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1.
Plant J ; 118(4): 927-939, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38525669

RESUMEN

Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Técnicas Biosensibles , Giberelinas , Protoplastos , Transducción de Señal , Giberelinas/metabolismo , Técnicas Biosensibles/métodos , Arabidopsis/metabolismo , Arabidopsis/genética , Protoplastos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética
2.
Glob Chang Biol ; 30(6): e17367, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38840430

RESUMEN

Wildfire activity is increasing globally. The resulting smoke plumes can travel hundreds to thousands of kilometers, reflecting or scattering sunlight and depositing particles within ecosystems. Several key physical, chemical, and biological processes in lakes are controlled by factors affected by smoke. The spatial and temporal scales of lake exposure to smoke are extensive and under-recognized. We introduce the concept of the lake smoke-day, or the number of days any given lake is exposed to smoke in any given fire season, and quantify the total lake smoke-day exposure in North America from 2019 to 2021. Because smoke can be transported at continental to intercontinental scales, even regions that may not typically experience direct burning of landscapes by wildfire are at risk of smoke exposure. We found that 99.3% of North America was covered by smoke, affecting a total of 1,333,687 lakes ≥10 ha. An incredible 98.9% of lakes experienced at least 10 smoke-days a year, with 89.6% of lakes receiving over 30 lake smoke-days, and lakes in some regions experiencing up to 4 months of cumulative smoke-days. Herein we review the mechanisms through which smoke and ash can affect lakes by altering the amount and spectral composition of incoming solar radiation and depositing carbon, nutrients, or toxic compounds that could alter chemical conditions and impact biota. We develop a conceptual framework that synthesizes known and theoretical impacts of smoke on lakes to guide future research. Finally, we identify emerging research priorities that can help us better understand how lakes will be affected by smoke as wildfire activity increases due to climate change and other anthropogenic activities.


Asunto(s)
Ecosistema , Lagos , Humo , Incendios Forestales , Humo/análisis , América del Norte , Monitoreo del Ambiente
3.
Rev Esp Enferm Dig ; 2024 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-38501803

RESUMEN

This paper describes the clinical case of a patient diagnosed with diffuse large B-cell lymphoma with splenic involvement, focusing on the role of abdominal ultrasound (US) in this context. The patient experienced dyspepsia and progressive asthenia over several months so an abdominal US was performed. The US showed multiple heterogeneous and hypoechogenic focal splenic lesions together with a moderate left pleural effusion. Lymphoma was suspected due to these findings and the diagnosis was then confirmed with the performance of a CT scan and a biopsy that revealed the presence of a diffuse large B-cell lymphoma with metastatic disease. Chemotherapy containing rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone was administered and the patient is currently on clinical remission. This study highlights the role of abdominal ultrasound as a useful technique in the non-invasive assessment of this entity, considering an appropriate clinical context and mainly after detecting multiple hypoechogenic splenic lesions in the US.

4.
Nat Methods ; 17(7): 717-725, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32601426

RESUMEN

Optogenetics is the genetic approach for controlling cellular processes with light. It provides spatiotemporal, quantitative and reversible control over biological signaling and metabolic processes, overcoming limitations of chemically inducible systems. However, optogenetics lags in plant research because ambient light required for growth leads to undesired system activation. We solved this issue by developing plant usable light-switch elements (PULSE), an optogenetic tool for reversibly controlling gene expression in plants under ambient light. PULSE combines a blue-light-regulated repressor with a red-light-inducible switch. Gene expression is only activated under red light and remains inactive under white light or in darkness. Supported by a quantitative mathematical model, we characterized PULSE in protoplasts and achieved high induction rates, and we combined it with CRISPR-Cas9-based technologies to target synthetic signaling and developmental pathways. We applied PULSE to control immune responses in plant leaves and generated Arabidopsis transgenic plants. PULSE opens broad experimental avenues in plant research and biotechnology.


