 71% to 77%. In the case of distorted and/or doubtful particles, a correct detection of 72% is attained; these particles are the main source of differences between experts (a difference of 48% between both interpretations).

This kind of algorithm is "tailored" for one virus family particle, in this case, the polyomavirus; it can be parameterized for different working magnifications, but cannot be applied to different virus, that has different texture, structure and typical radiometry characteristics. We believe that similar results can be obtained in other virus families with the necessary adaptations.
Intrinsically fluorescent silica nanocontainers: a promising theranostic platform
Authors: Rodrigues A.S. , Ribeiro T. , Fernandes F. , Farinha J.P.S. , Baleizão C.
Published: August 2013

A large decrease in the side effects of a drug can be obtained if it is efficiently delivered in a timely manner and in the needed location only. By combining therapeutic and diagnostic (theranostic) functionalities with targeting capabilities and large surface areas, nanoparticles provide an ideal vehicle for personalized medicine.

The main objective of our work is to develop hybrid Mesoporous Silica Nanoparticles (MSNs) for theranostics, carrying fluorescent beacons for traceability and imaging, featuring a smart release control mechanism, able to accommodate large drug loads and to deliver their cargo on demand to a desired location. This communication focus on the preparation of the fluorescent MSNs, and characterization by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and laser scanning fluorescence confocal microscopy (LSFCM).

Mesoporous Silica Nanoparticles (MSNs) with well-defined and controllable particle morphology are exceptional supports/nanocontainers for molecules and polymers.This class of materials are characterized by an ordered pore system of 2-8nm diameter, pore volumes above 1mL/g and particle size from 40nm to several hundred nanometers. The preparation of fluorescent hybrid MSNs involves the presence of a fluorescent molecule during particle synthesis, which becomes aligned with the pores, thus impervious to aggregation and self-quenching effects. The MSNs external surface can be selectively functionalized to immobilize polymers or (bio)molecules for possible targeting or sensing, and the pore is available for solvent diffusion, allowing the incorporation of different molecules.

We prepared monodispersed hybrid MSNs incorporating a fluorescent perylenediimide (PDI) derivative in the wall structure. The MSN-PDI were characterized by TEM (Figure 1), SEM and fluorescence emission spectroscopy. LSFCM images of the MSN-PDI after incubation in HEK293 cells show the internalization of the nanoparticles (Figure 2). These new hybrid nanoparticles, after surface-functionalization with stimuli-responsive gate systems, open possibilities for the development of traceable drug delivery systems.

This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal) and COMPETE (FEDER) within projects PTDC/CTM/101627/2008 and PEst-OE/CTM/LA0024/2011. T.R. and F.F. also thank FCT for Ph.D. (SFRH/BD/64702/2009) and Pos-Doc (SFRH/BPD/64320/2009) grants.
Deformable models as a tool for biometric and histopathological applications
Authors: López M.A.G. , Posada N.G. , Gadelha J.R. , Morgado F.
Published: August 2013

Mathematical morphology is a novel geometry-based technique for image processing and analysis, originally developed to process binary images, based on the use of simple concepts from set theory and geometry such as set inclusion, intersection, union, complementation, and translation Geometry is used to represent object shape, physics inflict constraints on how the shape may vary over space and time, and optimal approximation theory make available the formal underpinnings of mechanisms for fitting the models to measured data. Deformable curve, surface, and solid models gained popularity after they were proposed by Terzopoulos for use in computer vision and computer graphics in the mid 1980’s introducing the theory of continuous (multidimensional) deformable models in a Lagrangian dynamics setting, based on deformation energies in the form of generalized splines. The deformable model that has attracted the most attention to date is popularly known as “snakes”, planar deformable contours that are useful in several image analysis tasks. They are often used to approximate the locations and shapes of object boundaries in images based on the reasonable assumption that boundaries are piecewise continuous or smooth.

This work presents a novel approach since it joins (and, indeed, reinforces) the index framework with the evaluation of the same biological samples by a suitable combination of deformable models. Nucleus contour is identified through Active Shape Models techniques, and cytoplasm contour’s detected through parametric Snakes, with prior image preprocessing based on statistical and mathematical morphology techniques. Morphometric parameters such as nucleus and cytoplasm area and ratio between them are then easily computed. Biometry was performed using an ocular micrometer and nucleus/cytoplasm ratios were obtained characterizing each of the three identified stages: Immature, Vitellogenic and Mature. This resulted in a collection of tools, called morphological operators, which are eminently suited for the analysis of shape and structure in binary images. Acartia tonsa was used as model to establish an index for oocyte maturity determination based in citometry and histochemical evaluation of gonadic masses. It was validated the application of a methodology with a realistic background and a new, more accurate and ecologically realistic index for oocyte staging emerged.
A Photobleaching/Photoblinking analytical model for LSFCM imaging
Authors: Sanches J.M. , Rodrigues I.
Published: August 2013

