Abstract: A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.

Palavras-Chave: chromatography; ion exchange chromatography; animal tissues; secretion; skin; sample preparation; viscosity; amphibians; hydrolases

MARIANO, DOUGLAS O.C.; MESSIAS, MARCELA Di G.; PREZOTTO-NETO, JOSE P.; SPENCER, PATRICK J.; PIMENTA, DANIEL C. Biochemical analyses of proteins from duttaphrynus melanostictus (Bufo melanostictus) skin secretion: soluble protein retrieval from a viscous matrix by ion-exchange batch sample preparation. Protein Journal, v. 37, n. 4, p. 380-389, 2018. DOI: 10.1007/s10930-018-9780-z. Disponível em: http://repositorio.ipen.br/handle/123456789/29320. Acesso em: $DATA.

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Abstract: Background Tetanus toxin blocks the release of the inhibitory neurotransmitters in the central nervous system and causes tetanus and its main form of prevention is through vaccination. The vaccine is produced by inactivation of tetanus toxin with formaldehyde, which may cause side effects. An alternative way is the use of ionizing radiation for inactivation of the toxin and also to improve the potential immunogenic response and to reduce the post-vaccination side effects. Therefore, the aim of this study was to characterize the tetanus toxin structure after different doses of ionizing radiation of 60Co. Methods Irradiated and native tetanus toxin was characterized by SDS PAGE in reducing and non-reducing conditions and MALD-TOF. Enzymatic activity was measured by FRET substrate. Also, antigenic properties were assessed by ELISA and Western Blot data. Results Characterization analysis revealed gradual modification on the tetanus toxin structure according to doses increase. Also, fragmentation and possible aggregations of the protein fragments were observed in higher doses. In the analysis of peptide preservation by enzymatic digestion and mass spectrometry, there was a slight modification in the identification up to the dose of 4 kGy. At subsequent doses, peptide identification was minimal. The analysis of the enzymatic activity by fluorescence showed 35 % attenuation in the activity even at higher doses. In the antigenic evaluation, anti-tetanus toxin antibodies were detected against the irradiated toxins at the different doses, with a gradual decrease as the dose increased, but remaining at satisfactory levels. Conclusion Ionizing radiation promoted structural changes in the tetanus toxin such as fragmentation and/or aggregation and attenuation of enzymatic activity as the dose increased, but antigenic recognition of the toxin remained at good levels indicating its possible use as an immunogen. However, studies of enzymatic activity of tetanus toxin irradiated with doses above 8 kGy should be further analyzed.

Palavras-Chave: cobalt 60; detoxification; enzyme activity; gamma radiation; irradiation; neurology; radiation dose units; tetanus; toxins; vaccines

SARTORI, GISELLE P.; COSTA, ANDREA da; MACARINI, FERNANDO L. dos S.; MARIANO, DOUGLAS O.C.; PIMENTA, DANIEL C.; SPENCER, PATRICK J.; NALI, LUIZ H. da S.; GALISTEO JUNIOR, ANDRES J. Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation. Journal of Venomous Animals and Toxins including Tropical Diseases, v. 27, p. 1-13, 2021. DOI: 10.1590/1678-9199-JVATITD-2020-0140. Disponível em: http://repositorio.ipen.br/handle/123456789/32051. Acesso em: $DATA.

