Optimization of parameters for obtaining callus, suspension, and root cultures of meadowsweet (filipendula ulmaria) to isolate the largest number of biologically active substances with geroprotective properties

Abstract The study of biologically active substances-secondary metabolites of plants that exhibit geroprotective properties is an actual and popular direction in medicine to prevent early aging. This work aims to select the cultivation parameters for obtaining in vitro cell cultures of meadowsweet containing the largest amount of biologically active substances (BAS) for their further extraction as candidate substances for geroprotectors. To specify the effectiveness of the selected cell culture cultivation parameters, biomass growth for callus and root cultures, growth index, specific growth rate, and viability for suspension cultures was carried out. The study results made it possible to select the nutrient media for the cultivation of cell cultures of meadowsweet. It has been found that the greater the antioxidant activity of the extracts, the greater the antimicrobial properties it exhibits. In this study, cell cultures in vitro and alcohol extracts from the plant Filipendula ulmaria were considered as raw materials rich in candidate substances for geroprotectors. According to the data obtained, the plant is rich in hydroxybenzoic and salicylic acids, spireoside, avicularin, and hyperoside.

Resumo O estudo de substâncias biologicamente ativas metabólitos secundários de plantas que apresentam propriedades geroprotetoras é uma tendência atual e popular no campo da medicina para a prevenção do envelhecimento precoce. O objetivo deste trabalho foi selecionar os parâmetros de cultivo para obtenção de culturas celulares in vitro de Ulmária contendo a maior quantidade de substâncias biologicamente ativas (SBA), para sua posterior extração como substâncias candidatas a serem geroprotetoras. Para especificar a eficácia dos parâmetros selecionados de cultivo em cultura de células, foi realizada a análise de crescimento de biomassa para culturas de calos e raízes, índice de crescimento, taxa de crescimento específica e viabilidade para culturas em suspensão. Os resultados do estudo possibilitaram a seleção do meio nutriente para o cultivo de células de Ulmária. Verificou-se que, quanto maior a atividade antioxidante dos extratos, maiores eram as propriedades antimicrobianas exibidas. Neste estudo, culturas celulares in vitro e extratos alcoólicos da planta Filipendula ulmaria foram considerados matérias-primas ricas em substâncias candidatas a serem geroprotetoras. De acordo com os dados obtidos, a planta é rica em ácidos hidroxibenzoico e salicílico, espirosídeo, avicularina e hiperosídeo.

Iceberg lettuce cultivated in different systems of planting and sources of fertilizer

Abstract Organic fertilization is a cheaper and highly effective option for profitability and consequent improvement of the soil's physical, chemical, and biological structure. Thus, the objective of this work was to evaluate different types of fertilization: organic (poultry shed litter), mineral, and leaf path on yield parameters of lettuce grown in various types of planting. The treatments consisted of using two planting systems (P1 - Line and P2 - quincunxes) and mineral and organic fertilizers (A1 - mineral fertilization; A2 - mineral fertilization + leaf fertilization; A3 - organic fertilization with poultry shed litter and A4 - fertilization organic + mineral). The experimental units consisted of 36 and 52 plants, respectively, for treatments P1 and P2, and all central plants of the experimental unit were evaluated. Heart height, fresh mass, and leaf number were observed. The mineral and mineral + leaf treatments did not differentiate, either in line or in quincunxes. The treatment that stood out about the analyzed variables was the organic fertilization and quincunxes planting system, reflecting a more significant number of lettuce plants and better use of the area.

Resumo A adubação orgânica é uma opção mais barata e de grande eficácia em relação à rentabilidade e consequente melhoria da estrutura física, química e biológica do solo. Desta forma, o objetivo deste trabalho foi avaliar diferentes tipos de adubação: orgânica (cama de frango), mineral e via foliar sobre parâmetros de produtividade de alface cultivada em diferentes tipos de plantio. Os tratamentos consistiram na utilização de dois sistemas de plantio (P1 - Linha e P2 - Quincôncio) e adubações minerais e orgânicas (A1 - adubação mineral; A2 - adubação mineral + adubação foliar; A3 - adubação orgânica com cama de aviário e A4 - adubação orgânica + mineral). As unidades experimentais foram compostas por 36 e 52 plantas, respectivamente, para os tratamentos em linha e em quincôncio, sendo avaliadas todas as plantas centrais da unidade experimental. Foram observados a altura do coração, massa fresca e número de folhas. Os tratamentos mineral e mineral + foliar não diferenciaram entre si, tanto em linha quanto em quincôncio. O tratamento que se destacou em relação às variáveis analisadas foi aquele baseado na adubação orgânica e sistema de plantio em quincôncio, refletindo em maior número de pés de alface e melhor aproveitamento da área.

Optimization of parameters for obtaining callus, suspension, and root cultures of meadowsweet (filipendula ulmaria) to isolate the largest number of biologically active substances with geroprotective properties / Otimização de parâmetros para obtenção de culturas de calos, suspensão e raiz de Ulmária (Filipendula ulmaria) para isolar o maior número de substâncias biologicamente ativas com propriedades geroprotetoras

The study of biologically active substances-secondary metabolites of plants that exhibit geroprotective properties is an actual and popular direction in medicine to prevent early aging. This work aims to select the cultivation parameters for obtaining in vitro cell cultures of meadowsweet containing the largest amount of biologically active substances (BAS) for their further extraction as candidate substances for geroprotectors. To specify the effectiveness of the selected cell culture cultivation parameters, biomass growth for callus and root cultures, growth index, specific growth rate, and viability for suspension cultures was carried out. The study results made it possible to select the nutrient media for the cultivation of cell cultures of meadowsweet. It has been found that the greater the antioxidant activity of the extracts, the greater the antimicrobial properties it exhibits. In this study, cell cultures in vitro and alcohol extracts from the plant Filipendula ulmaria were considered as raw materials rich in candidate substances for geroprotectors. According to the data obtained, the plant is rich in hydroxybenzoic and salicylic acids, spireoside, avicularin, and hyperoside.

