Search results for: euploid embryos

Authors: Iva Blazkova, Amitava Moulick, Vedran Milosavljevic, Pavel Kopel, Marketa Vaculovicova, Vojtech Adam, Rene Kizek

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Doxorubicin is one of the most commonly used and the most effective chemotherapeutic drugs. This antracycline drug isolated from the bacteria Streptomyces peuceticus var. caesius is sold under the trade name Adriamycin (hydroxydaunomycin, hydroxydaunorubicin). Doxorubicin is used in single therapy to treat hematological malignancies (blood cancers, leukaemia, lymphoma), many types of carcinoma (solid tumors) and soft tissue sarcomas. It has many serious side effects like nausea and vomiting, hair lost, myelosupression, oral mucositis, skin reactions and redness, but the most serious one is the cardiotoxicity. Because of the risk of heart attack and congestive heart failure, the total dose administered to patients has to be accurately monitored. With the aim to lower the side effects and to targeted delivery of doxorubicin into the tumor tissue, the different nanoparticles are studied. The drug can be bound on a surface of nanoparticle, encapsulated in the inner cavity, or incorporated into the structure of nanoparticle. Among others, carbon nanoparticles (graphene, carbon nanotubes, fullerenes) are highly studied. Besides the number of inorganic nanoparticles, a great potential exhibit also organic ones mainly lipid-based and polymeric nanoparticle. The aim of this work was to perform a toxicity study of free doxorubicin compared to doxorubicin conjugated with various nanotransporters. The effect of liposomes, fullerenes, graphene, and carbon nanotubes on the toxicity was analyzed. As a first step, the binding efficacy of between doxorubicin and the nanotransporter was determined. The highest efficacy was detected in case of liposomes (85% of applied drug was encapsulated) followed by graphene, carbon nanotubes and fullerenes. For the toxicological studies, the chicken embryos incubated under controlled conditions (37.5 °C, 45% rH, rotation every 2 hours) were used. In 7th developmental day of chicken embryos doxorubicin or doxorubicin-nanotransporter complex was applied on the chorioallantoic membrane of the eggs and the viability was analyzed every day till the 17th developmental day. Then the embryos were extracted from the shell and the distribution of doxorubicin in the body was analyzed by measurement of organs extracts using laser induce fluorescence detection. The chicken embryo mortality caused by free doxorubicin (30%) was significantly lowered by using the conjugation with nanomaterials. The highest accumulation of doxorubicin and doxorubicin nanotransporter complexes was observed in the liver tissue

Keywords: doxorubicin, chicken embryos, nanotransporters, toxicity

Procedia PDF Downloads 449

Authors: Hasan Basri Jumin

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Protoplasts isolated from embryogenic callus of Murraya paniculata (L. Jack.) were cultured in MT (Murashige and Tucker, 1969) basal medium containing 5% sucrose supplemented with kinetin, malt extract (ME) and 0.6 M sorbitol. About 85% of the surviving protoplasts formed a cell wall within 6 d of culture and the first cell division was observed 7 days after isolation. The highest plating effi¬ciency was obtained on MT basal medium containing 5% sucrose supplemented with 0.01 mg 1-1 kinetin 600 mg 1-1 ME, MT basal medium containing 5% sucrose and supplemented with 0.01 mg 1-1 Indole-acetic-acid (IAA) was found to be a medium suitable for the development somatic embryos into heart-shaped somatic embryos. The highest percentage of shoot formation was obtained using 0.1 mg 1-1 Indole-acitic-acid (IAA) 0..1 mg 1-1 gibberellic acid (GA3). In this investigation 40 plants were survived and grew normally in the soil. After two months maitained in the soil plants formed flower and flower developed into fruits on the soil treated with BA.

Keywords: gibberellic-acid, indole-acetic-acid, protoplast, precocious-flowering, somatic-embryo

Procedia PDF Downloads 345

Authors: Valbona Sota, Efigjeni Kongjika

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Wild almond is a woody species, which is difficult to propagate either generatively by seed or by vegetative methods (grafting or cuttings) and also considered as Endangered (EN) in Albania based on IUCN criteria. As a wild relative of cultivated fruit trees, this species represents a source of genetic variability and can be very important in breeding programs and cultivation. For this reason, it would be of interest to use an effective method of in vitro mid-term conservation, which involves strategies to slow plant growth through physicochemical alterations of in vitro growth conditions. Multiplication of wild almond was carried out using zygotic embryos, as primary explants, with the purpose to develop a successful propagation protocol. Results showed that zygotic embryos can proliferate through direct or indirect organogenesis. During subculture, stage was obtained a great number of new plantlets identical to mother plants derived from the zygotic embryos. All in vitro plantlets obtained from subcultures underwent in vitro conservation by minimal growth in low temperature (4ºC) and darkness. The efficiency of this technique was evaluated for 3, 6, and 10 months of conservation period. Maintenance in these conditions reduced micro cuttings growth. Survival and regeneration rates for each period were evaluated and resulted that the maximal time of conservation without subculture on 4ºC was 10 months, but survival and regeneration rates were significantly reduced, specifically 15.6% and 7.6%. An optimal period of conservation in these conditions can be considered the 5-6 months storage, which can lead to 60-50% of survival and regeneration rates. This protocol may be beneficial for mass propagation, mid-term conservation, and for genetic manipulation of wild almond.

Keywords: micropropagation, minimal growth, storage, wild almond

Procedia PDF Downloads 128

Authors: Suneeta Gireesh Panicker

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Aim: Aminopeptidase P (APP) is an enzyme that has specificity for proline, can specifically cleave Xaa-Proline peptides and is a metallo-aminopeptidase. The bonds nearby to the imino acid proline are tough to cleave by many peptidases, but APP can specifically break peptide bonds engaged with proline. Membrane-bound form and a cytosolic form are the two forms in which this enzyme exists. The exact physiological function of APP remains unclear and hence the present work attempts to determine it. Methods: In the present study, the expression pattern of cytosolic Aminopeptidase P (DAP) was determined in all the embryonic stages and larval stages of wild-type Drosophila by using polyclonal monospecific antibodies. To show the presence of DAP RNA in embryonic and larval stages, RNA in situ hybridization was performed. DAP promoter-LacZ fusion reporter gene vector was used to construct transgenic embryos to study the regulation pattern of DAP. To study the DAP expression profile, a transgenic fly consisting of a DAP promoter with β-gal and GFP reporter genes in front of it was constructed. Results: DAP protein expression was observed in neuroectodermal cells, posterior midgut primordium, proctodeum, ventral neuroblast and primordial stomatogastric nervous system. It was observed in the ventral cord and midgut in stage 12. The completely developed embryos showed the intense occurrence of it in the ventral cord and gut region. The eye-antennal disc, wing disc and leg disc also showed the presence of DAP protein. LacZ expression in transgenic embryos also showed the same pattern. Conclusion: Similar to various known multiple-functional proteins, DAP could be one with different functions at different stages and in different cells. Data presented here designates DAP functions in the early embryonic and imaginal dics differentiation and development, suggesting that it may be required for the metabolism of proteins like neuropeptides and tachykinins.