Laser scanning fluorescence confocal microscope (LSFCM) imaging is an extensively used modality in biological research. However, these images present low signal to noise ratio and a time intensity decay effect due to the so called photoblinking/photobleaching (PBPB) phenomenon that corresponds to an intensity fading of a fluorescent probe along the time, as shown in Figure 1. This effect is caused by quantum phenomena associated with the electronic excitation and photochemical reactions among the fluorescent and the surrounding molecules induced by the incident radiation that temporarily or irreversibly destroy their ability to fluoresce. Since illumination is needed to excite and observe the tagging fluorescent proteins in the specimen and all the fluorophores will eventually photobleach upon extended excitation, the acquisition of this type of images becomes a hard task for long exposures.

There are in the literature several proposals to model this fading effects and among them the single and double exponential are the most used. However, simple and tractable theoretical models based on the physics of the observation process to support these empirical laws are not available. In this work, that theoretical model, supported on the underlying physics of the process, is derived to describe the PBPB effect (see Figure 2).

From a fluorescence point of view, tagging molecules can be in three main states (see Figure 3), i) ON-state, where they are able to fluoresce and be observed, ii) OFF-state, where they are temporarily not able to fluoresce and therefore are not visible and finally at the iii) BLEACHED-state where they become permanently OFF. Here, a continuous time differential equation dynamic model is proposed to describe the number of molecules at the ON-state, nON, along the time. The model is based on the underlying quantum mechanic physics theory of the observation process associated with this type of images and the common empirical weighted sum of two decaying exponentials (DExp), usually used in the literature, is derived from the model. The parameters βON and βOff are the transitions rate from and to the ON-state respectively and ξis the decay rate associated with the transitions for the permanent BLEACHED state.

Experiments with synthetic and real data are presented to validate the PBPB model and estimate the physical variables associated with the process. Intensity decay from real data and the corresponding theoretical curve are compared and displayed in Figure 4.

This work was supported by the FCT project [PEst-OE/EEI/LA0009/2011]. The authors thank Dr. José Rino and Profa Carmo Fonseca, from the Molecular Medicine Institute (IMM) of Lisbon, for providing biological technical support and the real data used in this paper.
The Gastropod Statolith Elemental Composition by EMPA
Authors: Galante-Oliveira S. , Marçal R. , Guimarães F. , Machado J. , Barroso C.M.
Published: August 2013

Gastropods statoliths are biogenic structures of CaCO3, analogous to fish otoliths. Their application in sclerochronology is recent and may be advantageous comparing to the most traditional organisms used hitherto. Moreover, since elements bind within calcified structures, statoliths theoretically incorporate chemical information from the surrounding environment over time. So, besides being used for age estimation and growth studies, statoliths elemental fingerprinting is recognized as natural tag to track larval dispersal and may also be considered as a potential marker of environmental change and/or pollution. However there is a lack of information on the elemental composition of adult gastropod statoliths. Here we characterize Nassariusreticulatus (Gastropoda: Caenogastropoda) statoliths elemental composition by electron microprobe analysis (EMPA).

One statolith from each of 3 adults was prepared for EMPA (Figure 1) and showed the same structural pattern: a nucleus circled by a metamorphic ring and, in addition to this core, 4 other rings delineating 5 increments (Figure 1F). General composition was assessed by Energy and Wavelength Dispersive Spectroscopy revealing C, Ca, O, Sr, Na, S and Mg as the most representative elements. These were then quantified in 3 points per increment. Carbon was not determined since surfaces were C-coated and the amount of oxides of Ca, Sr, Na, S and Mg was estimated from the valence. In addition, Ba – trace element in statoliths of some gastropods larvae – and Al, Cu, Hg, Sn – known as environmental pollutants – were also sought in one of the statoliths. For such, mean values for Ca, Sr, Na, S, Mg and O were set as the sample matrix. Standards and mean detection limits by element and statolith are indicated in Table 1.

Concentrations of Ca, O, Na, Sr and S varied between 3.7x105-4.2x105, 1.5x105-1.7x105, 2.5x103-6.1x103, 1.3x103-8.9x103 and 4.7x102-1.3x103 ppm, respectively; Mg, which was not always detected, ranged from 2.0x102-8.6x102 ppm. No significant differences in these elements mean concentrations were found between increments, except Na in one statolith between increments 1 and 5 (higher at the edge; Dunn’s test s=2.014, p>0.05). Thus the general elemental composition is nearly constant throughout lifespan (i.e. along statoliths radiuses). Regarding trace elements: Sn was never detected; Al and Hg were at 22.2% of the punctual analyses conducted while Cu was at 66.7% and Ba at 88.9%. Concentrations of Al, Hg, Cu and Ba varied between 55-71, 176-180, 183-302 and 647-969 ppm, respectively.