O estudo de substâncias biologicamente ativas - metabólitos secundários de plantas que apresentam propriedades geroprotetoras - é uma tendência atual e popular no campo da medicina para a prevenção do envelhecimento precoce. O objetivo deste trabalho foi selecionar os parâmetros de cultivo para obtenção de culturas celulares in vitro de Ulmária contendo a maior quantidade de substâncias biologicamente ativas (SBA), para sua posterior extração como substâncias candidatas a serem geroprotetoras. Para especificar a eficácia dos parâmetros selecionados de cultivo em cultura de células, foi realizada a análise de crescimento de biomassa para culturas de calos e raízes, índice de crescimento, taxa de crescimento específica e viabilidade para culturas em suspensão. Os resultados do estudo possibilitaram a seleção do meio nutriente para o cultivo de células de Ulmária. Verificou-se que, quanto maior a atividade antioxidante dos extratos, maiores eram as propriedades antimicrobianas exibidas. Neste estudo, culturas celulares in vitro e extratos alcoólicos da planta Filipendula ulmaria foram considerados matérias-primas ricas em substâncias candidatas a serem geroprotetoras. De acordo com os dados obtidos, a planta é rica em ácidos hidroxibenzoico e salicílico, espirosídeo, avicularina e hiperosídeo.

Iceberg lettuce cultivated in different systems of planting and sources of fertilizer / Alface americana cultivada em diferentes sistemas de plantio e fontes de fertilizantes

Organic fertilization is a cheaper and highly effective option for profitability and consequent improvement of the soil's physical, chemical, and biological structure. Thus, the objective of this work was to evaluate different types of fertilization: organic (poultry shed litter), mineral, and leaf path on yield parameters of lettuce grown in various types of planting. The treatments consisted of using two planting systems (P1 - Line and P2 - quincunxes) and mineral and organic fertilizers (A1 - mineral fertilization; A2 - mineral fertilization + leaf fertilization; A3 - organic fertilization with poultry shed litter and A4 - fertilization organic + mineral). The experimental units consisted of 36 and 52 plants, respectively, for treatments P1 and P2, and all central plants of the experimental unit were evaluated. Heart height, fresh mass, and leaf number were observed. The mineral and mineral + leaf treatments did not differentiate, either in line or in quincunxes. The treatment that stood out about the analyzed variables was the organic fertilization and quincunxes planting system, reflecting a more significant number of lettuce plants and better use of the area.

A adubação orgânica é uma opção mais barata e de grande eficácia em relação à rentabilidade e consequente melhoria da estrutura física, química e biológica do solo. Desta forma, o objetivo deste trabalho foi avaliar diferentes tipos de adubação: orgânica (cama de frango), mineral e via foliar sobre parâmetros de produtividade de alface cultivada em diferentes tipos de plantio. Os tratamentos consistiram na utilização de dois sistemas de plantio (P1 - Linha e P2 - Quincôncio) e adubações minerais e orgânicas (A1 - adubação mineral; A2 - adubação mineral + adubação foliar; A3 - adubação orgânica com cama de aviário e A4 - adubação orgânica + mineral). As unidades experimentais foram compostas por 36 e 52 plantas, respectivamente, para os tratamentos em linha e em quincôncio, sendo avaliadas todas as plantas centrais da unidade experimental. Foram observados a altura do coração, massa fresca e número de folhas. Os tratamentos mineral e mineral + foliar não diferenciaram entre si, tanto em linha quanto em quincôncio. O tratamento que se destacou em relação às variáveis analisadas foi aquele baseado na adubação orgânica e sistema de plantio em quincôncio, refletindo em maior número de pés de alface e melhor aproveitamento da área.

Probiotic Properties of Lactic Acid Bacteria Isolated from Spontaneously Fermented Kunun-Zaki

This study was conducted to investigate the probiotic properties of twenty lactic acid bacteria (LAB) previously isolated and identified from naturally fermented kunun-zaki (a Nigerian fermented cereal beverage). The probiotic properties of the twenty lactic acid bacteria (LAB) isolates were assessed using different standard methods and agar well diffusion method. The results showed that most of the isolates tolerated acidic pH and survived at 30-40oC. All the tested LAB isolates grew at 1% NaCl concentration and remarkably, six LAB isolates {L. brevis (3) and L. plantarum (3)} exhibited good growth at higher NaCl concentrations (10-15%). The highest percentage of cellular auto-aggregation was observed in L plantarum (84.86%) and the lowest was obtained in L. brevis (27.44%). All the twenty LAB isolates tested in-vitro in this study grew in bile salt; fermented glucose; produced good aroma; produced no hemolysis revealing they were not pathogenic and produced antagonistic activity against selected pathogens (Staphylococcus aureus, Salmonella typhi, Shigella. dysenteriae and Escherichia coli) which made them suitable and safe for human consumption as potential probiotics and for industrial purposes.

STUDY OF OVIPOSITION IN CARYEDON SERRATUS ON PHASEOLUS VULGARIS

Control of insect pest is a serious problem, as the use of different chemical pesticides to control them is injurious to human health and environment. Thorough knowledge of their biology is quite important to manage them. During present investigations oviposition in Caryedon serratus have been observed on Phaseolus vulgaris for the first time. The female C. serratus preferred to lay eggs near the depression as convex surface of the seed of P. vulgaris. Maximum number of eggs per seed was 15. Maximum egg laying occurred during the first 6-days of oviposition under all three temperatures (25C, 28C and 34C). The peak of egg laying occurred on 3-day at 25C and 34C, however, it occurred on 5-day at 28C. C. serratus prefer laying eggs in the depression, may possibly be due to two reasons, first, the seed coat may be weakest at this point, second, eggs laid in depression may be protected. Most of egg laying activity by day-6, helps the female to conserve its energy for greater longevity.