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Abstract: Glass-ceramics based on the Li2O-SiO2 system have been extensively used as restorative dental materials due to their excellent chemical durability, aesthetics, inertness in the buccal environment, and high fracture strength; but they are not bioactive. On the other hand, all known bioactive glasses show ability to bond to bone, teeth and cartilage coupled to osteoconduction and osteoinduction, but their fracture strength and toughness are rather low. The aim of this study is to develop and evaluate the in vitro biocompatibility of a new type of (bioactive and strong) lithia-silica glass-ceramic. For these purposes, two types of glass-ceramics based on a multicomponent lithia-silica system were studied: lithium metasilicate (LM) and lithium disilicate (LD). The in vitro bioactivity study was conducted in a SBF solution, before and after different times of immersion; the new materials were analyzed by XRD, FTIR, and SEM. Some samples were subjected to in vitro biodegradation tests to quantify the release of lithium and the weight loss. Cytotoxicity, adhesion, and cell proliferation on different samples were examined by using the Methyl Tetrazolium salt (MTS) and Alizarin Red. For ~40 vol% crystallinity, lithium metasilicate was detected as the major phase, whereas for ~80 vol% crystallinity, lithium disilicate was the major phase. The LD proved to be strong (3p-bending strength of 233 ± 12 MPa) and bioactive after 14 days of immersion in SBF. In terms of lithium ion release, the LD was outside the toxic range (> 8.3 ppm). The LM and LD are not cytotoxic. The LD shows the best cellular adhesion and proliferation, leading to the formation of a mineralized matrix after 21 days. These results clearly suggest that the new LD brand is strong and highly biocompatible and warrants further study.

Palavras-Chave: biology; compatibility; dentistry; teeth; biological regeneration; in vitro; crystallization; lithium; ceramics; glass

DAGUANO, JULIANA K.M.B.; MILESI, MARIANA T.B.; RODAS, ANDREA C.D.; WEBER, ALINE F.; SARKIS, JORGE E.S.; HORTELLANI, MARCOS A.; ZANOTTO, EDGAR D. In vitro biocompatibility of new bioactive lithia-silica glass-ceramics. Materials Science & Engineering C, v. 94, p. 117-125, 2019. DOI: 10.1016/j.msec.2018.09.006. Disponível em: http://repositorio.ipen.br/handle/123456789/29994. Acesso em: $DATA.

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Abstract: The NUMEN (NUclear Matrix Elements for Neutrinoless double beta decay) project was recently proposed with the aim to investigate the nuclear response to Double Charge Exchange reactions for all the isotopes explored by present and future studies of 0νββ decay. The expected level of radiation in the NUMEN experiment imposes severe limitations on the average lifetime of the electronic devices. During the experiments, it is expected that the electronic devices will be exposed to about 105 neutrons/cm2/s according to FLUKA simulations. This paper investigates the reliability of a System On Module (SOM) under neutron radiation. The tests were performed using thermal, epithermal, and fast neutrons produced by the Instituto de Pesquisas Energéticas e Nucleares 4.5MWNuclear Research Reactor. The results show that the National Instruments SOM is robust to neutron radiation for the proposed applications in the NUMEN project.

Palavras-Chave: nuclear matrix; neutrons; modulation; silver nitrates; calculation methods; neutron beams; charge exchange

LO PRESTI, DOMENICO; MEDINA, NILBERTO H.; GUAZZELLI, MARCILEI A.; MORALLES, MAURICIO; AGUIAR, VITOR A.P.; OLIVEIRA, JOSE R.B.; ADDED, NEMITALA; MACCHIONE, EDUARDO L.A.; SIQUEIRA, PAULO de T.D.; ZAHN, GUILHERME; GENEZINI, FREDERICO; BONANNO, DANILO; GALLO, GIUSEPPE; RUSSO, SALVATORE; SGOUROS, ONOUFRIOS; MUOIO, ANNAMARIA; PANDOLA, LUCIANO; CAPPUZZELLO, FRANCESCO. Neutron radiation effects on an electronic system on module. Review of Scientific Instruments, v. 91, n. 8, p. 083301-1 - 083301-5, 2020. DOI: 10.1063/5.0010968. Disponível em: http://repositorio.ipen.br/handle/123456789/31481. Acesso em: $DATA.