Keywords: aminopeptidase P, in situ hybridization, transgenic fly, embryonic stages

Procedia PDF Downloads 85

Authors: Hasan Basri Jumin

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The in vitro flowering of orange jessamine plantlets derived from protoplast was affected by the manipulation of plant growth regulators, sugar and light conditions. MT basal medium containing 5% sucrose and supplemented with 0.001 mg 1-1 indole-acetic-acid was found to be a suitable medium for development of globular somatic embryos derived from protoplasts to form heart-shaped somatic embryos with cotyledon-like structures. The highest percentage (85 %) of flowering was achieved with plantlet on half-strength MT basal medium containing 5% sucrose and 0.001 mg1-1 indole-acetic-acid in light. Exposure to darkness for more than 3 weeks followed by re-exposure to light reduced flowering. Flowering required a 10-day exposure to indole-acetic-acid. Photoperiod with 18 h and 79.4 µmol m-2 s-1 light intensity promoted in vitro flowering in high frequencies. The sucrose treatment affected the flower bud size distribution. Flower buds originating from plantlet derived from protoplasts developed into normal flowers.

Keywords: indole-acetc-acid, light-intensity, Murraya-paniculata, photoperiod, plantlet, Zeatin

Procedia PDF Downloads 418

Authors: S. Padma, D. H. Tejavathi

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Somatic embryogenesis is a remarkable illustration of the dictum of plant totipotency where developmental reconstruction of somatic cells takes place towards the embryogenic pathway. It recapitulates the morphological and developmental process that occurs in zygotic embryogenesis. S. androgynous commonly called as multivitamin plant. The leaves are consumed as green leafy vegetable by the Southeast Asian communities due to their rich nutritional profile. Despite being a good nutritional vegetable with proteins, vitamins, minerals, amino acids, it is warned for excessive intake due to the presence of alkoloid called papaverine. Papaverine at higher concentrations is toxic and leads to a syndrome called Bronchiolitis Obliterans. In the present study, morphogenetic potential of shoot tip, leaf and nodal explants of Sauropus androgynous was investigated to develop and enhance the reliable plant regeneration protocol via somatic embryogenesis. Somatic embryos were derived directly from the embryogenic callus derived from shoot tip, node and leaf cultures on Phillips and Collins (L2) medium supplemented with NAA at various concentrations ranging from 5.3 µM/l to 26.85 µM/l within two months of inoculation. Thus obtained embryos were sub cultured to modified L2 media supplemented with increased vitamin level for the further growth. Somatic embryos with well-developed cotyledons were transferred to normal and modified L2 basal medium for conversion. The plantlets thus obtained were subjected to brief acclimatization before transferring them to land. About 95% of survival rate was recorded. The augmentation process of culturing various explants through somatic embryogenesis using synthetic medium with various plant growth regulators under controlled conditions have aggrandized the commercial production of Sauropus making it easily available over the conventional propagation methods. In addition, regeneration process through somatic embryogenesis has ameliorated the development of desired character in Sauropus with low papaverine content thereby providing a valuable resource to the food and pharmaceutical industry. Based on this research, plant tissue culture techniques have shown promise for economical and convenient application in Sauropus androgynous breeding.

Keywords: L2 medium, multivitamin plant, NAA, papaverine

Procedia PDF Downloads 207

Authors: Okafor C. Uche, Agbo P. Ejiofor, Okezie C. Eziuche

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This study provides a regeneration protocol for Treculia africana Decne (an endangered plant) through embryo culture. Mature zygotic embryos of T. africana were excised from the seeds aseptically and cultured on varied strengths (full, half and quarter) of Murashige and Skoog (MS) basal medium supplemented. All treatments experienced 100±0.00 percent sprouting except for half and quarter strengths. Plantlets in MS full strength had the highest fresh weight, leaf area, and longest shoot length when compared to other treatments. All explants in full, half, quarter strengths and control had the same number of leaves and sprout rate. Between the treatments, there was a significant difference (P>0.05) in their effect on the length of shoot and root, number of adventitious root, leaf area, and fresh weight. Full strength had the highest mean value in all the above-mentioned parameters and differed significantly (P>0.05) from others except in shoot length, number of adventitious roots, and root length where it did not differ (P<0.05) from half strength. The result of this study indicates that full strength MS basal medium offers a better option for the optimum growth for Treculia africana regeneration in vitro.

Keywords: medium strengths, Murashige and Skoog, Treculia africana, zygotic embryos

Procedia PDF Downloads 253

Authors: Mohammed Abdelsabour-Khalaf, F. Yusuf , B Brand-Saberi

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Purpose: Applications of nanotechnology nowadays extended to include a wide range of scientific areas such electron micrscopy and gene expression. The aim of the current study was to investigate the developmental expression pattern of genes involved in human glomerulo-nephropathies associated with massive proteinuria and podocyte differentiation using the chicken mesonephros as a model system. Method: We performed in situ hybridization using chicken specific mRNA probes for genes expressed in the early nephron and slit diaphragm genes. The probes used were cNeph1, cNeph2, cSim1, cLmx1b, and cAtoh8. Chicken embryos from Hamburger Hamilton developmental stage HH19 (E3) to HH 34 (E9) were used for the in situ hybridization (ISH). ISH was performed on whole mount embryos which were sectioned by vibratome. Results: Our result show that Neph1, Neph2, Sim1. Lmx1b and Atoh8 genes are dynamically expressed during nephron morphogenesis and Neph1 and Atoh8 are also specifically expressed in the podocytes during late stages of differentiation. Conclusion: We conclude from our results that the genes implicated in congenital and acquired glomerulo-nephropathies like Neph1 and Neph2 are dynamically expressed during mesonephros development pointing towards a role in the formation of the filtration barrier and the differentiation of the mesonephric podocytes. Thus the avian mesonephros could serve as a model to study human kidney diseases.

Keywords: mesonephros, chicken embryo, gene expression, immunohistochemistry

Procedia PDF Downloads 620

Authors: Katalin Gombos, Bence Gálik, Krisztina Ildikó Kalács, Krisztina Gödöny, Ákos Várnagy, József Bódis, Attila Gyenesei, Gábor L. Kovács

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Although non-invasive pre-implantation genetic testing for aneuploidy (NIPGT-A) is potentially appropriate to assess chromosomal ploidy of the embryo, practical application of it in a routine IVF center has not been started in the absence of a recommendation. We developed a comprehensive workflow for a clinically applicable strategy for NIPGT-A based on next-generation sequencing (NGS) technology. We performed MALBAC whole genome amplification and NGS on spent blastocyst culture media of Day 3 embryos fertilized with intra-cytoplasmic sperm injection (ICSI). Spent embryonic culture media of morphologically good quality score embryos were enrolled in further analysis with the blank culture media as background control. Chromosomal abnormalities were identified by an optimized bioinformatics pipeline applying a copy number variation (CNV) detecting algorithm. We demonstrate a comprehensive workflow covering both wet- and dry-lab procedures supporting a clinically applicable strategy for NIPGT-A. It can be carried out within 48 h which is critical for the same-cycle blastocyst transfer, but also suitable for “freeze all” and “elective frozen embryo” strategies. The described integrated approach of non-invasive evaluation of embryonic DNA content of the culture media can potentially supplement existing pre-implantation genetic screening methods.

