An Efficient Agrobacterium-Mediated Genetic Transformation Method for Solanum betaceum Cav. Embryogenic Callus.

Somatic embryogenesis in Solanum betaceum (tamarillo) has proven to be an effective model system for studying morphogenesis, since optimized plant regeneration protocols are available, and embryogenic competent cell lines can be induced from different explants. Nevertheless, an efficient genetic transformation system for embryogenic callus (EC) has not yet been implemented for this species. Here, an optimized faster protocol of genetic transformation using Agrobacterium tumefaciens is described for EC. The sensitivity of EC to three antibiotics was determined, and kanamycin proved to be the best selective agent for tamarillo callus. Two Agrobacterium strains, EHA105 and LBA4404, both harboring the p35SGUSINT plasmid, carrying the reporter gene for ß-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII), were used to test the efficiency of the process. To increase the success of the genetic transformation, a cold-shock treatment, coconut water, polyvinylpyrrolidone and an appropriate selection schedule based on antibiotic resistance were employed. The genetic transformation was evaluated by GUS assay and PCR-based techniques, and a 100% efficiency rate was confirmed in the kanamycin-resistant EC clumps. Genetic transformation with the EHA105 strain resulted in higher values for gus insertion in the genome. The protocol presented provides a useful tool for functional gene analysis and biotechnology approaches.

An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing.

Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.

Regulatory non-coding RNAs: Emerging roles during plant cell reprogramming and in vitro regeneration.

Plant regeneration is a well-known capacity of plants occurring either in vivo or in vitro. This potential is the basis for plant micropropagation and genetic transformation as well as a useful system to analyse different aspects of plant development. Recent studies have proven that RNA species with no protein-coding capacity are key regulators of cellular function and essential for cell reprogramming. In this review, the current knowledge on the role of several ncRNAs in plant regeneration processes is summarized, with a focus on cell fate reprogramming. Moreover, the involvement/impact of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and small-interfering RNAs (siRNAs) in the regulatory networks of cell dedifferentiation, proliferation and differentiation is also analysed. A deeper understanding of plant ncRNAs in somatic cell reprogramming will allow a better modulation of in vitro regeneration processes such as organogenesis and somatic embryogenesis.

Small Non-Coding RNAs at the Crossroads of Regulatory Pathways Controlling Somatic Embryogenesis in Seed Plants.

Small non-coding RNAs (sncRNAs) are molecules with important regulatory functions during development and environmental responses across all groups of terrestrial plants. In seed plants, the development of a mature embryo from the zygote follows a synchronized cell division sequence, and growth and differentiation events regulated by highly regulated gene expression. However, given the distinct features of the initial stages of embryogenesis in gymnosperms and angiosperms, it is relevant to investigate to what extent such differences emerge from differential regulation mediated by sncRNAs. Within these, the microRNAs (miRNAs) are the best characterized class, and while many miRNAs are conserved and significantly represented across angiosperms and other seed plants during embryogenesis, some miRNA families are specific to some plant lineages. Being a model to study zygotic embryogenesis and a relevant biotechnological tool, we systematized the current knowledge on the presence and characterization of miRNAs in somatic embryogenesis (SE) of seed plants, pinpointing the miRNAs that have been reported to be associated with SE in angiosperm and gymnosperm species. We start by conducting an overview of sncRNA expression profiles in the embryonic tissues of seed plants. We then highlight the miRNAs described as being involved in the different stages of the SE process, from its induction to the full maturation of the somatic embryos, adding references to zygotic embryogenesis when relevant, as a contribution towards a better understanding of miRNA-mediated regulation of SE.

Alterações dos níveis séricos de creatinina, ácido úrico, creatina kinase e da taxa de filtração glomerular em corredores de "rua" / Changes in serum creatinine, uric acid, creatine kinase and glomerular filtration in street runners

As estratégias adotadas pelos corredores de "rua", durante as provas, sofrem interferência da distância da corrida e dos níveis técnico e físico dos competidores. Assim,o objetivo deste estudo foi de examinar os efeitos bioquímicos da Creatinina (C), Ácido Úrico (AU), Creatina Kinase (CK) e da Taxa de Filtração Glomerular (TFG) provocados por uma prova de corrida de "rua" de 6 (seis) Km. Participaram n=(15) atletas masculinos (40,53±8,65 anos) separados em 3 grupos: Grupo 1 Melhores Tempos (G1MT) n=5; Grupo 2 Tempos Intermediários (G2TI) n =5; Grupo 3 Piores Tempos (G3PT) n=5. Foram coletadas amostras de sangue 30 min antes e imediatamente após a corrida. Os dados foram analisados através da ANOVA Two-Way, Wilcoxon e Mann Whitney. Consideraram-se os níveis significativos (p<0,05). Os resultados permitem concluir que ocorreram aumentos significativos intragrupos nas atividades séricas de (C) no G1MT pré: 1,18±0,04 mg.dL-¹ pós: 1,60±0,15 mg.dL-¹; G2TI pré: 1,04±0,15 mg.dL-¹ pós: 1,56 ±0,21 mg.dL-¹; G3PT pré 1,08±0,13 mg.dL-¹ pós 1,52±0,32 mg.dL-¹ e no (AU) G1MT pré: 3,80±0,75 mg.dL-¹ pós 4,56±0,94 mg.dL-¹; G2TI pré 4,36±1,62 mg.dL-¹ pós 5,0 ±1,69 mg.dL-¹; G3PT pré 4,62±1,08 mg.dL-¹ pós: 5,42±0,86 mg.dL-¹, enquanto a (CK) e a (TFG) não apresentaram diferença significativa.