Asunto(s)
Regulación de la Expresión Génica de las Plantas , Luz , Optogenética , Arabidopsis/genética , Arabidopsis/inmunología , Sistemas CRISPR-Cas/genética , Modelos Teóricos , Plantas Modificadas Genéticamente
5.
Ecotoxicology ; 31(6): 1035-1043, 2022 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-35831720

RESUMEN

Chloroquine (CQ) has been widely used for many years against malaria and various viral diseases. Its important use and high potential to being persistent make it of particular concern for ecotoxicological studies. Here, we evaluated the toxicity of CQ alone and in combination with copper (Cu) to the euryhaline rotifer Proales similis. All experiments were carried out using chronic toxicity reproductive five-day tests and an application factor (AF) of 0.05, 0.1, 0.3, and 0.5 by multiplying the 24-h LC50 values of CQ (4250 µg/L) and Cu (68 µg/L), which were administered in solution. The rate of population increase (r, d-1) ranged from 0.50 to 52 (controls); 0.20 to 0.40 (CQ); 0.09 to 0.43 (Cu); and -0.03 to 0.30 (CQ-Cu) and showed significant decrease as the concentration of both chemicals in the medium increased. Almost all tested mixtures induced synergistic effects, mainly as the AF increased. We found that the presence of Cu intensifies the vulnerability of organisms to CQ and vice versa. These results stress the potential hazard that these combined chemicals may have on the aquatic systems. This research suggests that P. similis is sensitive to CQ as other standardized zooplankton species and may serve as a potential test species in the risk assessment of emerging pollutants in marine environments.


Asunto(s)
Cobre , Rotíferos , Animales , Cloroquina/toxicidad , Cobre/toxicidad , Dosificación Letal Mediana , Pruebas de Toxicidad Crónica
6.
Int J Mol Sci ; 23(22)2022 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-36430856

RESUMEN

Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune and degenerative disease with axonal damage and demyelination as its main features. Its dual neurological and autoimmune nature makes it a disease that is difficult to treat. Treatments that simultaneously stop the immune response while protecting and repairing the nervous system are urgent. That is of utmost importance for the primary progressive multiple sclerosis (PPMS), a rare and severe variant of MS, characterized by worsening neurological function from the onset of symptoms. In this sense, inhibitors of glycogen synthase kinase 3ß (GSK3ß) and phosphodiesterase 7 (PDE7) have recently shown great therapeutic potential for the treatment of demyelinating diseases. Here we investigated a dual inhibitor of these two targets, the small molecule VP3.15, in a preclinical model, which resembles primary-progressive MS (PPMS), the Theiler's mouse encephalomyelitis virus-induced demyelinated disease (TMEV-IDD). In our study, VP3.15 ameliorates the disease course improving motor deficits of infected mice. Chronic treatment with VP3.15 also showed significant efficacy in the immunomodulation process, as well as in the proliferation and differentiation of oligodendroglial precursors, improving the preservation of myelin and axonal integrity. Therefore, our results support a treatment with the safe VP3.15 as an integrative therapeutic strategy for the treatment of PPMS.


Asunto(s)
Esclerosis Múltiple Crónica Progresiva , Esclerosis Múltiple , Theilovirus , Animales , Ratones , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7 , Esclerosis Múltiple/tratamiento farmacológico , Glucógeno Sintasa Quinasa 3 beta , Esclerosis Múltiple Crónica Progresiva/tratamiento farmacológico , Modelos Animales de Enfermedad
7.
Crit Care ; 25(1): 369, 2021 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-34774087

