Drought-inducible promoters in sugarcane roots ("Saccharum" spp.)

Orientador: Marcelo Menossi TeixeiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A seca é um dos principais fatores limitantes na produtividade de espécies agrícolas e seus efeitos tendem a ser mais intensos em algumas áreas atualmente utilizadas pela agricultura, segundo previsões climáticas. Nesse contexto, a compreensão dos mecanismos de resposta ao déficit hídrico apresenta grande importância para o desenvolvimento de cultivares mais produtivos e tolerantes a condições climáticas adversas. Recentemente, a manipulação de características do sistema radicular tem ganhado destaque como oportunidade para o melhoramento genético vegetal. Em paralelo, é sabido que o uso de promotores induzidos ou órgão específicos é uma estratégia interessante para um controle mais fino da expressão de transgenes. Dessa forma, o objetivo deste trabalho foi caracterizar a atividade de promotores de seis genes induzidos por seca e/ou raiz-específicos e seu potencial biotecnológico utilizando plantas transgênicas de cana-de-açúcar. Construções contendo as sequências promotoras fusionadas ao gene "GUS" foram utilizadas para rastrear sua atividade regulatória. Perfis de indução por seca e expressão preferencial em raízes foram detecados nos eventos transgenicos via qPCR do gene "GUS". Entretanto, a atividade enzimática da proteína repórter não foi observada em plantas submetidas ao ensaio histoquímico do GUS, indicando um provável mecanismo de silenciamento pós-transcricional. Para testar essa hipótese e propor uma solução, uma nova construção foi gerada contendo o promotor "ScDehydrin1" ("ScDHN1") controlando a transcrição do gene "scoGUS", uma versão codificante do gene "GUS" com otimização de códons para cana-de-açúcar. O uso do gene repórter "scoGUS" levou a detecção do atividade do promotor "ScDHN1" em condições de estresse osmótico enquanto que, nas mesmas condições, não houve atividade em plantas contendo a sequência GUS nativa de Escherichia coli. Associado a caracterização do promotor "ScDHN1", realizamos a identificação genômica da família deidrina em cana além da caracterização do perfil transcricional de seus membros em resposta a seca. Os dados indicam que membros da familiam deidrinas devem exercer funções protetivas em cana durante deficit hidrico. Por fim, novas sequências promotoras de cana-de-açúcar com perfil de indução por seca e/ou preferencial em raízes foram caracterizados visando seu uso biotecnológico. Os dados gerados nesse trabalho também serão úteis para estudos básicos com sequências regulatórias em cana, em que a otimização de códons de genes repórteres parece exercer papel fundamentalAbstract: Drought is one of the main limiting factors for agricultural productivity and its effects are likely to be more intense in some land areas, according to climate change models. In this context, the understanding of regulatory mechanisms activated by plants in response to adverse environmental conditions may help to develop stress tolerant crops. Recently, genetic changes in the root system have been highlighted as an opportunity for plant breeding. Additionally, the use of inducible and organ specific promoters may help in the fine tuning of transgene expression. Therefore, the aim of this study was to characterize the regulatory activity of promoters from genes induced by drought and/or root-specific and their biotechnological potential in sugarcane transgenic plants. The promoter sequences were fused to "GUS" gene reporter to track their regulatory activity. Induction by drought and root-preferential regulation were detected in transgenic events via qPCR of the GUS transcript. However, no GUS activity was detected in plants submitted to GUS histochemical assay, indicating a probable post-transcriptional silencing event. In order to test this hypothesis and solve this issue, we generated a new construction composed by the promoter "ScDHN1" controlling the transcription of "scoGUS" gene, a codon-optimized GUS sequence for sugarcane. The use of "scoGUS" enabled to detect the promoter "ScDHN1" activity in plantlets under osmotic stress in vitro, while in the same conditions no activity was detected for plantlets harboring the native "GUS" gene from Escherichia coli. We also performed a genomic identification of the dehydrin family in sugarcane, and the influence of drought on the transcriptional regulation of "ScDHN" genes. Our data support that dehydrin members might play protective roles in sugarcane under hydric deficit. Lastly, new sugarcane promoter sequences displaying drought-inducible and/or root-preferential profile were characterized aiming biotechnology applications. The data provided in this work will also be useful for basic studies with regulatory sequence in sugarcane, in which the use of codon-optimized reporter genes is essentialDoutoradoGenetica Vegetal e MelhoramentoDoutor em Genetica e Biologia Molecular2016/05501-6FAPES

New insertion method of transnasal ileus tube for small bowel obstruction: Anterior balloon method.