EMPA proved to be a useful technique in sclerochronology, allowing the characterization of the overall composition of gastropods statoliths and the quantification of elements in specific locations with high resolution.

This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the grant SFRH/BPD/70368/2010.
Are squid statoliths hollow during embryogenesis?
Authors: Rosa R. , Dionísio G. , Bensimon-Brito A. , Jacinto A. , Pimentel M.S. , Trübenbach K. , Moreno A. , Barquinha P. , Diniz M.
Published: August 2013

Squid statoliths are paired calcareous concretions essentially composed of calcium carbonate crystallised as aragoniteand found inside cranial fluid-filled cavities, the statocysts - the main organ of detection of gravity and movement. Statolith use as an ageing tool was validated in the 1980s, when the daily periodicity of the growth increments was confirmed by chemically marking these structures in animals maintained in aquaria. Although statolith analysis has brought major advances in the knowledge of squid population-dynamics, little is known about the processes involved in statolith’s calcification and increment deposition (e.g. during embryogenesis). According to Villanueva (2000), embryonic statolith development involves the formation of structures such as the focus, nucleus, postnuclear zone and natal ring.

The aim of this study was to investigatethe microstructure of the embryonic statolith, considering all main structures,in recently-hatched squids using optical (fluorescence) microscopy, scanning electron microscopy (SEM) and confocal microscopy.

Recently-spawned egg masses of the European squid, Loligo vulgaris, were collected in the west coast of Portugal. After hatching at 13-15ºC, statoliths were removed from the paralarvae andpreserved at -20ºC. Prior to confocal analysis, statoliths were submitted to Alizarin red S (ARS) staining (5% of ARS in 0.1% KOH) to verify the presence of calcium content. In addition, images from optical microscopy and scanning electron microscopy (SEM) were acquired.

The findings obtained by auto-fluorescence observation (Figure 1 A, B) and SEM (Figure 1 C), show that the embryonic statolith is hollow in some of the area between the nucleus and the natal ring. ARS stained samples analysed in confocal microscopy (Figure 2 A-G), confirmed the previous observation.

This surprising structural feature in the origin of CaCO3 sensorial structures has never been described before and suggests a production, with minimum of material and energy expenditure. This is fundamentally interesting not only in biological terms but also considering biotemplating orbiomimetics approaches (i.e. synthetic approaches), where hollow calcium carbonate structures have attracted considerable attention owing to their unique structural, optical, and surface properties that lead them to a wide range of applications, including as templates for functional architecture composite materials.

The Portuguese Foundation for Science and Technology (FCT) supported this study through project grant PTDC/BIA-BEC/103266/2008 to R. Rosa.

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Histology and histochemistry of the reproductive potential of Acartia clausi (copepoda: calanoida)
Authors: Morgado F. , Terdalkar S. , Gadelha J.R. , Pereira M.L.
Published: August 2013

The seasonal fluctuations in zooplankton densities in temperate climates have been long known and the multiplicity of performed studies identified a vast number of factors responsible for these phenomena, such as changes in the physico-chemical factors and other such types of environmental forces governing them. Acartia clausi is a euryaline temperate-boreal species very common in the Portuguese coastal ecosystems, in both estuarine and coastal waters. It is usually described as a temperate water species of neritic calanoid copepod, which is associated with warmer water regions, and as a result becomes more abundant in the summer months, reaching a biomass maximum during the months of July and August. Growth and egg production have been studied extensively in some Acartia species. In the present study histology and histochemistry were selected to determine the reproductive potential of A. clausi.

The ovigerous females were identified with a binocular microscope, isolated and fixed in the Bouin’s solution for histological (5 om thickness, mounting and Haematoxylin - Eosin staining) and histochemical analysis (Periodic Acid Schiff method (PAS) with Haematoxylin as a counter stain for the identification of the carbohydrate content and vitellogenic oocytes. The size of the oocytes was evaluated through measurements made with a micrometer.

The microscopic studies and Image analysis indicated that, in the month of September, the majority of the oocytes were immature and had reduced or almost negligible carbohydrate contents with very few vitellogenic oocytes (Figure 1 A and B), while the specimens from the month of March exhibited a large difference in the oocyte dimensions. These were mature and more vitellogenic and occupied almost half the volume of the body (Figure 1 C, D and E). This shows that, during the month of September, the environmental conditions are not favorable for the maturation of gonads in these species while in the month of March they proliferate and the species shows a high degree of reproductive potential.