Keywords: next generation sequencing, in vitro fertilization, embryo assessment, non-invasive pre-implantation genetic testing

Procedia PDF Downloads 156

Authors: Magdalena Kowalska, Weronika Rupik

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The pancreas is an important organ present in all vertebrate species. It contains two different tissues, exocrine and endocrine, that act as two glands in one. The development and differentiation of the pancreas in reptiles is poorly known in comparison to other vertebrates. Therefore, the aim of this study was to investigate the particular steps concerning the differentiation of the pancreas in the grass snake (Natrix natrix) embryos. For this, histological methods (including hematoxylin and eosin, and Heidenhain's AZAN staining), transmission electron microscopy and three-dimensional (3D) reconstructions from serial paraffin sections were used. The results of this study indicated that the first step of pancreas development in Natrix was the connection of the two pancreatic buds: dorsal and ventral one. Then, duct walls in both buds started to be remodeled from the multilayered to single-layered epithelium. This remodeling started in the dorsal bud and was simultaneously with the differentiation of the duct lumens which occurred by the cavition. During this process, the cells that had no contact with the mesenchyme underwent cell death named anoikis. These findings indicated that the walls of ducts in the embryonic pancreas of the grass snake were initially formed by the abundant principal and single endocrine cells. Later the basal and goblet cells differentiated. Among the endocrine cells, as the first the B and A cells differentiated, then the D and PP cells. The next step of the pancreatic development was the withdrawing of the endocrine cells from the duct walls to form the pancreatic islets. The endocrine cells and islets were found only in the dorsal part of the pancreas in Natrix embryos what is different than in other vertebrate species. The islets were formed mainly by the A cells. Simultaneously, with the differentiation of the endocrine pancreas, the acinar tissue started to differentiate. The source of the acinar cells were pancreatic ducts similar as in other vertebrates. The acini formation began at the proximal part of the pancreas and went towards the caudal direction. Differentiating pancreatic ducts developed into the branched system that can be divided into extralobular, intralobular, and intercalated ducts, similarly as in other vertebrate species. However, the pattern of branching was different. In conclusions, particular steps of the pancreas differentiation in the grass snake were different than in other vertebrates. It can be supposed that these differences are related to the specific topography of the snake’s internal organs and their taxonomy position. All specimens used in the study were captured according to the Polish regulations concerning the protection of wild species. Permission was granted by the Local Ethics Commission in Katowice (41/2010; 87/2015) and the Regional Directorate for Environmental Protection in Katowice (WPN.6401.257.2015.DC).

Keywords: embryogenesis, organogenesis, pancreas, Squamata

Procedia PDF Downloads 170

Authors: Angelica Alvarez-Lee, Sergio Martinez-Diaz, Jose Luis Garcia-Corona, Humberto Lanz-Mendoza

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World aquaculture is always looking for improvements to achieve productions with high yields avoiding the infection by pathogenic agents. The best way to achieve this is to know the biological model to create alternative treatments that could be applied in the hatcheries, which results in greater economic gains and improvements in human public health. In the last decade, immunomodulation in shrimp culture with probiotics, organic acids and different carbon sources has gained great interest, mainly in larval and juvenile stages. Immune priming is associated with a strong protective effect against a later pathogen challenge. This work provides another perspective about immunostimulation from spawning until hatching. The stimulation happens during development embryos and generates resistance to infection by pathogenic bacteria. Massive spawnings of white shrimp L. vannamei were obtained and placed in experimental units with 700 mL of sterile seawater at 30 °C, salinity of 28 ppm and continuous aeration at a density of 8 embryos.mL⁻¹. The immunostimulating effect of three death strains of non-pathogenic bacterial (Escherichia coli, Staphylococcus aureus and Bacillus subtilis) and a pathogenic strain for white shrimp (Vibrio parahaemolyticus) was evaluated. The strains killed by heat were adjusted to O.D. 0.5, at A 600 nm, and directly added to the seawater of each unit at a ratio of 1/100 (v/v). A control group of embryos without inoculum of dead bacteria was kept under the same physicochemical conditions as the rest of the treatments throughout the experiment and used as reference. The duration of the stimulus was 12 hours, then, the larvae that hatched were collected, counted and transferred to a new experimental unit (same physicochemical conditions but at a salinity of 28 ppm) to carry out a challenge of infection against the pathogen V. parahaemolyticus, adding directly to seawater an amount 1/100 (v/v) of the live strain adjusted to an OD 0.5; at A 600 nm. Subsequently, 24 hrs after infection, nauplii survival was evaluated. The results of this work shows that, after 24 hrs, the hatching rates of immunostimulated shrimp embryos with the dead strains of B. subtillis and V. parahaemolyticus are significantly higher compared to the rest of the treatments and the control. Furthermore, survival of L. vanammei after a challenge of infection of 24 hrs against the live strain of V. parahaemolyticus is greater (P < 0.05) in the larvae immunostimulated during the embryonic development with the dead strains B. subtillis and V. parahaemolyticus, followed by those that were treated with E. coli. In summary superficial antigens can stimulate the development cells to promote hatching and can have normal development in agreeing with the optical observations, plus exist a differential response effect between each treatment post-infection. This research provides evidence of the immunostimulant effect of death pathogenic and non-pathogenic bacterial strains in the rate of hatching and oversight of shrimp L. vannamei during embryonic and larval development. This research continues evaluating the effect of these death strains on the expression of genes related to the defense priming in larvae of L. vannamei that come from massive spawning in hatcheries before and after the infection challenge against V. parahaemolyticus.

Keywords: immunostimulation, L. vannamei, hatching, survival

Procedia PDF Downloads 142

Authors: Sara Ignoto, Elena M. Scalisi, Carmen Sica, Martina Contino, Greta Ferruggia, Antonio Salvaggio, Santi C. Pavone, Gino Sorbello, Loreto Di Donato, Roberta Pecoraro, Maria V. Brundo

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The new fifth generation technology (5G), which should favor high data-rate connections (1Gbps) and latency times lower than the current ones (<1ms), has the characteristic of working on different frequency bands of the radio wave spectrum (700 MHz, 3.6-3.8 GHz and 26.5-27.5 GHz), thus also exploiting higher frequencies than previous mobile radio generations (1G-4G). The higher frequency waves, however, have a lower capacity to propagate in free space and therefore, in order to guarantee the capillary coverage of the territory for high reliability applications, it will be necessary to install a large number of repeaters. Following the introduction of this new technology, there has been growing concern in recent years about the possible harmful effects on human health and several studies were published using several animal models. This study aimed to observe the possible short-term effects induced by 5G-millimeter waves on heartbeat of early life stages of Danio rerio using DanioScope software (Noldus). DanioScope is the complete toolbox for measurements on zebrafish embryos and larvae. The effect of substances can be measured on the developing zebrafish embryo by a range of parameters: earliest activity of the embryo’s tail, activity of the developing heart, speed of blood flowing through the vein, length and diameters of body parts. Activity measurements, cardiovascular data, blood flow data and morphometric parameters can be combined in one single tool. Obtained data are elaborate and provided by the software both numerical as well as graphical. The experiments were performed at 27 GHz by a no commercial high gain pyramidal horn antenna. According to OECD guidelines, exposure to 5G-millimeter waves was tested by fish embryo toxicity test within 96 hours post fertilization, Observations were recorded every 24h, until the end of the short-term test (96h). The results have showed an increase of heartbeat rate on exposed embryos at 48h hpf than control group, but this increase has not been shown at 72-96 h hpf. Nowadays, there is a scant of literature data about this topic, so these results could be useful to approach new studies and also to evaluate potential cardiotoxic effects of mobile radiofrequency.