BACKGROUND:Small bowel obstruction (SBO) is usually caused by postoperative adhesions and malignant disease, and decompression is effective for SBO. Our previous case report suggested that a new transnasal ileus tube insertion method, the anterior balloon method (ABM), could achieve decompression for adhesive SBO. AIMS:The study aimed to investigate the effectiveness of a new method for inserting transnasal ileus tubes in patients with SBO. METHODS:Altogether, 134 patients with small bowel obstruction treated from January 2011 to December 2017 were reviewed. The patients were categorized into two groups: those with the new method that inserts an anterior balloon (ABM group: 52 patients, 2014-2017) versus those with the ordinary insertion method (OIM group: 82 patients, 2011-2014). RESULTS:The patients' characteristics and symptoms on admission were similar in the ABM and OIM groups. Adhesions were the main cause of ileus in the two groups. The insertion time duration was significantly shorter in the ABM group than in OIM group (28.4 ± 9.1 vs. 33.5 ± 13.0 min; p = 0.01). The ABM group also had significantly longer tubes than OIM group (222.4 ± 32.2 vs. 157.4 ± 31.7 cm; p < 0.001), which resulted in a significantly shorter time until clinical symptoms were relieved in ABM group. There were no significant differences in adverse events between the two groups. CONCLUSIONS:The ABM group had shorter insertion duration and longer tubes than those of OIM group. The ABM might become a preferred therapeutic choice to achieve decompression in patients with SBO

Safety and Efficacy of the Noncessation Method of Antithrombotic Agents after Emergency Endoscopic Hemostasis in Patients with Nonvariceal Upper Gastrointestinal Bleeding: A Multicenter Pilot Study

Background and Aims. The present study aimed to clarify the safety and efficacy of the noncessation method of antithrombotic agents after emergency endoscopic hemostasis in patients with nonvariceal upper gastrointestinal bleeding (UGIB). Methods. In this multicenter, prospective, pilot study, we performed emergency endoscopic hemostasis for nonvariceal UGIB in patients taking antithrombotic agents and resumed the medications without a cessation period (group A). The clinical characteristics, types of antithrombotic agents, UGIB etiology, treatment outcome, and adverse events were evaluated. We used propensity score matching to compare treatment outcomes and adverse events with our previous cohort (group B) in whom antithrombotic agents were transiently discontinued after emergency endoscopic hemostasis. Results. Forty-three consecutive patients were prospectively enrolled. The main antithrombotic agents were low-dose aspirin and direct oral anticoagulants; 11 patients (25.6%) were taking multiple antithrombotics. Peptic ulcers were the main cause of bleeding (95.4%). Endoscopic hemostasis was successful in all patients and the incidence of rebleeding within a month was 7.0%. Propensity score matching created 40 matched pairs. Endoscopic hemostasis was performed by soft coagulation significantly more frequently in group A than in group B (97.5% versus 60.0%, P < 0.001). Neither the rebleeding rate within a month nor thromboembolic event rate was different between the two groups. However, the mean duration of hospitalization was significantly shorter in group A than in group B (8.6 ± 5.2 d versus 14.4 ± 7.1 d, P < 0.001). Conclusions. Antithrombotic agents possibly can be continued after successful emergency endoscopic hemostasis for nonvariceal UGIB

Comparison of sedation between the endoscopy room and operation room during endoscopic submucosal dissection for neoplasms in the upper gastrointestinal tract

Abstract Background The present study was performed to compare the safety of sedation during endoscopic submucosal dissection (ESD) in the endoscopy room versus operation room. Methods In total, 297 patients with gastrointestinal tumors who underwent ESD from January 2011 to December 2016 were retrospectively reviewed. The patients were divided into two groups: those who underwent ESD in the endoscopy room without propofol (Group E) versus operation room with propofol (Group O). The patient, tumor, and procedure characteristics; adverse events; and treatment outcomes were compared between the two groups. Results The patient and tumor characteristics, including age (73.6 ± 8.2 vs. 72.5 ± 9.1 years), comorbidities, and tumor size and histology, were not different between Groups E and O. The ESD procedure time was comparable between Groups E and O (105.4 ± 70.4 vs. 106.5 ± 64.4 min), and the anesthesia time was equivalent (138.3 ± 78.1 vs. 148.4 ± 68.8 min). There were no significant differences in adverse events between the two groups. During the ESD procedure, desaturation occurred significantly more often in Group E than O (12.9% vs. 4.0%, P = 0.021, odds ratio: 3.53, 95% CI: 1.17–14.4). The recovery time after ESD was significantly longer in Group E than O (180 (100–360) vs. 90 (0–180) min, P < 0.001). Conclusions A decreased desaturation rate and shorter recovery time after ESD were the advantages of sedation in the operation room with propofol compared with sedation in the endoscopy room. These findings warrant further exploration of the advantages of safe and effective ESD for upper gastrointestinal neoplasms in the operation room