A Clinical Analysis of Phlebolymphedema and Its Relationship with Physical Activity and Disability

Background: The clinical features and epidemiological data about patients with phebolymphedema or lymphedema related to Chronic Venous Disease in Mexico is limited and has been understudied; at the same time, the relationships between its clinical features, physical activity level and disability remained unknown. Methods: This is a longitudinal cohort study based on the analysis of clinical data of 90 patients gathered between 2021 and 2022. The statistical analysis was carried out using the software SPSS version 25 and GrandhPad Prism 8; a descriptive analysis was developed using measures of central tendency for the variables of a quantitative nature and frequency distribution for those categorical variables. The behavior of the variables was revealed through the Shapiro-Wilk statistic. The mean difference analysis was carried out with the Student’s T for independent samples. To identify the effect of gender, age, and severity of the disease on the study variables, a three-way analysis of variance was obtained with a Sidak comparison analysis. For the associations between qualitative and dichotomous nature variables, the Chi-Square statistic was obtained along with the odds ratio to determine the intensity of the relationships found. Results: A total of 90 patients were included in the analysis; 71% (64) were female and 29% (26) were male; with a mean age of 62.7 years old (± 30.5). A mean BMI of 33.2, 79.9% (77) of patients reached overweight and obesity ranges. 50% (45) of patients reported disability to perform one or more daily life activities related to the disease’s condition (signs, symptoms, volume). Only 12% (10) of all patients performed at or above the minimum physical activity recommended for their population group, and 88% (80) of patients had no physical activity or performed under the proper population group’s recommendation of minimal physical activity. It was found that the practice of physical activity, the number of compromised segments, the stage of the pathology, and the presence of cardiological and metabolic antecedents provide a statistically significant association with disability. Notable statistical difference among sex as a risk factor was not found. Regarding the BMI, the only differences were observed in the level of severity of the disease, regardless of the presence of any disability (p=0.006), evidencing that the greater the severity, the higher the BMI in both men and women. About physical activity, it is observed that inactivity or minimal practice of physical activity is a risk factor for referred disability (p<0.05), since it is prolonged that the non-practice of physical activity increases by 230% the chances of referring a disability associated to the disease compared to those people who referred to practice minimal or above minimal physical activity; the presence of a cardiological and/or metabolic history is related to an increase of about 150% to 180% of presenting disability compared to patients who do not have these pathologies. Conclusion: This study shows clinical and epidemiological features of phlebolymphedema and their relationship with the level of physical activity and reported disability of 90 patients. Further studies are needed to improve and broaden the understanding of the clinical characteristics of phlebolymphedema and its correlations.

Reversal of Hypertension and Amelioration of Oxidative Stress by Persea americana and Allium sativum in Experimentally-Induced Hypertensive Wistar Rats

Aim: This study evaluated the antihypertensive and antioxidant effects of hexane, ethyl acetate and methanol extracts of Persea americana and Allium sativum in experimentally-induced hypertensive Wistar rats. Study Design: Experimental Research. Place and Duration of Study: Department of Veterinary Pharmacology and Toxicology, University of Ibadan (Animal House), between May 2019 and December 2019. Methodology: The experiment was carried out in 85 rats randomly divided into 17 groups. Group 1 were normotensive rats while hypertension was induced in groups 2-16 by unilateral nephrectomy and inclusion of NaCl (1%) in drinking water. Group 17 had abdominal incision without nephrectomy (sham). Treatment groups were administered P. americana or A. sativum extracts at 20 mg/kg or 50 mg/kg dose and standard antihypertensives; lisinopril or hydrochlorothiazide. Results: Results of the experiment showed treatment of hypertensive rats with 50 mg/kg of A. sativum hexane and P. americana methanol extract caused the most significant decrease in blood pressure compared to normotensive rats. Various extracts of these two plants elevated antioxidants levels (GPx, GST, GSH and SOD) in the brain, heart, kidney and liver significantly while H2O2 and MDA were significantly decreased compared to untreated hypertensive rats. NO, an important neurotransmitter for normal endothelial function was also restored in the extract-treated rat, as a deficiency contributes greatly to the development of hypertension. Conclusion: The study concluded that P. americana and A. sativum do not only lower blood pressure. The plants also inhibited generation of free radicals by enhancing the antioxidant system and mopped up generated free radicals demonstrated by decline in H2O2 and MDA levels. These plants have been shown in this study to contain potential drug candidates which can be proposed for treatment of hypertension.

Role of cyclooxygenases 1 and 2 in the maintenance of colonic mucosal integrity in an experimental colitis model

The role of cyclooxygenase (COXs) isoforms in maintaining colonic mucosal integrity is not fully understood. This study aimed to evaluate the role of COX-1 and -2 on colonic mucosal integrity in an experimental colitis model. Colitis was induced in Wistar rats by intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (20 mg + 50% ethanol). The control group (sham group) received saline only. After 7, 14, or 28 days, colonic samples were removed, and macroscopic lesion scores, wet weight, myeloperoxidase activity, and transepithelial electrical resistance (TER) were determined. In other rat groups, colonic samples from the sham group and a 7th day post-colitis group were mounted in Üssing chambers with the luminal side exposed to a buffer solution (control), acetylsalicylic acid (ASA), SC-560 (COX-1 inhibitor), or celecoxib (COX-2 inhibitor). TER and epithelial permeability to fluorescein were measured. The 7th day colitis group had higher macroscopic damage scores, wet weight, and myeloperoxidase activity and lower basal TER than the sham, 14th day colitis, and 28th day colitis groups. Inhibition of COX-1 but not COX-2 significantly decreased TER and increased permeability to fluorescein in the 7th day post-colitis group compared to the sham group. Additionally, ASA decreased the colonic mucosal integrity on day seven post-colitis compared to the sham group. A decrease in the colonic mucosa integrity in the experimental colitis model can be aggravated only by the inhibition of COX-1, which demonstrated the importance of this enzyme in the maintenance of colonic mucosal integrity.

Cellulite cervico-faciale à l'hôpital national Ignace Deen de Conakry: résultats d'une étude de 5 ans / Cervico-facial cellulitis at the Ignace Deen hospital in Conakry: results of a 5-year study

Introduction : La cellulite cervico-faciale est une diffusion cellulaire d'un foyer infectieux vers les tissus cellulo-adipeux du cou et de la face, avec une prédominance des anaérobies et des germes commensaux de la flore orale. L'objectif de ce travail était de contribuer à l'amélioration de la prise en charge de cellulites cervico-faciales à l'Hôpital National Ignace Deen. Méthodes : Il s'est agi d'une étude rétro-prospective de type descriptif sur une période de cinq (5) ans (01 janvier 2017 au 31 décembre 2021) réalisée aux services d'ORLCCF et d'Odontostomatologie/CMF de l'Hôpital National Ignace Deen de Conakry. Résultats : 97 patients qui présentaient une cellulite cervico-faciale, soit une fréquence de 4,36% ont été inclus. La tranche d'âge de 21 à 30 ans était la plus représentée soit 40,21%. L'âge moyen était de 33 ,15 ± 13,97 ans avec des extrêmes de 1 et 65 ans. Le sex-ratio était de 1,30. La tuméfaction douloureuse était le principal motif de consultation soit 98,97%. Les Anti-Inflammatoires Non Stéroïdiens étaient le facteur le plus présent chez 85 patients soit 78,35%. L'état général était peu satisfaisant chez 94 patients soit 96,91%. Une cellulite suppurée a été retrouvée chez 82,47% patients. Le traitement était médico-chirurgical chez 85 patients soit 87,63%. L'évolution était bonne chez 90 patients soit 92,78%. Conclusion : La cellulite cervico-faciale est une urgence médicochirurgicale qu'il faut savoir diagnostiquer et prendre en charge dans les meilleurs délais. Non traitée, elle peut engager le pronostic vital.