Keywords: Danio rerio, DanioScope, cardiotoxicity, millimeter waves.

Procedia PDF Downloads 163

Authors: Cecile Edel, Sandrine Giscard D'Estaing, Elsa Labrune, Jacqueline Lornage, Mehdi Benchaib

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Introduction: The use of incubator equipped with time-lapse technology in Artificial Reproductive Technology (ART) allows a continuous surveillance. With morphocinetic parameters, algorithms are available to predict the potential outcome of an embryo. However, the different proposed time-lapse algorithms do not take account the missing data, and then some embryos could not be classified. The aim of this work is to construct a predictive model even in the case of missing data. Materials and methods: Patients: A retrospective study was performed, in biology laboratory of reproduction at the hospital ‘Femme Mère Enfant’ (Lyon, France) between 1 May 2013 and 30 April 2015. Embryos (n= 557) obtained from couples (n=108) were cultured in a time-lapse incubator (Embryoscope®, Vitrolife, Goteborg, Sweden). Time-lapse incubator: The morphocinetic parameters obtained during the three first days of embryo life were used to build the predictive model. Predictive model: A quadratic regression was performed between the number of cells and time. N = a. T² + b. T + c. N: number of cells at T time (T in hours). The regression coefficients were calculated with Excel software (Microsoft, Redmond, WA, USA), a program with Visual Basic for Application (VBA) (Microsoft) was written for this purpose. The quadratic equation was used to find a value that allows to predict the blastocyst formation: the synthetize value. The area under the curve (AUC) obtained from the ROC curve was used to appreciate the performance of the regression coefficients and the synthetize value. A cut-off value has been calculated for each regression coefficient and for the synthetize value to obtain two groups where the difference of blastocyst formation rate according to the cut-off values was maximal. The data were analyzed with SPSS (IBM, Il, Chicago, USA). Results: Among the 557 embryos, 79.7% had reached the blastocyst stage. The synthetize value corresponds to the value calculated with time value equal to 99, the highest AUC was then obtained. The AUC for regression coefficient ‘a’ was 0.648 (p < 0.001), 0.363 (p < 0.001) for the regression coefficient ‘b’, 0.633 (p < 0.001) for the regression coefficient ‘c’, and 0.659 (p < 0.001) for the synthetize value. The results are presented as follow: blastocyst formation rate under cut-off value versus blastocyst rate formation above cut-off value. For the regression coefficient ‘a’ the optimum cut-off value was -1.14.10-3 (61.3% versus 84.3%, p < 0.001), 0.26 for the regression coefficient ‘b’ (83.9% versus 63.1%, p < 0.001), -4.4 for the regression coefficient ‘c’ (62.2% versus 83.1%, p < 0.001) and 8.89 for the synthetize value (58.6% versus 85.0%, p < 0.001). Conclusion: This quadratic regression allows to predict the outcome of an embryo even in case of missing data. Three regression coefficients and a synthetize value could represent the identity card of an embryo. ‘a’ regression coefficient represents the acceleration of cells division, ‘b’ regression coefficient represents the speed of cell division. We could hypothesize that ‘c’ regression coefficient could represent the intrinsic potential of an embryo. This intrinsic potential could be dependent from oocyte originating the embryo. These hypotheses should be confirmed by studies analyzing relationship between regression coefficients and ART parameters.

Keywords: ART procedure, blastocyst formation, time-lapse incubator, quadratic model

Procedia PDF Downloads 306

Authors: Jayeon Kim, Eunjung Yu, Taeki Yoon

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Most spontaneous miscarriage is believed to be a consequence of embryo aneuploidies. Transferring eukaryotic embryos selected by PGS is expected to decrease the miscarriage rate. Current PGS indications include advanced maternal age, recurrent pregnancy loss, repeated implantation failure. Recently, use of PGS for healthy women without above indications for the purpose of improving in vitro fertilization (IVF) outcomes is on the rise. However, it is still controversy about the beneficial effect of PGS in this population, especially, in women with a history of no more than 2 miscarriages or miscarriage of eukaryotic abortus. This study aimed to investigate if karyotyping result of abortus is a good indicator of preimplantation genetic screening (PGS) in subsequent IVF cycle in women with a history of spontaneous abortion. A single-center retrospective cohort study was performed. Women who had spontaneous abortion(s) (less than 3) and dilatation and evacuation, and subsequent IVF from January 2016 to November 2016 were included. Their medical information was extracted from the charts. Clinical pregnancy was defined as presence of a gestational sac with fetal heart beat detected on ultrasound in week 7. Statistical analysis was performed using SPSS software. Total 234 women were included. 121 out of 234 (51.7%) underwent karyotyping of the abortus, and 113 did not have the abortus karyotyped. Embryo biopsy was performed on 3 or 5 days after oocyte retrieval, followed by embryo transfer (ET) on a fresh or frozen cycle. The biopsied materials were subjected to microarray comparative genomic hybridization. Clinical pregnancy rate per ET was compared between PGS and non-PGS group in each study group. Patients were grouped by two criteria: karyotype of the abortus from previous miscarriage (unknown fetal karyotype (n=89, Group 1), eukaryotic abortus (n=36, Group 2) or aneuploidy abortus (n=67, Group 3)), and pursuing PGS in subsequent IVF cycle (pursuing PGS (PGS group, n=105) or not pursuing PGS (non-PGS group, n=87)). The PGS group was significantly older and had higher number of retrieved oocytes and prior miscarriages compared to non-PGS group. There were no differences in BMI and AMH level between those two groups. In PGS group, the mean number of transferable embryos (eukaryotic embryo) was 1.3 ± 0.7, 1.5 ± 0.5 and 1.4 ± 0.5, respectively (p = 0.049). In 42 cases, ET was cancelled because all embryos biopsied turned out to be abnormal. In all three groups (group 1, 2, and 3), clinical pregnancy rates were not statistically different between PGS and non-PGS group (Group 1: 48.8% vs. 52.2% (p=0.858), Group 2: 70% vs. 73.1% (p=0.730), Group 3: 42.3% vs. 46.7% (p=0.640), in PGS and non-PGS group, respectively). In both groups who had miscarriage with eukaryotic and aneuploidy abortus, the clinical pregnancy rate between IVF cycles with and without PGS was not different. When we compare miscarriage and ongoing pregnancy rate, there were no significant differences between PGS and non-PGS group in all three groups. Our results show that the routine application of PGS in women who had less than 3 miscarriages would not be beneficial, even in cases that previous miscarriage had been caused by fetal aneuploidy.