RESUMEN

BACKGROUND: Extracorporeal cardiopulmonary resuscitation (E-CPR) is used for the treatment of refractory cardiac arrest. However, the optimal target to reach for mean arterial pressure (MAP) remains to be determined. We hypothesized that MAP levels critically modify cerebral hemodynamics during E-CPR and tested two distinct targets (65-75 vs 80-90 mmHg) in a porcine model. METHODS: Pigs were submitted to 15 min of untreated ventricular fibrillation followed by 30 min of E-CPR. Defibrillations were then delivered until return of spontaneous circulation (ROSC). Extracorporeal circulation was initially set to an average flow of 40 ml/kg/min. The dose of epinephrine was set to reach a standard or a high MAP target level (65-75 vs 80-90 mmHg, respectively). Animals were followed during 120-min after ROSC. RESULTS: Six animals were included in both groups. During E-CPR, high MAP improved carotid blood flow as compared to standard MAP. After ROSC, this was conversely decreased in high versus standard MAP, while intra-cranial pressure was superior. The pressure reactivity index (PRx), which is the correlation coefficient between arterial blood pressure and intracranial pressure, also demonstrated inverted patterns of alteration according to MAP levels during E-CPR and after ROSC. In standard-MAP, PRx was transiently positive during E-CPR before returning to negative values after ROSC, demonstrating a reversible alteration of cerebral autoregulation during E-CPR. In high-MAP, PRx was negative during E-CPR but became sustainably positive after ROSC, demonstrating a prolonged alteration in cerebral autoregulation after ROSC. It was associated with a significant decrease in cerebral oxygen consumption in high- versus standard-MAP after ROSC. CONCLUSIONS: During early E-CPR, MAP target above 80 mmHg is associated with higher carotid blood flow and improved cerebral autoregulation. This pattern is inverted after ROSC with a better hemodynamic status with standard versus high-MAP.


Asunto(s)
Presión Arterial , Reanimación Cardiopulmonar , Circulación Cerebrovascular , Oxigenación por Membrana Extracorpórea , Animales , Presión Arterial/fisiología , Reanimación Cardiopulmonar/métodos , Circulación Cerebrovascular/fisiología , Hemodinámica , Porcinos , Resultado del Tratamiento
8.
Int J Mol Sci ; 22(5)2021 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-33671012

RESUMEN

The need for remyelinating drugs is essential for healing disabling diseases such as multiple sclerosis (MS). One of the reasons for the lack of this class of therapies is the impossibility to monitor remyelination in vivo, which is of utmost importance to perform effective clinical trials. Here, we show how optical coherence tomography (OCT), a cheap and non-invasive technique commonly used in ophthalmology, may be used to assess remyelination in vivo in MS patients. Our pioneer approach validates OCT as a technique to study remyelination of the optic nerve and reflects what is occurring in non-accessible central nervous system (CNS) structures, like the spinal cord. In this study we used the orally bioavailable small molecule VP3.15, confirming its therapeutical potential as a neuroprotective, anti-inflammatory, and probably remyelinating drug for MS. Altogether, our results confirm the usefulness of OCT to monitor the efficacy of remyelinating therapies in vivo and underscore the relevance of VP3.15 as a potential disease modifying drug for MS therapy.


Asunto(s)
Esclerosis Múltiple/tratamiento farmacológico , Nervio Óptico/efectos de los fármacos , Remielinización , Bibliotecas de Moléculas Pequeñas/farmacología , Tomografía de Coherencia Óptica/métodos , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/diagnóstico por imagen , Esclerosis Múltiple/patología , Neuroprotección , Nervio Óptico/diagnóstico por imagen , Nervio Óptico/patología
9.
New Phytol ; 225(1): 250-267, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31487399

RESUMEN

The key basic helix-loop-helix (bHLH) transcription factor in iron (Fe) uptake, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), is controlled by multiple signaling pathways, important to adjust Fe acquisition to growth and environmental constraints. FIT protein exists in active and inactive protein pools, and phosphorylation of serine Ser272 in the C-terminus, a regulatory domain of FIT, provides a trigger for FIT activation. Here, we use phospho-mutant activity assays and study phospho-mimicking and phospho-dead mutations of three additional predicted phosphorylation sites, namely at Ser221 and at tyrosines Tyr238 and Tyr278, besides Ser 272. Phospho-mutations at these sites affect FIT activities in yeast, plant, and mammalian cells. The diverse array of cellular phenotypes is seen at the level of cellular localization, nuclear mobility, homodimerization, and dimerization with the FIT-activating partner bHLH039, promoter transactivation, and protein stability. Phospho-mimicking Tyr mutations of FIT disturb fit mutant plant complementation. Taken together, we provide evidence that FIT is activated through Ser and deactivated through Tyr site phosphorylation. We therefore propose that FIT activity is regulated by alternative phosphorylation pathways.


Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Bioensayo/métodos , Mutación/genética , Secuencia de Aminoácidos , Animales , Proteínas de Arabidopsis/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Células CHO , Cricetinae , Cricetulus , Modelos Biológicos , Fosforilación , Fosfotirosina/metabolismo , Multimerización de Proteína , Estabilidad Proteica , Activación Transcripcional/genética
10.
Cytokine ; 126: 154913, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31731048

RESUMEN

Given the role of host defense peptides (HDPs) in the defensive response against mycobacteria, we analyzed the circulating levels of LL-37, ß-defensin-2 and -3 in newly diagnosed patients with pulmonary (PTB) or pleural tuberculosis (PLTB) in whom measurements of pleural fluids were also performed. Severe PTB patients displayed higher circulating amounts of ß-defensin-3, statistically different from controls, further decreasing upon antimycobacterial treatment. LL-37 concentrations appeared within the normal range at diagnosis, but tended to increase during treatment, becoming statistically upon its completion in moderate cases. PLTB patients revealed decreased levels of ß-defensin-2 in presence of increased amounts of ß-defensin-3 and LL-37; in their plasma or pleural fluids. Considering the immune-endocrine dysregulation of tuberculosis, we also performed correlation analysis detecting positive associations between levels of cortisol, IL-6 and ß-defensin-3 in plasma from untreated severe patients as did their dehydroepiandrosterone and LL-37 values. Increased presence of ß-defensins, may represent an attempt to improve defensive mechanisms; which also take part in the inflammatory reaction accompanying TB, reinforced by the association with immune-endocrine mediators. The divergent profile of PLTB patients, decreased ß-defensin-2 but increased ß-defensin-3 and LL-37 levels, suggests a differential role of these HDPs in a situation characterized for its better protective response.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/sangre , Mycobacterium tuberculosis/inmunología , Tuberculosis Pleural/patología , Tuberculosis Pulmonar/patología , beta-Defensinas/sangre , Adulto , Deshidroepiandrosterona/sangre , Femenino , Humanos , Hidrocortisona/sangre , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Tuberculosis Pleural/sangre , Tuberculosis Pulmonar/sangre , Adulto Joven , Catelicidinas
11.
Nucleic Acids Res ; 45(4): 2196-2209, 2017 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-28053117

RESUMEN

Modular DNA assembly simplifies multigene engineering in Plant Synthetic Biology. Furthermore, the recent adoption of a common syntax to facilitate the exchange of plant DNA parts (phytobricks) is a promising strategy to speed up genetic engineering. Following this lead, here, we present a platform for plant biodesign that incorporates functional descriptions of phytobricks obtained under pre-defined experimental conditions, and systematically registers the resulting information as metadata for documentation. To facilitate the handling of functional descriptions, we developed a new version (v3.0) of the GoldenBraid (GB) webtool that integrates the experimental data and displays it in the form of datasheets. We report the use of the Luciferase/Renilla (Luc/Ren) transient agroinfiltration assay in Nicotiana benthamiana as a standard to estimate relative transcriptional activities conferred by regulatory phytobricks, and show the consistency and reproducibility of this method in the characterization of a synthetic phytobrick based on the CaMV35S promoter. Furthermore, we illustrate the potential for combinatorial optimization and incremental innovation of the GB3.0 platform in two separate examples, (i) the development of a collection of orthogonal transcriptional regulators based on phiC31 integrase and (ii) the design of a small genetic circuit that connects a glucocorticoid switch to a MYB/bHLH transcriptional activation module.