Introduction: Cervico-facial cellulitis is a cellular diffusion of an infection to the cellulo-adipose tissues of the neck and face, with a predominance of anaerobic and commensal germs of oral flora. The objective of this work was to contribute improving the management of cervicofacial cellulitis at Ignace Deen National Hospital. Methods: This was a five (5) year (01 January 2017 to 31 December 2021) descriptive retro-prospective study conducted at the ENT-RTC and Odontostomatology / FJA departments of the Ignace Deen National Hospital in Conakry. Results: 97 patients who presented cervico-facial cellulitis, i.e. a frequency of 4.36%, were included. The 21- 30 age group was the most represented, either 40.21%. The average age was 33.15 ± 13.97 years with extremes of 1 and 65 years. The sex ratio was 1.30. Painful swelling was the main reason for consultation, 98.97%. Non- Steroidal Anti-inflammatory Drugs were the most common factor in 85 of our patients, either 78.35%. The overall condition was unsatisfactory in 94 of our patients, either 96.91%. Suppurated cellulitis was found in 82.47% of our patients. Treatment was medico-surgical in 85 patients, either 87.63%. The evolution was good in 90 patients, either 92.78%. Conclusion: Cervico-facial cellulitis is a medical and surgical emergency that must be diagnosed and managed as soon as possible. If left untreated, it can trigger a vital prognosis.

Trachéotomie chirurgicale chez l'enfant au centre hospitalier universitaire de Bouaké à propos de 32 cas / Surgical tracheotomy in children at the university hospital center of Bouake about 32 cases

Objectif : déterminer les indications de la trachéotomie chirurgicale chez l'enfant, décrire la technique et les suites opératoires. Matériel et méthode : il s'agit d'une étude descriptive rétrospective portant sur les trachéotomies pédiatriques réalisées au CHU de Bouaké de janvier 2016 à décembre 2022. Les indications de la trachéotomie, la technique opératoire et l'évolution ont été étudiées. Le test exact de Fisher a été utilisé pour évaluer le lien entre le motif de la trachéotomie et les pathologies au seuil α de 5%. Résultats : pendant la période d'étude, 32 enfants ont été trachéotomisés. Leur âge variait entre 9 mois et 15 ans avec un âge moyen de 7,2 ans. Il s'agissait de 22 garçons et 10 filles. L'impossibilité d'intuber le patient (65,62%) et la dyspnée laryngée sévère (25%) constituaient les motifs de la trachéotomie. Les corps étrangers laryngo-trachéaux et la papillomatose laryngée représentaient respectivement 37,5% et 21,87% des pathologies. Nous n'avons pas noté de lien significatif entre le motif de la trachéotomie et les pathologies (p=1,000). L'intervention a été réalisée sous sédation dans 78,12% des cas. L'ouverture trachéale était en forme de « H ¼ et de siège sous-isthmique. Les suites opératoires étaient simples chez 93,75% des patients. La décanulation a été effective dans 24 cas (75%). Conclusion : les principaux motifs de la trachéotomie dans notre expérience sont l'impossibilité d'intuber le patient et la dyspnée laryngée sévère. Les pathologies étaient dominées par les corps étrangers laryngo-trachéaux et la papillomatose laryngée. Les suites opératoires étaient généralement satisfaisantes.

Objective: to determine the indications for surgical tracheotomy in children, to describe the technique and the postoperative course. Material and method: This is a retrospective descriptive study of pediatric tracheotomies performed at the Bouaké University Hospital from January 2016 to December 2022. The indications for tracheotomy, the operating technique and the evolution were studied. Fisher's exact test was used to assess the link between the reason for the tracheotomy and the pathologies at the α threshold of 5%. Results: during the study period, 32 children were tracheotomized. Their age varied between 9 months and 15 years with an average age of 7.2 years. They were 22 boys and 10 girls. The impossibility of intubating the patient (65.62%) and severe laryngeal dyspnea (25%) were the reasons for the tracheotomy. Laryngo-tracheal foreign bodies and laryngeal papillomatosis accounted for 37.5% and 21.87% of pathologies respectively. We did not note any significant link between the reason for the tracheotomy and the pathologies (p=1,000). The intervention was performed under sedation in 78.12% of cases. The tracheal opening was "H" shaped and sub-isthmic. The postoperative course was simple in 93.75% of patients. Decanulation was effective in 24 cases (75%). Conclusion: the main reasons for tracheotomy in our experience are the impossibility of intubating the patient and severe laryngeal dyspnea. The pathologies were dominated by laryngo-tracheal foreign bodies and laryngeal papillomatosis. The postoperative course was generally satisfactory

Knowledge and management of WEEE by independent repairers in the city of Bouake in a context of health and environmental risk, 2022

Studies on waste management in Côte d'Ivoire are mostly focused on household and solid waste. It is appropriate to take a sustained look at the management of electrical and electronic equipment waste to better guide policies in this area. This work is a cross-sectional study covering the period from April 1, 2022 to June 30, 2022. The aim was to determine the knowledge and practices of the independent electrical and electronic repairers of the city of Bouaké in terms of electrical and electronic equipment waste (WEEE) management. A comprehensive sample of 307 people was interviewed by means of a questionnaire. The repairers interviewed had already heard of WEEE in 47.2% of cases. They had a good level of knowledge of WEEE in 85.5% of cases. In the final model in multivariate analysis, only the possession of a radio set at home had a significant influence on knowledge of WEEE. Repairers were unaware of the existence of D3E management legislation in 60.7% of cases; apart from humans (84.8%) who were most exposed to the dangers of inappropriate management of WEEE, other entities (air, soil, water, animals) would be polluted according to 36.6, 35.2, 4.1, and 3.4% of repairers, respectively. The most feared type of harm reported is injury (95.65%). Subjects dumped WEEE in a municipal landfill in 32.4% of cases. Sale, empowerment of pre-collection agents, and handing over to a recycling professional, cumulatively represent the most common practice (66.9%).