Keywords: preimplantation genetic diagnosis, miscarriage, kpryotyping, in vitro fertilization

Procedia PDF Downloads 181

Authors: Judith Correa-Gomez, Cristina Garcia-De la Pena, Veronica Avila-Rodriguez, David R. Aguillon-Gutierrez

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Kemp's ridley (Lepidochelys kempii) is the smallest species of sea turtle. It nests on the beaches of the Gulf of Mexico during summer. To date, there is no information about congenital anomalies in this species, which could be an important factor to be considered as a survival threat. The aim of this study was to determine congenital anomalies in dead embryos and hatchlings of Kemp's ridley sea turtle during 2020 nesting season. Fieldwork was conducted at the 'Campamento Tortugero Barra Norte', on the shores of Tuxpan, Veracruz, Mexico. A total of 95 nests were evaluated, from which 223 dead embryos and hatchlings were collected. Anomalies were detected by detailed physical examinations. Photographs of each anomaly were taken. From the 223 dead turtles, 213 (95%) showed a congenital anomaly. A total of 53 types of congenital anomalies were found: 22 types on the head region, 21 on the carapace region, 6 on the flipper region, and 4 regarding the entire body. The most prevalent anomaly in the head region was the presence of prefrontal supernumerary scales (42%, 93 occurrences). On the carapace region, the most common anomaly was the presence of supernumerary gular scales (59%, 131 occurrences). The two most common anomalies on the flipper region were amelia in fore flippers and rear bifurcation of flippers (0.9%, 2 occurrences each). The most common anomaly involving the entire body was hypomelanism (35%, 79 occurrences). These results agree with the recent studies on congenital malformations on sea turtles, being the head and the carapace regions the ones with the highest number of congenital anomalies. It is unknown whether the reported anomalies can be related to the death of these individuals. However, it is necessary to develop embryological studies in this species. To our best knowledge, this is the first worldwide report on Kemp’s ridley sea turtle anomalies.

Keywords: Amelia, hypomelanism, morphology, supernumerary scales

Procedia PDF Downloads 160

Authors: Titis Indah Adi Rahayu

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Angiogenesis is the process of generating new capillary from pre-existing blood vessels. VEGFA is a major regulator in angiogenesis that binds and activates two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1/KDR) which regulate pathological and physiological angiogenesis. Disruption of VEGFA and VEGFR2 regulation lead to many diseases. The study of α-Mangostin (derivate of xanthone) as anti-oxidant and anti inflammation has been explored recently and both of them have relation to vasculature however the effect of α-Mangostin in blood vessel formation in healthy tissue in vivo has not been studied. Zebrafish is a powerful model in studying angiogenesis and shared many advantages that is a viable whole animal model for screening small molecules that affect blood vessel formation. Therefore the aim of this study is to evaluate angiogenic activity of α-Mangostin in healthy tissue in vivo in zebrafish embryo in relation of patterning blood vessel. Blood vessel patterning is highly characteristic in the developing of zebrafish embryo and the subintestinal vessel (SIV) can be stained and visualized microscopically as a primary screen for α-Mangostin that effect angiogenesis. The zebrafish embryos are divided into 2 groups. Group one consists of the zebrafish embryos at 1 dpf for 4 days which are tested to α-Mangostin in several concentration 2 µM, 4 µM, 6 µM, 8 µM and 10 µM whereas in group two the zebrafish larva at 4 dpf are exposed to α-Mangostin 1,75 µM, 2,3 µM, 2,9 µM, 3,8 µM dan 5 µM for 2 days. DMSO is served as a control for each group. The level expression of vegfa and vegfr2 are observed quantitatively using real time q-PCR and patterning of SIV are then analized via alkaline phospatase staining. Result shows that the level expression of vegfa and vegfr2 is repressed quantitatively as shown in real time q-PCR in the group of 1-4 days of α-Mangostin exposure where it is increased in the group of 4-6 days of α-Mangostin exposure. The result is then compared to alkaline phospatase staining of SIV using stereo microscope. It indicates that α-Mangostin does not disturb the patterning of SIV formation in zebrafish.

Keywords: angiogenesis, Danio rerio, α-Mangostin, SIV, vegfa, vegfr2

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Authors: Kairat Uteulin, Azhar Iskakova, Serik Mukhambetzhanov, Bayan Yesbolayeva, Gabit Bari, Aslan Zheksenbai, Kabyl Zhambakin, Chingis Dzhabykbayev, Vladimir Piven, Izbasar Rakhimbaiev

Abstract:

To explore the potential for in vitro rapid regeneration of Russian dandelion (Taraxacum kok-saghyz Rodin), different concentrations of 6-Benzylaminopurine (BAP), 2,4-Dichlorophenoxyacetic acid (2.4-D) and BAP combined with Indole-3-acetic acid (IAA) were evaluated for their effects on the induction of somatic embryos from leaf, seed stem and root explants. Different explants were cultured on MS medium supplemented with various concentrations (0, 0.5, 1, 1.5, 2, 2.5 and 3 mg/l) of each kind of hormone. Callus induction percentage, fresh weight, color and texture of the callus were assessed after 14 and 28 days of culture. The optimum medium for the proliferation of embryogenic calli from leaf and root explants was MS supplemented with 2.5 mg/L BAP and 0.5 mg/L 2.4-D. Concentrations of 2.5 mg/L BAP and 1.5 mg/L IAA also had a remarkable effect on root and stem explants. The best concentration to produce callus from stem explants was 0.5 mg/L BAP and 1 mg/L IAA. Results of mean comparison showed that BAP and 2.4-D were more effective on different explants than BAP and IAA. Results of the double staining method proved that somatic embryogenesis occurred in the most concentrations of BAP and 2.4-D. Under microscopic observations, the different developmental stages of the embryos (globular, heart, torpedo and cotyledonary) were revealed together in callus cells, indicating that the most tested hormone combinations were effective for somatic embryogenesis formation in this species. Seed explants formed torpedo and cotyledonary stages faster than leaf and root explants in the most combinations. Most calli from seed explants were cream colored and friable, while calli were compact and light green from leaf and root explants. Some combinations gave direct regeneration and (3 mg/L BAP and 2 mg/L IAA) in seed explants and (0.5 mg/L BAP and 2.5 mg/L IAA) in leaf explants had the highest number of shoots with average of 21 and 27 shoots per callus. The developed protocol established the production of different callus types from seed, leaf, and root explants and plant regeneration through somatic embryogenesis.

Keywords: taraxacum kok-saghyz Rodin, callus, somatic embryogenesis

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Authors: Hadi S. Hosseini, Larry A. Taber

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In the embryo, heart is initially a simple tubular structure that undergoes complex morphological changes as it transforms into a four-chambered pump. This work focuses on mechanisms that create heart tube (HT). The early embryo is composed of three relatively flat primary germ layers called endoderm, mesoderm, and ectoderm. Precardiac cells located within bilateral regions of the mesoderm called heart fields (HFs) fold and fuse along the embryonic midline to create the HT. The right and left halves of this plate fold symmetrically to bring their upper edges into contact along the midline, where they fuse. In a region near the fusion line, these layers then separate to generate the primitive HT and foregut, which then extend vertically. The anterior intestinal portal (AIP) is the opening at the caudal end of the foregut, which descends as the HT lengthens. The biomechanical mechanisms that drive this folding are poorly understood. Our central hypothesis is that folding is caused by differences in growth between the endoderm and mesoderm while subsequent extension is driven by contraction along the AIP. The feasibility of this hypothesis is examined using experiments with chick embryos and finite-element modeling (FEM). Fertilized white Leghorn chicken eggs were incubated for approximately 22-33 hours until appropriate Hamburger and Hamilton stage (HH5 to HH9) was reached. To inhibit contraction, embryos were cultured in media containing blebbistatin (myosin II inhibitor) for 18h. Three-dimensional models were created using ABAQUS (D. S. Simulia). The initial geometry consists of a flat plate including two layers representing the mesoderm and endoderm. Tissue was considered as a nonlinear elastic material with growth and contraction (negative growth) simulated using a theory, in which the total deformation gradient is given by F=F^*.G, where G is growth tensor and F* is the elastic deformation gradient tensor. In embryos exposed to blebbistatin, initial folding and AIP descension occurred normally. However, after HFs partially fused to create the upper part of the HT, fusion, and AIP descension stopped, and the HT failed to grow longer. These results suggest that cytoskeletal contraction is required only for the later stages of HT formation. In the model, a larger biaxial growth rate in the mesoderm compared to the endoderm causes the bilayered plate to bend ventrally, as the upper edge moves toward the midline, where it 'fuses' with the other half . This folding creates the upper section of the HT, as well as the foregut pocket bordered by the AIP. After this phase completes by stage HH7, contraction along the arch-shaped AIP pulls the lower edge of the plate downward, stretching the two layers. Results given by model are in reasonable agreement with experimental data for the shape of HT, as well as patterns of stress and strain. In conclusion, results of our study support our hypothesis for the creation of the heart tube.