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Abstract: Amphibians are, currently, considered the first vertebrates that had performed the aquatic to terrestrial transition during evolution; therefore, water balance and dehydration control were prerequisites for such environment conquering. Among anurans, Phyllomedusa is a well-studied genus, due to its peptide-rich skin secretion. Here, we have analyzed the skin secretion of Phyllomedusa distincta targeting the proteins present in the skin secretion. The major soluble protein was chromatographically isolated and utilized to immunize rabbits. Through proteomics approaches, we were able to identify such protein as being the diacylglycerol O-acyltransferase 2 (DGAT2), a crucial enzyme involved in lipid synthesis and in the skin water balance. Immunohistochemistry assays revealed the protein tissular distribution for different animal species, belonging to different branches of the phylogenetic tree. Specifically, there was positivity to the anti-DGAT2 on Amphibians’ skin, and no antibody recognition on fish and mammals’ skins. The DGAT2 multiple sequence alignment reveals some degree of conservation throughout the genera; however, there is a different cysteine pattern among them. Molecular modeling analyses corroborate that the different cysteine pattern leads to distinct 3D structures, explaining the different antibody recognition. Moreover, the protein phylogenetic analyses place the Xenopus DGAT2 (the available amphibian representative) next to the Coelacanthus enzyme, which have led the authors to term this a ‘paleo-protein’. DGAT2 would be, therefore, an ancient protein, crucial to the terrestrial environment conquest, with a unique folding—as indicated by the molecular models and immunohistochemistry analyses—a consequence of the different cysteine pattern but with conserved biological function.

Palavras-Chave: amphibians; peptides; skin; proteins; secretion; chemical analysis; temperature control

SCIANI, JULIANA M.; NEVES, ADRIANA; VASSÃO, RUTH C.; SPENCER, PATRICK; ANTONIAZZI, MARTA M.; JARED, CARLOS; PIMENTA, DANIEL C. The amphibian diacylglycerol O-acyltransferase 2 (DGAT2): a ‘paleoprotein’ with conserved function but unique folding. Protein Journal, v. 38, n. 1, p. 83-94, 2019. DOI: 10.1007/s10930-019-09814-x. Disponível em: http://repositorio.ipen.br/handle/123456789/30037. Acesso em: $DATA.

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Abstract: The International Atomic Energy Agency (IAEA) has been encouraging the use of passive cooling systems in new designs of nuclear power plants. Next nuclear reactor generations are intended to have simpler and robust safety resources. Natural Circulation based systems hold an undoubtedly prominent position among these. The study of limiting conditions of these systems has led to instability behavior analysis where many different two-phase flow patterns are present. Void fraction is a key parameter in thermal transfer analysis of these flow instability conditions. This work presents a new method to estimate void fraction from images captured of an experimental two-phase flow circuit. The method integrates a set of Artificial Neural Networks with a modified Randomized Hough Transform to make multiple scans over acquired images, using crescent-sized masks. This method was called Randomized Hough Transform with Neural Network (RHTN). Each different mask size is chosen according with bubble sizes, which are the main ‘objects of interest’ in this image analysis. Images are segmented using fuzzy inference with different parameters adjusted based on acquisition focus. Void fraction calculation considers the volume of the imaged geometrical section of flow inside cylindrical glass tubes considering the acquisition depth-of-field used. The bubble volume is estimated based on geometrical parameters inferred for each detected bubble. The image database is obtained from experiments performed on a vertical two-phase flow circuit made of cylindrical glass where flow-patterns visualization is possible. The results have shown that the estimation method had good agreement with increasing void fraction experimental values. RHTN has been very efficient as bubble detector with very low ‘false-positive’ cases (< 0.004%) due robustness obtained through integration between Artificial Neural Networks with Randomized Hough Transforms.

Palavras-Chave: two-phase flow; void fraction; neural networks; image processing; bubbles; fuzzy logic; transformations; nuclear power plants; pattern recognition; natural convection; randomness

SERRA, PEDRO L.S.; MASOTTI, PAULO H.F.; ROCHA, MARCELO S.; ANDRADE, DELVONEI A. de; TORRES, WALMIR M.; MESQUITA, ROBERTO N. de. Two-phase flow void fraction estimation based on bubble image segmentation using Randomized Hough Transform with Neural Network (RHTN). Progress in Nuclear Energy, v. 118, p. 1-21, 2020. DOI: 10.1016/j.pnucene.2019.103133. Disponível em: http://repositorio.ipen.br/handle/123456789/30353. Acesso em: $DATA.

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