Acaricidal, larvacidal, and repellent activity of green synthesized silver nanoparticles against Hyalomma dromedarii

@#We aimed at determination of acaricidal, larvacidal, and repellent activities of green synthesized silver nanoparticles (SNP) against Hyalomma dromedarii as one of the most common ticks in camels. SNP were green synthesized by reducing Lupinus albus extract through the precipitation technique. The acaricidal, larvicidal, and repellent activity of SNP against H. dromedarii was studied through the adult immersion test (AIT), the larval packet test (LPT), the vertical movement behavior of tick’s larvae method, anti-acetylcholinesterase (AChE) activity, and oxidative enzyme activity. The green synthesized SNP displayed a spherical form with a size ranging from 25–90 nm; whereas the most distribution of particles size was reported at 50-65 nm. SNP dose-dependently (p<0.001) increased the mortality rate of H. dromedarii adult; whereas at 16 and 32 µg/mL completely killed the adult females. Treatment of exposure of H. dromedarii adult to SNP markedly (p<0.001) declined the mean number, weight, and hatchability of eggs. Treatment of H. dromedarii larvae with SNP reduced the viability rate of larvae with the LC50 and LC90 values of 3.1 and 6.9 µg/mL, respectively. Exposure of H. dromedarii larvae to SNP, especially at ½ LC50 and LC50, markedly (p<0.001) increased the oxidative stress and declined the level of antioxidant enzymes in H. dromedarii larvae; whereas, markedly suppressed the AChE activity of the larvae stage of H. dromedarii in comparison to the control group. These results showed that SNP green synthesized by L. albus extract had promising acaricidal, larvicidal and repellent activity against H. dromedarii adults and larvae as a dose-dependent response. SNP also considrably decreased the level of acetylcholinesterase and antioxidant activity and also provokes oxidative stress in H. dromedarii larvae. However, more investigation must be designed to clear the accurate mechanisms and the efficacy of SNP in practical use.

Antileishmanial effects, cellular mechanisms, and cytotoxicity of Elettaria cardamomum essential oil against Leishmania major infection

@#Leishmaniasis is an infectious disease with various clinical manifestations. We studied the therapeutic effects of Elettaria cardamomum essential oil (ECEO) against Leishmania major infection. In vitro effects of ECEO against L. major were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and macrophage assays. Nitric oxide (NO) production, infection inhibition in macrophages, and the apoptotic activity of ECEO in treated parasites were also measured. By calculating the 50% cytotoxic concentrations (CC50), we studied the cytotoxicity effects of ECEO on human macrophage cells (THP-1). The efficacy of ECEO for improving cutaneous leishmaniasis (CL) lesions in mice (BALB/c) was determined by evaluating the size of lesions and the number of amastigotes before and after four weeks of treatment. The effects of ECEO on liver and kidney function in the tested mice were also evaluated. ECEO dose-dependently (p<0.001) inhibited the viability and the mean number of promastigotes and amastigote forms of L. tropica. Four weeks of treatment with ECEO at the doses of 2.5 and 5 mg/kg/ day significantly (p<0.001) improved the CL lesions and reduced the number of parasites in the infected mice. ECEO significantly increased NO production, apoptosis induction, and infection rate in parasites. The CC50 value for ECEO and MA was 303.4 µg/mL and 835.2 µg/mL, respectively. In the mice receiving ECEO at the doses of 2.5 and 5 mg/kg/day for 28 days, no significant change was reported between the serum level of liver enzymes and kidney factors when compared with the control group. ECEO displayed promising efficacy in parasite reduction in vitro and in the animal model. ECEO can thus be used as an alternative medicine to treat CL.

Mortality of the cotton boll weevil in drip and sprinkler irrigated cotton crops

Abstract The cotton boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is a key cotton crop pest in Brazil. Adverse climatic factors, such as high temperatures and low soil moisture, dehydrate oviposited cotton squares (bud flowers) on the ground and cause high mortality of its offspring within these plant structures. The objective of this research was to evaluate the mortality of the cotton boll weevil in drip and sprinkler irrigated cotton crops. The experimental design was in randomized blocks with two treatments: drip (T1) and sprinkler (T2, control) irrigated cotton crops with sixteen replications. Each parcel had one emergence cage, installed between two cotton rows per irrigation system, with 37 cotton squares with opened oviposition punctures and yellowish bracts, to capture adult cotton boll weevils. The average number of boll weevils that emerged from the cotton squares and the causes of mortality at different development stages were determined per treatment. Third-generation life tables of the boll weevil were prepared using the natural mortality data in drip and sprinkler irrigation treatments and plus actual, apparent and indispensable mortality rates and the lethality of each mortality cause. We conclude that the application of water directly to the root zone of the plants in a targeted manner, using the drip irrigation system, can cause high mortality of the cotton boll weevil immature stages inside cotton squares fallen on the ground. This is because the cotton squares fallen on the drier and hotter soil between the rows of drip-irrigated cotton dehydrates causing the boll weevils to die. This is important because it can reduce its population density of the pest and, consequently, the number of applications of chemical insecticides for its control. Thus, contributing to increase the viability of cotton production, mainly in areas of the Brazilian semiarid region where the cotton is cultivated in organic system.