Keywords: heart tube formation, FEM, chick embryo, biomechanics

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Authors: Zelikha Labbani

Abstract:

Many biotechnology methods have been used in plant breeding programs. The in vitro isolated microspore culture is the one of these methods. For durum wheat, the use of this technology has been limited for a long time due to the low number of embryos produced and also most regeneration plants are albina. The objective of this paper is to show that using isolated microspores culture on durum wheat is possible due to the development of the new methods using the new pretreatment of the microspores before their isolation and cultivation.

Keywords: isolated microspore culture, pretreatments, in vitro embryogenesis, plant breeding program

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Authors: Roberta Pecoraro, Elena Maria Scalisi

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Engineered Nanoparticles (ENPs) and Nanomaterials (ENMs) concern an active research area and a sector in full expansion. They have physical-chemical characteristics and small size that improve their performance compared to common materials. Due to the increase in their production and their subsequent release into the environment, new strategies are emerging to assess risk of nanomaterials. NPs can be released into the environment through aquatic systems by human activities and exert toxicity on living organisms. We evaluated the potential toxic effect of cerium oxide (CeO2) nanoparticles because it’s used in different fields due to its peculiar properties. In order to assess nanoparticles toxicity, Fish Embryo Toxicity (FET) test was performed. Powders of CeO2 NPs supplied by the CNR-IMM of Catania are indicated as CeO2 type 1 (as-prepared) and CeO2 type 2 (modified), while CeO2 type 3 (commercial) is supplied by Sigma-Aldrich. Starting from a stock solution (0.001g/10 ml dilution water) of each type of CeO2 NPs, the other concentration solutions were obtained adding 1 ml of the stock solution to 9 ml of dilution water, leading to three different solutions of concentration (10-4, 10-5, 10-6 g/ml). All the solutions have been sonicated to avoid natural tendency of NPs to aggregate and sediment. FET test was performed according to the OECD guidelines for testing chemicals using our internal protocol procedure. A number of eight selected fertilized eggs were placed in each becher filled with 5 ml of each concentration of the three types of CeO2 NPs; control samples were incubated only with dilution water. Replication was performed for each concentration. During the exposure period, we observed four endpoints (embryo coagulation, lack of formation of somites, failure to lift the yolk bag, no heartbeat) by a stereomicroscope every 24 hours. Immunohistochemical analysis on treated larvae was performed to evaluate the expression of metallothioneins (MTs), Heat Shock Proteins 70 (HSP70) and 7-ethoxyresorufin-O-diethylase (EROD). Our results have not shown evident alterations on embryonic development because all embryos completed the development and the hatching of the eggs, started around the 48th hour after exposure, took place within the last observation at 72 hours. A good reactivity, both in the embryos and in the newly hatched larvae, was found. The presence of heartbeat has also been observed in embryos with reduced mobility confirming their viability. A higher expression of EROD biomarker was observed in the larvae exposed to the three types of CeO2, showing a clear difference with the control. A weak positivity was found for MTs biomarker in treated larvae as well as in the control. HSP70 are expressed homogeneously in all the type of nanoparticles tested but not too much greater than control. Our results are in agreement with other studies in the literature, in which the exposure of Danio rerio larvae to other metal oxide nanoparticles does not show adverse effects on survival and hatching time. Further studies are necessary to clarify the role of these NPs and also to solve conflicting opinions.

Keywords: Danio rerio, endpoints, fish embryo toxicity test, metallic nanoparticles

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Authors: Fitria D. Ayuningtyas, Anggraini Barlian

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Green turtle (Chelonia mydas) is one of TSD (Temperature-dependent Sex Determination, TSD) animals which sex is determined by the egg’s incubation temperature. GSD (Genotypic Sex Determination) homologous genes such as Wilms’ Tumor (Wt1) and Forkhead Box L2 (FoxL2) play a role in TSD animal sex determination process. Wt1 plays a role in both male pathway, as a transcription factor for Sf1 gene and in female pathway, as a transcription factor for Dax1. FoxL2 plays a role specifically in female sex determination, and known as transcriptional factor for Aromatase gene. Until now, research on the pattern of Wt1 and FoxL2 genes expression in C.mydas has not been conducted yet. The aim of this research is to know the pattern of Wt1 and FoxL2 genes expression in Mesonephros-Gonad (MG) complexes of Chelonia mydas embryos incubated in masculinizing temperature (MT) and feminizing temperature (FT). Eggs of C.mydas incubated in 3 different stage of TSP (Thermosensitive Period) at masculinizing temperature (26±10C, MT) and feminizing temperature (31±10C FT). Mesonefros-gonad complexes were isolated at Pre-TSP stage (FT at days 14th, MT at days 24th), TSP stage (FT at days 24th, MT at days 36th) and differentiated stage (FT at days 40th, MT at days 58th). RNA from mesonephros-gonad (MG) complexes were converted into cDNA by RT-PCR process, and the pattern of Wt1 and FoxL2 genes expression is analyzed by quantitative Real Time PCR (qPCR) method, β-actin gene is used as an internal control. The pattern of Wt1 gene expression in Pre-TSP stage was almost the same between MG complexes incubated at MT or FT, while TSP and differentiation stage, the pattern of Wt1 gene expression in MG complexes incubated at MT or FT was increased. Wt1 gene expression of MG complexes that incubated at FT was higher than at MT. There was a difference pattern between Wt1 gene expression in this research compared to the previous research in protein level. It could be assumed that the difference caused by post-transcriptional regulation mechanisms before mRNA of Wt1 gene translated into protein structure. The pattern of FoxL2 gene expression in Pre-TSP stage was almost the same between MG complexes that incubated at MT and FT, and increased in both TSP and differentiated stage. The FoxL2 gene expression in MG complexes that incubated in FT is higher than MT on TSP and differentiated stage. Based on the results of this research, it can be assumed that Wt1 and FoxL2 gene were expressed in MG complexes that incubated both at MT and FT since Pre-TSP stage. The pattern of Wt1 gene expression was increased in every stage of gonadal development, and so do the pattern of FoxL2 gene expression. Wt1 and FoxL2 gene expressions were higher in MG complexes incubated at FT than MT.