Asunto(s)
Biología Computacional/métodos , ADN de Plantas , Plantas/genética , Plantas/metabolismo , Programas Informáticos , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Regiones Promotoras Genéticas , Protoplastos/metabolismo , Transcripción Genética , Interfaz Usuario-Computador , Navegador Web
12.
J Basic Microbiol ; 59(1): 38-53, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30320901

RESUMEN

Quorum-sensing (QS) mechanisms are important in intra- and inter-specific communication among bacteria. We investigated QS mechanisms in Bradyrhizobium japonicum strain CPAC 15 and Azospirillum brasilense strains Ab-V5 and Ab-V6, used in commercial co-inoculants for the soybean crop in Brazil. A transconjugant of CPAC 15-QS with partial inactivation of N-acyl-homoserine lactones (AHLs) was obtained and several parameters were evaluated; in vitro, CPAC 15 and the transconjugant differed in growth, but not in biofilm formation, and no differences were observed in the symbiotic performance in vivo. The genome of CPAC 15 carries functional luxI and luxR genes and low amounts of three AHL molecules were detected: 3-OH-C12-AHL, 3-OH-C14-AHL, and 3-oxo-C14-AHL. Multiple copies of luxR-like genes, but not of luxI are present in the genomes of Ab-V5 and Ab-V6, and differences in gene expression were observed when the strains were co-cultured with B. japonicum; we may infer that the luxR-genes of A. brasilense may perceive the AHL molecules of B. japonicum. Soybean symbiotic performance was improved especially by co-inoculation with Ab-V6, which, contrarily to Ab-V5, did not respond to the AHLs of CPAC 15. We concluded that A. brasilense Ab-V5, but not Ab-V6, responded to the QS signals of CPAC 15, and that the synergistic interaction may be credited, at least partially, to the QS interaction. In addition, we confirmed inter- and intra-species QS communication between B. japonicum and A. brasilense and, for Azospirillum, at the strain level, impacting several steps of the symbiosis, from cell growth to plant nodulation and growth.


Asunto(s)
Acil-Butirolactonas/metabolismo , Azospirillum brasilense/fisiología , Bradyrhizobium/fisiología , Glycine max/microbiología , Interacciones Microbianas/fisiología , Percepción de Quorum/fisiología , Simbiosis/fisiología , Azospirillum brasilense/genética , Azospirillum brasilense/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Bradyrhizobium/genética , Bradyrhizobium/crecimiento & desarrollo , Brasil , Técnicas de Cocultivo , Regulación Bacteriana de la Expresión Génica , Percepción de Quorum/genética , Proteínas Represoras/genética , Simbiosis/genética , Transactivadores/genética , Factores de Transcripción/genética
13.
Rev Esp Enferm Dig ; 111(6): 467-470, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-31117799

RESUMEN

INTRODUCTION AND OBJECTIVES: quadruple therapy with bismuth is recommended as a first line treatment for Helicobacter pylori (H. pylori) infection. The aim of this study was to evaluate the compliance, adverse effects and effectiveness of this treatment with the new galenic three-in-one capsule formulation containing bismuth subcitrate, metronidazole and tetracycline (Pylera®). METHODS: a prospective, non-controlled, single center observational study was performed in a cohort of 200 consecutive patients with an untreated H. pylori infection; 58% were female. The subjects were treated for ten days with Pylera® of three capsules four times daily with meals and a proton pump inhibitor taken before breakfast and dinner. The Pylera® capsule contains 140 mg of bismuth subcitrate, 125 mg of metronidazole and 125 mg of tetracycline. The compliance and adverse effects of the treatment were evaluated via telephone contact and via an interview during the clinical revision. Eradication of infection was controlled for at least four weeks after treatment termination via the urea breath test, the stool antigen test with monoclonal antibodies or by histology. RESULTS: treatment compliance was observed in 96% (192/200) of the patients. Only 28.5% (57/200) of the patients experienced adverse effects, which led to abandoning the treatment in only seven subjects. Severe adverse effects developed in only one case due to Clostridium difficile infection. The effectiveness based on intention to treat was 91.5% (183/200, 95% CI: 87.1-96.8) and per protocol was 95.2% (182/191, 95% CI: 90.9-98.9). CONCLUSIONS: in our experience, Pylera® is an effective and safe treatment that should be considered as a first line therapeutic option for the eradication of H. pylori infection.