Resumo O bicudo-do-algodoeiro, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), é uma praga-chave da cultura do algodão no Brasil. Fatores climáticos adversos, como altas temperaturas e baixa umidade do solo, desidratam os botões florais de algodão ovipositados caídos ao solo e causam alta mortalidade de seus descendentes dentro dessas estruturas vegetais.O objetivo desta pesquisa foi avaliar a mortalidade do bicudo-do-algodoeiro em lavouras de algodão irrigadas por gotejamento e aspersão. O delineamento experimental foi em blocos casualizados com dois tratamentos: cultivo de algodão irrigado por gotejamento (T1) e aspersão (T2, testemunha) com dezesseis repetições. Cada parcela possuía uma gaiola de emergência, instalada entre duas fileiras de algodão por sistema de irrigação, com 37 botões florais de algodão com orifícios de oviposição, brácteas abertas e amarelecidas, para captura do bicudo adulto. O número médio de bicudos que emergiu dos botões florais de algodão e as causas de mortalidade, em diferentes estágios de desenvolvimento, foram determinados por tratamento. As tabelas de vida de terceira geração do bicudo-do-algodoeiro foram preparadas usando os dados de mortalidade natural em tratamentos de irrigação por gotejamento e aspersão e as taxas de mortalidade reais, aparentes e indispensáveis e a letalidade de cada causa de mortalidade. Concluímos que a aplicação de água diretamente na zona radicular das plantas de forma direcionada, utilizando o sistema de irrigação por gotejamento, pode causar altas mortalidade dos estágios imaturos do bicudo-do-algodoeiro nos botões florais de algodão caídos no solo. Isso ocorre porque os botões florais de algodão caídos no solo mais seco e mais quente entre as fileiras do algodão irrigado por gotejamento desidratam, causando a morte dos bicudos. Isso é importante por poder reduzir a densidade populacional desse inseto e, consequentemente, o número de aplicações de inseticidas químicos para seu controle. Assim, contribuindo para aumentar a viabilidade da produção de algodão, principalmente em áreas do semiárido brasileiro em sistema orgânico.

Do fall armyworms Metaflumizone resistante populations affect the activity of Trichogramma pretiosum?

Abstract The possible interference of resistant pests populations to insecticides in natural enemies in the action thas not been clarified yet. Thus, this study aimed to evaluate Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) performance on Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) eggs with resistance frequency to the Metaflumizone over six generations of product exposure. Egg cards (2.0 x 7.0 cm) containing eggs from two populations of S. frugiperda, (resistant to Metaflumizone and the other susceptible), were exposed to T. pretiosum females for 24 hours in free-choice and no-choice testing in three generations (G1, G4, and G6). A completely randomized experimental design was used with 25 replications, each consisting of an egg card (experimental unit) containing 20 eggs. The parameters evaluated were: parasitism (%), emergence (%), sex ratio, number of emerged parasitoids per egg and males/females longevity. ANOVA and Tukey test (P 0.05) were applied on the results. Results showed a reduction in parasitism [41.0% (G1) and 28.4% (G4)], egg emergence (17.5%) and parasitoids/egg [16.2 (G4) and 17.2 (G6)] in eggs originating from the population with resistance frequency. Females emerging from G6 populations eggs without exposure to Metaflumizone had greater longevity (3.5 days more) than the resistant population. The sex ratio and male longevity were not affected. The results indicate a reduction in T. pretiosum activity if S. frugiperda populations have some frequency of resistance to Metaflumizone.

Resumo A possível interferência de populações de pragas resistentes na ação de inimigos naturais ainda não foi esclarecida. Assim, este trabalho teve como objetivo avaliar o desempenho de Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) em ovos de Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) com frequência de resistência à Metaflumizona ao longo de seis gerações de exposição ao produto. Cartelas (2,0 x 7,0 cm) com ovos de duas populações de S. frugiperda, (resistente à Metaflumizona e outra suscetível), foram expostas às fêmeas de T. pretiosum por 24 horas em condições de livre escolha e sem chance de escolha por três gerações (G1, G4 e G6). O delineamento experimental foi inteiramente casualizado com 25 repetições, sendo cada repetição composta por uma cartela (unidade experimental) contendo 20 ovos. Os parâmetros avaliados foram: parasitismo (%), emergência (%), razão sexual, número de parasitoides emergidos por ovo e longevidade de machos e fêmeas. ANOVA e teste de Tukey (P 0,05) foram aplicados aos dados coletados. Os resultados mostraram redução do parasitismo [41,0% (G1) e 28,4% (G4)], emergência de ovos (17,5%) e parasitoides/ovo [16,2 (G4) e 17,2 (G6)] em ovos oriundos da população com frequência de resistência. As fêmeas emergidas de ovos da população G6 sem exposição à Metaflumizona, tiveram maior longevidade (3,5 dias a mais) do que a população exposta ao inseticida. A razão sexual e a longevidade de machos não foram afetadas. Os resultados indicam uma redução na atividade de T. pretiosum se as populações de S. frugiperda apresentarem alguma frequência de resistência à Metaflumizona.

Evaluation of indigenous plants extracts for mosquitocidal activity against different stages of Culex quinquefasciatus say (Diptera: Culicidae)

Abstract Being vector of West Nile Virus and falariasis the control of Culex quinquefasciatus is likely to be essential. Synthetic insecticide treatment is looking most effective for vectors mosquito control. However, these products are toxic to the environment and non-target organisms. Consequently, ecofriendly control of vectors mosquito is needed. In this regard botanical insecticide is looking more fruitful. Therefore, the present research aimed to investigate the effectiveness of methanolic extract and various fractions, including, n-hexane, ethyl-acetate, chloroform, and aqueous fraction, obtained from methanolic extract of Ailanthus altissima, Artemisia scoparia, and Justicia adhatoda using separating funnel against larval, pupal, and adult stages of Culex quinquefasciatus. The larvae and pupae of Culex quinquefasciatus were exposed to various concentrations (31.25-1000 ppm) of methanolic extract and its fractions for 24 hours of exposure period. For knock-down bioassay (filter paper impregnation bioassay) different concentration of the methanolic extract and its various fractions (i.e. 0.0625, 0.125, 0.25, 0.5 and 1mg/mL) were applied for 1 hour exposure period. The results were statistically analysed using standard deviation, probit analysis, and linear regression. The R2 values of larvae, pupae, and adult range from 0.4 to 0.99. The values of LC50 (concentration causing 50% mortality) for late 3rd instar larvae after 24 hours exposure period range from 93-1856.7 ppm, while LC90 values range from 424 -7635.5ppm. The values of LC50for pupae range form 1326.7-6818.4ppm and and values of LC90 range from 3667.3-17427.9ppm, respectively. The KDT50 range from 0.30 to 2.8% and KDT90 values range from1.2 to 110.8%, respectively. In conclusion, Justicia adhatoda may be effective for controlling populations of vector mosquito.