Keywords: chelonia mydas, FoxL2, gene expression, TSD, Wt1

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Authors: Zelikha Labbani

Abstract:

The in vitro isolated microspore culture is the most powerful androgenic pathway to produce doubled haploid plants in the short time. To deviate a microspore toward embryogenesis, a number of factors, different for each species, must concur at the same time and place. Once induced, the microspore undergoes numerous changes at different levels, from overall morphology to gene expression. Induction of microspore embryogenesis not only implies the expression of an embryogenic program, but also a stress-related cellular response and a repression of the gametophytic program to revert the microspore to a totipotent status. As haploid single cells, microspore became a strategy to achieve various objectives particularly in genetic engineering. In this communication we would show the most recent advances in the producing haploid embryos via in vitro isolated microspore culture.

Keywords: in vitro isolated microspore culture, success, haploid cells, bioinformatics, biomedicine

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Authors: Rachel Matar, Maxime Merheb

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Chemical drugs have been used for many centuries as the only way to cure diseases until the novel gene therapy has been created in 1960. Gene therapy is based on the insertion, correction, or inactivation of genes to treat people with genetic illness (1). Gene therapy has made wonders in Parkison’s, Alzheimer and multiple sclerosis. In addition to great promises in the healing of deadly diseases like many types of cancer and autoimmune diseases (2). This method implies the use of recombinant DNA technology with the help of different viral and non-viral vectors (3). It is nowadays used in somatic cells as well as embryos and gametes. Beside all the benefits of gene therapy, this technique is deemed by some opponents as an ethically unacceptable treatment as it implies playing with the genes of living organisms.

Keywords: gene therapy, genetic disease, cancer, multiple sclerosis

Procedia PDF Downloads 541

Authors: Zelikha Labbani

Abstract:

The In Vitro isolated micro spore culture is the most powerful androgenic pathway to produce doubled haploid plants in the short time. To deviate a micro spore toward embryogenesis, a number of factors, different for each species, must concur at the same time and place. Once induced, the micro spore undergoes numerous changes at different levels, from overall morphology to gene expression. Induction of micro spore embryogenesis not only implies the expression of an embryogenic program, but also a stress-related cellular response and a repression of the gametophytic program to revert the microspore to a totipotent status. As haploid single cells, micro spore became a strategy to achieve various objectives particularly in genetic engineering. In this study we would show the most recent advances in the producing haploid embryos via In Vitro isolated micro spore culture.

Keywords: haploid cells, In Vitro isolated microspore culture, success

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Authors: Elena Maria Scalisi

Abstract:

Currently, photodegradation by nanoparticles (NPs) is a common solution for wastewater treatment. Nanoparticles are efficient for removing organic and inorganic pollutants, heavy metals from wastewater and killing microorganisms through environmentally friendly. In this context, the major representative of photocatalytic technology for industrial wastewater treatment are TiO₂ nanoparticles (TiO₂-NPs). TiO₂-NPs have a strong catalytic activity that depends to their physicochemical properties. Thanks to their small size (between 1-100 nm), nanoparticles occupy less volume, then their surface area increases. The increase in the surface-to-volume ratio results in the increase of the particle surface energy, which improve their reactivity potential. However, these unique properties represent risks to the ecosystems and organisms when unintentionally TiO₂-NPs are release into the environment and absorbed by living organisms. Several studies confirm that there is a high level of interest concerning the safety of TiO₂-NPs in the aquatic environment, furthermore, ecotoxicological tools are useful to correctly evaluate their toxicity. In the current study, we aimed to characterize potential toxic effects of TiO₂-NP suspension to zebrafish during embryo-larval stages to evaluate parameters such as survival rates, malformation, hatching, the overall length of the larvae heartbeat, and biochemical biomarkers that reflect the acute toxicity and sublethal effects of TiO₂-NPs. Zebrafish embryos were exposed to titanium dioxide nanoparticles (TiO₂-NPs at 1mg/L, 2mg/L, and 4mg/L) from fertilization to the free swimming stage (144hpf). Every day, we recorded the toxicological endpoints, moreover, immunohistochemical analysis has been performed at the end of the exposure. In particular, we have evaluate the expression of the following biomarkers: Heat Shock Protein 70 (HSP70), Poly ADP-Ribose Polymerase-1 (PARP-1), Metallothioneins (MTs). Our results have shown that hatch ability, survival, and malformation rate were not affected by TiO₂ NPs at these exposure levels. However, TiO₂-NPs caused an increase of heartbeat and reduction of body length; at the same time, TiO₂-NPs have inducted the production of ROS and the expression of oxidative stress biomarkers HSP70 and PARP-1. Hight positivity for PARP-1 at all concentration tested was observed. As regards MT, positivity was found in the expression of this biomarker in the whole body of the embryo, with the exception of the end of the tail. Metallothioneins (MT) are biomarkers widely used in environmental monitoring programs for aquatic creatures. At the light of our results i.e. no death until the end of the experiment (144hpf), no malformation and expression of the biomarkers mentioned, it is evident that zebrafish larvae with their natural detoxification pathways are able to resist the presence of toxic substances and then they can tolerate the presence of metal concentrations. However, an excessive oxidative state can compromise cell function, therefore the uncontrolled release of nanoparticles into the environment is severe and must be constantly monitored.

Keywords: nanoparticles, embryo zebrafish, HSP70, PARP-1

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Authors: N. G. Klivleyeva, T. I. Glebova, G. V. Lukmanova, S. B. Bayseit, S. Z. Taubaeva, M. K. Kalkozhaeva

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The role of viral diseases in the general infectious disease incidence increases every year and requires special attention to the problem of interpreting the etiology of infectious agents. Influenza and acute respiratory viral infections are one of the most pressing public health issues. In the period 2012-2014, collection of 419 nasal swabs and 150 blood sera has been carried out in the patient care institutions of the various Kazakhstan regions from patients with symptoms of ARVI and pneumonia. Primary identification of biosamples for the presence of influenza viral antigens in enzyme immunoassay on nitrocellulose membrane gave positive results in 125 swabs (29.8%). Biosample screening in immunofluorescence test revealed the presence of influenza viral antigens against A/H1 in 63 samples (15.0%), A/H3 – in 70 samples (16.7%) and type B – in 9 samples (2.1%). As a result of primary infection, and successive passages in chick embryos and MDCK cell cultures, 38 HAAg were isolated from 419 samples with a clear cytopathic effect and hemagglutination titre in MDCK cell culture within 1:2-1:4, in CE - 1:8-1:256. The infectivity of isolates in chicken embryos were 3.5-6.5 lg EID50/0.2, in MDCK cell culture – 2.5-6.5 lg PFU/ml. Identification of 28 isolates was carried out in inhibition reactions of hemagglutinating activity and neuraminidase activity, showed their belonging to the influenza virus: 26 strains to A/H1N1, one - to A/H3N2, and one - to type B. Serological examination of blood sera for the presence of specific antibodies being an indirect evidence of the performed isolation and contributing to the timely interpretation of the disease etiology in the epidemics takes an important place in the comprehensive study of influenza viruses circulating among people. Serological analyzes were carried out in HAI assay using a kit consisting of 12 reference strains obtained from the WHO centre for reference and research on Influenza (CDC, Atlanta, USA) and three Kazakhstan (A/Almaty/347/09 (H1N1v), A/Almaty/462/11 (H3N2) and B/Almaty/414/10) human influenza viruses that are stored in the laboratory collection. The results of serological analysis of 150 blood sera showed that antihaemagglutinins against the A/H3N2 virus serosubtype were found in 46 samples (49.4%) out of 93 sera collected in 2012-2013. The antibody titres were within 1:160-1:320. 19 sera (20.4%) were seropositive against influenza A/H1N1 virus, the antibodies were observed in titres of 1:20-1:40. Six sera (6.4%) were positive against the influenza A/H1N1+A/H3N2 virus (mixed infection); the antibodies were recorded in titres of 1:20-1:40. Antihaemagglutinins against influenza type B virus were detected only in five sera (5.4%). The results of analysis of 57 sera collected in 2014 showed that antihaemagglutinins against A/H3N2 virus subtype were detected in 32 blood sera (56.1%) in titres of 1:160-1:640. Ten sera (17.5%) were seropositive against A/H1N1 virus; antihaemagglutinins against influenza type B virus were not detected. Therefore, virological and serological studies have shown that in Kazakhstan, as well as in the world, the influenza viruses A/H1N1, A/H3N2 and influenza B viruses were actively circulating during the epidemic seasons in 2012-2014.