This work forms a valid approach in understanding the population fluctuations and reproductive status in a key species of copepod showing a particular temporal variation associated with its reproductive strategy.
Application of Electron Backscatter Diffraction (EBSD) for Crystallographic Characterization of Aluminum-Doped Zinc Oxide Sputtered Films
Authors: Garcia C.B. , Ariza E. , Tavares C.J.
Published: August 2013

Zinc Oxide is a wide band-gap compound semiconductor that has been used in optoelectronic and photovoltaic applications due to its good electrical and optical properties. Aluminium has been an efficient n-type dopant for ZnO to produce low resistivity films and high transparency to visible light. In addition, the improvement of these properties also depends on the morphology, crystalline structure and deposition parameters. In this work, ZnO:Al films were produced by d.c. pulsed magnetron sputtering deposition from a ZnO ceramic target (2.0 wt% Al2O3) on glass substrates, at a temperature of 250 ºC.

The crystallographic orientation of aluminum doped zinc oxide (ZnO:Al) thin films has been studied by Electron Backscatter Diffraction (EBSD) technique. EBSD coupled with Scanning Electron Microscopy (SEM) is a powerful tool for the microstructural and crystallographic characterization of a wide range of materials.

The investigation by EBSD technique of such films presents some challenges since this analysis requires a flat and smooth surface. This is a necessary condition to avoid any shadow effects during the experiments performed with high tilting conditions (70º). This is also essential to ensure a good control of the three dimensional projection of the crystalline axes on the geometrical references related to the sample.

Crystalline texture is described by the inverse pole figure (IPF) maps (Figure 1). Through EBSD analysis it was observed that the external surface of the film presents a strong texture on the basal plane orientation (grains highlighted in red colour). Furthermore it was possible to verify that the grain size strongly depends on the deposition time (Figure 1 (a) and (b)). The electrical and optical film properties improve with increasing of the grain size, which can be mainly, attributed to the decrease in scattering grain boundaries which leads to an increasing in carrier mobility (Figure 2).

The authors kindly acknowledge the financial support from the Portuguese Foundation for Science and Technology (FCT) scientific program for the National Network of Electron Microscopy (RNME) EDE/1511/RME/2005.
Microstructural characterisation of a collection of Protohistoric bronzes
Authors: Figueiredo E. , Silva R.J.C. , Araújo M.F. , Vilaça R.
Published: August 2013

Microstructural characterisation of an archaeological collection of Protohistoric bronze artefacts attributed to around the VIII century B.C. and has been carried out using optical microscopy (OM) and scanning electron microscopy combined with energy dispersive spectroscopy (SEM-EDS). The collection was found in Medronhal cave (Condeixa-a-Nova, Portugal) and is composed by 1 fibula, 5 bracelets and 31 rings.

Small areas on the surfaces of the artefacts were cleaned from corrosion and were metallographically prepared by a manual polishing with several diamond suspensions in a cotton swab until 1 micron diamond size. OM observations were performed in bright field (BF) illumination and under polarized light (Pol), in unetched and etched conditions. SEM-EDS was performed without a conductive coating for a minimum external elemental interference.

OM observations allowed the study of the method and sequence of manufacture involved in the production of the various types of artefacts. Results show that the fibula and the bracelets have equiaxed (recrystallised) grain structures resulting from cyclic thermo-mechanical treatments performed to a pre-form cast bar until requested shape and surface finishing was attained. The rings, which are of diverse sizes, have various types of microstructures as a result of diverse manufacturing techniques. Some show a dendritic structure that resulted from the casting, and others show recrystallised structures that resulted from cycles of deformation and annealing procedures posterior to their casting (Figure 1).

SEM-EDS analysis allowed the study of the presence of inclusions in the metallic matrix and the study of corrosion in some artefacts (Figure 2). Different types of inclusions were identified, namely copper sulphides, tin oxide, and lead rich inclusions. Both copper sulphides and lead inclusions are commonly found in archaeological bronzes, and can be a result of copper ores impurities. Tin oxide, on the other hand, is not so common, and its presence can be understood as a result of preferential oxidation of tin regarding copper during a melting or alloying operation. The study of the interface alloy/corrosion showed the presence of chlorides in internal corrosion layers, which can probably be related to an aggressiveness of the burial environment.

This research work has been financed by the Portuguese Science Foundation (FCT) through the EarlyMetal project (PTDC/HIS-ARQ/110442/2008), the grant SFRH/BPD/73245/2010 (to EF) and the Strategic Project-LA25-2011-2012 (PEst-C/CTM/LA0025/2011) (to CENIMAT/I3N).