Resumo Por ser o vetor do vírus do Nilo Ocidental e da falaríase, o controle de Culex quinquefasciatus Say é provavelmente essencial. O tratamento com inseticida sintético parece ser mais eficaz para o controle dos mosquitos vetores. No entanto, esses produtos são tóxicos para o meio ambiente e organismos não visados. Consequentemente, o controle ecológico dos mosquitos vetores é necessário. Nesse sentido, o inseticida botânico parece mais produtivo. Portanto, a presente pesquisa teve como objetivo investigar a eficácia do extrato metanólico e de várias frações, incluindo n-hexano, acetato de etila, clorofórmio e fração aquosa, obtidos do extrato metanólico de Ailanthus altissima (Mill.) Swingle, Artemisia scoparia Waldst. & Kit. e Justicia adhatoda L. usando funil de separação contra os estágios larval, pupal e adulto de C. quinquefasciatus. As larvas e pupas de C. quinquefasciatus foram expostas a várias concentrações (31,25-1000 ppm) de extrato metanólico, e suas frações por 24 horas de período de exposição. Para o bioensaio knock-down (bioensaio de impregnação de papel de filtro), diferentes concentrações do extrato metanólico e suas várias frações (ou seja, 0,0625, 0,125, 0,25, 0,5 e 1 mg / mL) foram aplicadas por um período de exposição de 1 hora. Os resultados foram analisados estatisticamente usando desvio padrão, análise Probit e regressão linear. Os valores de R2 de larvas, pupas e adultos variaram de 0,4 a 0,99. Os valores de LC50 (concentração que causa 50% de mortalidade) para larvas de terceiro estádio tardio após 24 horas de período de exposição variaram de 93-1856,7 ppm, enquanto os valores de LC90 variaram de 424-7635,5ppm. Os valores de LC50 para pupas variaram de 1326,7-6818,4 ppm e os valores de LC90 variaram de 3667,3-17427,9 ppm, respectivamente. O KDT50 variou de 0,30 a 2,8% e os valores de KDT90 variaram de 1,2 a 110,8%, respectivamente. Por fim, a espécie J. adhatoda pôde ser eficaz para controlar populações de mosquitos vetores.

Evaluation of indigenous plants' extracts for mosquitocidal activity against different stages of Culex quinquefasciatus say (Diptera: Culicidae) / Avaliação de extratos de plantas indígenas quanto à atividade mosquitocida contra diferentes estágios de culex quinquefasciatus say (Diptera: Culicidae)

Abstract Being vector of West Nile Virus and falariasis the control of Culex quinquefasciatus is likely to be essential. Synthetic insecticide treatment is looking most effective for vectors mosquito control. However, these products are toxic to the environment and non-target organisms. Consequently, ecofriendly control of vectors mosquito is needed. In this regard botanical insecticide is looking more fruitful. Therefore, the present research aimed to investigate the effectiveness of methanolic extract and various fractions, including, n-hexane, ethyl-acetate, chloroform, and aqueous fraction, obtained from methanolic extract of Ailanthus altissima, Artemisia scoparia, and Justicia adhatoda using separating funnel against larval, pupal, and adult stages of Culex quinquefasciatus. The larvae and pupae of Culex quinquefasciatus were exposed to various concentrations (31.25-1000 ppm) of methanolic extract and its fractions for 24 hours of exposure period. For knock-down bioassay (filter paper impregnation bioassay) different concentration of the methanolic extract and its various fractions (i.e. 0.0625, 0.125, 0.25, 0.5 and 1mg/mL) were applied for 1 hour exposure period. The results were statistically analysed using standard deviation, probit analysis, and linear regression. The R2 values of larvae, pupae, and adult range from 0.4 to 0.99. The values of LC50 (concentration causing 50% mortality) for late 3rd instar larvae after 24 hours exposure period range from 93-1856.7 ppm, while LC90 values range from 424 -7635.5ppm. The values of LC50for pupae range form 1326.7-6818.4ppm and and values of LC90 range from 3667.3-17427.9ppm, respectively. The KDT50 range from 0.30 to 2.8% and KDT90 values range from1.2 to 110.8%, respectively. In conclusion, Justicia adhatoda may be effective for controlling populations of vector mosquito.

Resumo Por ser o vetor do vírus do Nilo Ocidental e da falaríase, o controle de Culex quinquefasciatus Say é provavelmente essencial. O tratamento com inseticida sintético parece ser mais eficaz para o controle dos mosquitos vetores. No entanto, esses produtos são tóxicos para o meio ambiente e organismos não visados. Consequentemente, o controle ecológico dos mosquitos vetores é necessário. Nesse sentido, o inseticida botânico parece mais produtivo. Portanto, a presente pesquisa teve como objetivo investigar a eficácia do extrato metanólico e de várias frações, incluindo n-hexano, acetato de etila, clorofórmio e fração aquosa, obtidos do extrato metanólico de Ailanthus altissima (Mill.) Swingle, Artemisia scoparia Waldst. & Kit. e Justicia adhatoda L. usando funil de separação contra os estágios larval, pupal e adulto de C. quinquefasciatus. As larvas e pupas de C. quinquefasciatus foram expostas a várias concentrações (31,25-1000 ppm) de extrato metanólico, e suas frações por 24 horas de período de exposição. Para o bioensaio knock-down (bioensaio de impregnação de papel de filtro), diferentes concentrações do extrato metanólico e suas várias frações (ou seja, 0,0625, 0,125, 0,25, 0,5 e 1 mg / mL) foram aplicadas por um período de exposição de 1 hora. Os resultados foram analisados ​​estatisticamente usando desvio padrão, análise Probit e regressão linear. Os valores de R2 de larvas, pupas e adultos variaram de 0,4 a 0,99. Os valores de LC50 (concentração que causa 50% de mortalidade) para larvas de terceiro estádio tardio após 24 horas de período de exposição variaram de 93-1856,7 ppm, enquanto os valores de LC90 variaram de 424-7635,5ppm. Os valores de LC50 para pupas variaram de 1326,7-6818,4 ppm e os valores de LC90 variaram de 3667,3-17427,9 ppm, respectivamente. O KDT50 variou de 0,30 a 2,8% e os valores de KDT90 variaram de 1,2 a 110,8%, respectivamente. Por fim, a espécie J. adhatoda pôde ser eficaz para controlar populações de mosquitos vetores.