Keywords: influenza, MDCK cell, serological analysis, virus

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Authors: Hasan Basri Jumin

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Protoplasts isolated from embryogenic callus of Citrus sinensis were electrically used with mesophyll protoplasts isolated from seedless Citrus relatives. Hybrid of somatic embryos plantlets was obtained after 7 months of culture. Somatic hybrid plants were regenerated into normal seedlings and successfully transferred to soil after strictly acclimatization in the glass pot. The somatic hybrid plants were obtained by screening on the basis of chromosomes count. The number of chromosome of root tip counting revealed plantlets tetraploids (2n = 4x = 36) and the other were diploids (2n = 2x = 18) morphologically resembling the mesophyll parent. This somatic hybrid will be utilized as a possible pollen parent for improving the Citrus sinensis. A complete protoplast-to-plant system of somatic hybrid was developed for Citrus sinensis and Citrus relatives which could facilitate the transfer of nuclear and cytoplasmic genes from this species into cultivated Citrus through protoplast fusion.

Keywords: chromosome, Murraya paniculata, protoplast fusion, somatic hybrid, tetrapoliod

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Authors: Saeid H. Mobini, Monika Lulsdorf, Thomas D. Warkentin

Abstract:

The length of the breeding cycle from seed to seed is a limiting factor in the development of improved homozygous lines for breeding or recombinant inbred lines (RILs) for genetic analysis. The objective of this research was to accelerate the production of field pea RILs through application of rapid generation technology (RGT). RGT is based on the principle of growing miniature plants in an artificial medium under controlled conditions, and allowing them to produce a few flowers which develop seeds that are harvested prior to normal seed maturity. We aimed to maintain population size and genetic diversity in regeneration cycles. The effects of flurprimidol (a gibberellin synthesis inhibitor), plant density, hydroponic system, scheduled fertilizer applications, artificial light spectrum, photoperiod, and light/dark temperature were evaluated in the development of RILs from a cross between cultivars CDC Dakota and CDC Amarillo. The main goal was to accelerate flowering while reducing maintenance and space costs. In addition, embryo rescue of immature seeds was tested for shortening the seed fill period. Data collected over seven generations included plant height, the percentage of plant survival, flowering rate, seed setting rate, the number of seeds per plant, and time from seed to seed. Applying 0.6 µM flurprimidol reduced the internode length. Plant height was decreased to approximately 32 cm allowing for higher plant density without a delay in flowering and seed setting rate. The three light systems (T5 fluorescent bulbs, LEDs, and High Pressure Sodium +Metal-halide lamp) evaluated did not differ significantly in terms of flowering time in field pea. Collectively, the combination of 0.6 µM flurprimidol, 217 plant. m-2, 20 h photoperiod, 21/16 oC light/dark temperature in a hydroponic system with vermiculite substrate, applying scheduled fertilizer application based on growth stage, and 500 µmole.m-2.s-1 light intensity using T5 bulbs resulted in 100% of plants flowering within 34 ± 3 days and 96.5% of plants completed seed setting in 68.2 ± 3.6 days, i.e., 30-45 days/generation faster than conventional single seed descent (SSD) methods. These regeneration cycles were reproducible consistently. Hence, RGT could double (5.3) generations per year, using 3% occupying space, compared to SSD (2-3 generation/year). Embryo rescue of immature seeds at 7-8 mm stage, using commercial fertilizer solutions (Holland’s Secret™) showed seed setting rate of 95%, while younger embryos had lower germination rate. Mature embryos had a seed setting rate of 96.5% without either hormones or sugar added. So, considering the higher cost of embryo rescue using a procedure which requires skill, additional materials, and expenses, it could be removed from RGT with a further cost saving, and the process could be stopped between generations if required.

Keywords: field pea, flowering, rapid regeneration, recombinant inbred lines, single seed descent

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Authors: Venoos Iman, Suzita Mohd Noor, Syam Mohan, Mohamad Ibrahim Noordin

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Girinimbine, a carbazole alkaloid was isolated from the stem bark and root of Murraya koenigii and its structure and purity was identified by HPLC and LC-MS. Here we report that Girinimbine strongly inhibit angiogenesis activity both in vitro and in vivo. MTT result showed that girinimbine inhibits cell proliferation of the HUVECS cell line in vitro. Result of endothelial cell invasion, migration, tube formation and wound healing assays also demonstrated significant time and does dependent inhibition by girinimbine. Moreover, girinibine mediates its anti-angiogenic activity through up- and down-regulation of angiogenic and anti-aniogenic proteins. Furthermore, anti-angiogenic potential of girinimbine was evidenced in vivo on zebrafish model. Girinimbine inhibited neo-vessels formation in zebrafish embryos during 24 hours exposure time. Together, these results demonstrated for the first time that girinimbine could effectively suppress angiogenesis and strongly suggest that it might be a novel angiogenesis inhibitor.

Keywords: anti-angiogenic, carbazole alkaloid, girinimbine, zebrafish

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Authors: Norsyamimi Hassim, Masturah Markom

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Supercritical fluid extraction (SFE) has been known as a sustainable and safe extraction technique for plant extraction due to the minimal usage of organic solvent. In this study, a scale-up process for the selected herbal plant (Phyllanthus niruri) was investigated by using supercritical carbon dioxide (SC-CO2) with food-grade (ethanol-water) cosolvent. The quantification of excess ethanol content in the final dry extracts was conducted to determine the safety of enriched extracts. The extraction yields obtained by scale-up SFE unit were not much different compared to the predicted extraction yields with an error of 2.92%. For component contents, the scale-up extracts showed comparable quality with laboratory-scale experiments. The final dry extract showed that the excess ethanol content was 1.56% g/g extract. The fish embryo toxicity test (FETT) on the zebrafish embryos showed no toxicity effects by the extract, where the LD50 value was found to be 505.71 µg/mL. Thus, it has been proven that SFE with food-grade cosolvent is a safe extraction technique for the production of bioactive compounds from P. niruri.

Keywords: scale-up, supercritical fluid extraction, enriched extract, toxicity, ethanol content

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