The strategies adopted by corridors "street" during the evidence from interference of the race distance and levels of technical and physical competition. The objective of this study was to examine the biochemical effects of Creatinine (C), Uric Acid (AU), Creatine Kinase (CK) and Glomerular Filtration Rate (GFR) caused by a test run of "street" of 6 (six) Km participated n=(15) male athletes (40.53 ± 8.65 years) divided into three groups: Group 1 Best Times (G1MT) n = 5, Group 2 Intermediate Times (G2TI) n = 5; Group 3 Times Worst (G3PT) n = 5. Blood samples were collected 30 min before and immediately after the race. Data were analyzed by Two-Way ANOVA, Wilcoxon and Mann Whitney test. It was considered significant levels (p<0.05). The results showed that there were significant increases in serum activities of intra-group (C) in G1MT before: 1,18±0,04 mg.dL-¹ after: 1.60±0.15 mg.dL-¹; G2TI before: 1,04±0,15 mg.dL-¹ after: 1,56±0,21 mg.dL-¹; G3PT before: 1,08±0,13 mg.dL-¹ after: 1,52±0,32 mg.dL-¹ and (AU) G1MT before: 3,80±0,75 mg.dL-¹ after: 4,56±0,94 mg.dL-¹; G2TI before: 4,36±1,62 mg.dL-¹ after: 5,0±1,69 mg.dL-¹; G3PT before: 4,62±1,08 mg.dL-¹ after: 5,42±0,86 mg.dL-¹, while (CK) and (GFR) showed no significant difference.

Lethal effects of abamectin on the aquatic organisms Daphnia similis, Chironomus xanthus and Danio rerio.

Abamectin is used as an acaricide and insecticide for fruits, vegetables and ornamental plants, as well as a parasiticide for animals. One of the major problems of applying pesticides to crops is the likelihood of contaminating aquatic ecosystems by drift or runoff. Therefore, toxicity tests in the laboratory are important tools to predict the effects of chemical substances in aquatic ecosystems. The aim of this study was to assess the potential hazards of abamectin to the freshwater biota and consequently the possible losses of ecological services in contaminated water bodies. For this purpose, we identified the toxicity of abamectin on daphnids, insects and fish. Abamectin was highly toxic, with an EC(50) 48 h for Daphnia similis of 5.1 ng L(-1), LC(50) 96 h for Chironomus xanthus of 2.67 µg L(-1) and LC(50) 48 h for Danio rerio of 33 µg L(-1).

Development of methodology for determination of pesticides residue in water by SPE/HPLC/DAD.

To boost crop yield, sugarcane growers are using increasing amounts of pesticides to combat insects and weeds. But residues of these compounds can pollute water resources, such as lakes, rivers and aquifers. The present paper reports the results of a study of water samples from the Feijão River, which is the source of drinking water for the city of São Carlos, São Paulo, Brazil. The samples were evaluated for the presence of four leading pesticides--ametryn, atrazine, diuron and fipronil--used on sugarcane, the dominant culture in the region. The samples were obtained from three points along the river: the headwaters, along the middle course of the river and just before the municipal water intake station. The pesticides were extracted from the water samples by solid-phase extraction (SPE) and then analyzed by liquid chromatography with diode array detection (LC-DAD). The analytical method was validated by traditional methods, obtaining recovery values between 90 and 95%, with precision deviations inferior to 2.56%, correlation coefficients above 0.99 and detection and quantification limits varying from 0.02 to 0.05 mg L(-1) and 0.07 to 0.17 mg L(-1), respectively. No presence of residues of the pesticides was detected in the samples, considering the detection limits of the method employed.

Influence of optic disc size on the diagnostic performance of macular ganglion cell complex and peripapillary retinal nerve fiber layer analyses in glaucoma.

AIM: To evaluate the influence of optic disc size on the diagnostic accuracy of macular ganglion cell complex (GCC) and conventional peripapillary retinal nerve fiber layer (pRNFL) analyses provided by spectral domain optical coherence tomography (SD-OCT) in glaucoma. METHODS: Eighty-two glaucoma patients and 30 healthy subjects were included. All patients underwent GCC (7 × 7 mm macular grid, consisting of RNFL, ganglion cell and inner plexiform layers) and pRNFL thickness measurement (3.45 mm circular scan) by SD-OCT. One eye was randomly selected for analysis. Initially, receiver operating characteristic (ROC) curves were generated for different GCC and pRNFL parameters. The effect of disc area on the diagnostic accuracy of these parameters was evaluated using a logistic ROC regression model. Subsequently, 1.5, 2.0, and 2.5 mm(2) disc sizes were arbitrarily chosen (based on data distribution) and the predicted areas under the ROC curves (AUCs) and sensitivities were compared at fixed specificities for each. RESULTS: Average mean deviation index for glaucomatous eyes was -5.3 ± 5.2 dB. Similar AUCs were found for the best pRNFL (average thickness = 0.872) and GCC parameters (average thickness = 0.824; P = 0.19). The coefficient representing disc area in the ROC regression model was not statistically significant for average pRNFL thickness (-0.176) or average GCC thickness (0.088; P ≥ 0.56). AUCs for fixed disc areas (1.5, 2.0, and 2.5 mm(2)) were 0.904, 0.891, and 0.875 for average pRNFL thickness and 0.834, 0.842, and 0.851 for average GCC thickness, respectively. The highest sensitivities - at 80% specificity for average pRNFL (84.5%) and GCC thicknesses (74.5%) - were found with disc sizes fixed at 1.5 mm(2) and 2.5 mm(2). CONCLUSION: Diagnostic accuracy was similar between pRNFL and GCC thickness parameters. Although not statistically significant, there was a trend for a better diagnostic accuracy of pRNFL thickness measurement in cases of smaller discs. For GCC analysis, an inverse effect was observed.