Down-regulation of complement genes in lipopolysaccharidechallenged zebrafish (Danio rerio) larvae exposed to Indonesian propolis / Regulação negativa dos genes do complemento em larvas de peixe-zebra (Danio rerio) desafiadas com lipopolissacarídeo expostas à própolis indonésia

Although propolis has been reported for having anti-inflammatory activities, its effects on complement system has not been much studied. This research was conducted to find out the effects of Indonesian propolis on the expression levels of C3, C1r/s, Bf, MBL, and C6 in zebrafish larvae which were induced by lipopolysaccharide (LPS). Counting of macrophages migrating to yolk sac and liver histology were carried out. Larvae were divided into four groups: CON (cultured in E3 medium only), LPS (cultured in a medium containing 0.5 μg/L LPS), LPSIBU (cultured in a medium containing LPS, and then treated with 100 μg/L ibuprofen for 24 hours), and LPSPRO (cultured in a medium containing LPS, and then immersed in 14,000 μg/L propolis for 24 hours) groups. The results showed that complement gene expression in larvae from the LPSIBU and LPSPRO groups were generally lower than in larvae from the LPS group. The number of macrophage migrations to the yolk in the LPSPRO group was also lower than in the LPS group. Histological structure of liver in all groups were considered normal. This study shows that Indonesian propolis has the potential to be used as an alternative to the substitution of NSAIDs.

Embora a própolis tenha sido relatada por ter atividade anti-inflamatória, seus efeitos no sistema complemento, uma parte do sistema imunológico inato, não foram muito estudados. Esta pesquisa foi conduzida para descobrir os efeitos da própolis da Indonésia nos níveis de expressão de C3, C1r/s, Bf, MBL e C6 em larvas de peixe-zebra induzidas por lipopolissacarídeo (LPS). Foram realizadas contagens de macrófagos que migram para o saco vitelino e histologia do fígado. As larvas foram divididas em quatro grupos: CON (cultivadas apenas em meio E3), LPS (cultivadas em meio contendo 0,5 μg/L de LPS), LPSIBU (cultivadas em meio contendo LPS e, em seguida, tratadas com 100 μg/L de ibuprofeno por 24 horas) e LPSPRO (cultivado em meio contendo LPS, e então imerso em própolis 14,000 μg/L por 24 horas). Os resultados mostraram que a expressão do gene do complemento em larvas dos grupos LPSIBU e LPSPRO foi geralmente menor que em larvas do grupo LPS. O número de migrações de macrófagos para a gema no grupo LPSPRO também foi menor que no grupo LPS. A estrutura histológica do fígado em todos os grupos foi considerada normal. Este estudo mostra que a própolis indonésia tem potencial para ser utilizada como alternativa na substituição dos AINEs (anti-inflamatórios não esteroides).

Mortality of the cotton boll weevil in drip and sprinkler irrigated cotton crops / Mortalidade do bicudo-do-algodoeiro em lavouras de algodão irrigadas por gotejamento e aspersão

The cotton boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is a key cotton crop pest in Brazil. Adverse climatic factors, such as high temperatures and low soil moisture, dehydrate oviposited cotton squares (bud flowers) on the ground and cause high mortality of its offspring within these plant structures. The objective of this research was to evaluate the mortality of the cotton boll weevil in drip and sprinkler irrigated cotton crops. The experimental was in included randomized blocks with two treatments: drip (T1) and sprinkler (T2, control) irrigated cotton crops with sixteen replications. Each parcel had one emergence cage, installed between two cotton rows per irrigation system, with 37 cotton squares with opened oviposition punctures and yellowish bracts, to capture adult cotton boll weevils. The average number of boll weevils that emerged from the cotton squares and the causes of mortality at different development stages were determined per treatment. Third-generation life tables of the boll weevil were prepared using the natural mortality data in drip and sprinkler irrigation treatments and plus actual, apparent and indispensable mortality rates and the lethality of each mortality cause. We conclude that the application of water directly to the root zone of the plants in a targeted manner, using the drip irrigation system, can cause high mortality of the cotton boll weevil immature stages inside cotton squares fallen on the ground. This is because the cotton squares fallen on the drier and hotter soil between the rows of drip-irrigated cotton dehydrates causing the boll weevils to die. This is important because it can reduce its population density of the pest and, consequently, the number of applications of chemical insecticides for its control. Thus, contributing to increase the viability of cotton production, mainly in areas of the Brazilian semiarid region where the cotton is cultivated in organic system.

O bicudo-do-algodoeiro, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), é uma praga-chave da cultura do algodão no Brasil. Fatores climáticos adversos, como altas temperaturas e baixa umidade do solo, desidratam os botões florais de algodão ovipositados caídos ao solo e causam alta mortalidade de seus descendentes dentro dessas estruturas vegetais. O objetivo desta pesquisa foi avaliar a mortalidade do bicudo-do-algodoeiro em lavouras de algodão irrigadas por gotejamento e aspersão. O delineamento experimental foi em blocos casualizados com dois tratamentos: cultivo de algodão irrigado por gotejamento (T1) e aspersão (T2, testemunha) com dezesseis repetições. Cada parcela possuía uma gaiola de emergência, instalada entre duas fileiras de algodão por sistema de irrigação, com 37 botões florais de algodão com orifícios de oviposição, brácteas abertas e amarelecidas, para captura do bicudo adulto. O número médio de bicudos que emergiu dos botões florais de algodão e as causas de mortalidade, em diferentes estágios de desenvolvimento, foram determinados por tratamento. As tabelas de vida de terceira geração do bicudo-do-algodoeiro foram preparadas usando os dados de mortalidade natural em tratamentos de irrigação por gotejamento e aspersão e as taxas de mortalidade reais, aparentes e indispensáveis e a letalidade de cada causa de mortalidade. Concluímos que a aplicação de água diretamente na zona radicular das plantas de forma direcionada, utilizando o sistema de irrigação por gotejamento, pode causar altas mortalidade dos estágios imaturos do bicudo-do-algodoeiro nos botões florais de algodão caídos no solo. Isso ocorre porque os botões florais de algodão caídos no solo mais seco e mais quente entre as fileiras do algodão irrigado por gotejamento desidratam, causando a morte dos bicudos. Isso é importante por poder reduzir a densidade populacional desse inseto e, consequentemente, o número [...].