Asunto(s)
Antiinfecciosos/administración & dosificación , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori , Cumplimiento de la Medicación/estadística & datos numéricos , Metronidazol/administración & dosificación , Compuestos Organometálicos/administración & dosificación , Tetraciclina/administración & dosificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antiinfecciosos/efectos adversos , Cápsulas , Combinación de Medicamentos , Femenino , Humanos , Masculino , Metronidazol/efectos adversos , Persona de Mediana Edad , Compuestos Organometálicos/efectos adversos , Estudios Prospectivos , Tetraciclina/efectos adversos , Resultado del Tratamiento , Adulto Joven
14.
Arch Microbiol ; 200(8): 1191-1203, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29881875

RESUMEN

We investigated the effects of Azospirillum brasilense strains Ab-V5 and Ab-V6 in the induction of mechanisms of systemic acquired resistance (SAR) and induced system resistance (ISR) on maize (Zea mays L.) plants. Under normal growth conditions, the treatments consisted of the standard inoculation of cells at sowing, and leaf spray of cells or their metabolites at the V2.5 growth stage; under saline stress (170 mM NaCl), the treatment consisted of standard single and co-inoculation of A. brasilense and Rhizobium tropici. The main compounds in the Azospirillum metabolites were identified as indole-3-acetic acid (IAA) and salicylic acid (SA). Under normal conditions, A. brasilense cells applied at sowing or by leaf spray increased the activities of catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) in leaves, and of ascorbate peroxidase (APX) in roots; however, interestingly, in general the highest activities were observed by leaf spray of metabolites. Under normal conditions, the highest levels of salicylic acid (SA) and jasmonic acid (JA) were achieved in leaves by leaf spray of metabolites, of SA in roots by leaf spray of cells, and of JA in roots by standard inoculation and leaf spray of metabolites. Under saline stress, plant protection occurred via SA and abscisic acid (ABA), but not JA. In general, inoculation resulted in further increases in SA in leaves and roots, and ABA in leaves. We hypothesize that A. brasilense confers protection to maize plants by simultaneous induction of JA and SA pathways, and, under saline stressing conditions, by SA and ABA pathways.


Asunto(s)
Antioxidantes/metabolismo , Azospirillum brasilense/metabolismo , Zea mays/metabolismo , Ácido Abscísico/metabolismo , Catalasa/metabolismo , Ciclopentanos/metabolismo , Ácidos Indolacéticos/metabolismo , Malondialdehído/metabolismo , Oxilipinas/metabolismo , Hojas de la Planta/enzimología , Raíces de Plantas/enzimología , Ácido Salicílico/metabolismo , Estrés Fisiológico , Superóxido Dismutasa/metabolismo , Zea mays/enzimología , Zea mays/microbiología
15.
Anal Bioanal Chem ; 409(5): 1207-1220, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27815614

RESUMEN

A molecularly imprinted polymer (MIP) selective for cannabinoids [Δ9-tetrahydrocannabinol (Δ9-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THC-COOH), and 11-hydroxy-Δ9-tetrahydrocannabinol (Δ9-THC-OH)] has been synthesized, fully characterized, and applied to the assessment of plasma and urine analysis of marijuana abuse by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Δ9-THC-COOH was used as a template molecule, whereas ethylene glycol dimethacrylate (EGDMA) was used as a functional monomer, divinylbenzene (DVB) as a cross-linker, and 2,2'-azobisisobutyronitrile (AIBN) as an initiator. The prepared MIP was found to be highly selective for cannabinoids typically found in blood and urine, and also for cannabinol (CBN) and cannabidiol (CBD). MIP beads (50 mg) were loaded inside a cone-shaped device made of a polypropylene (PP) membrane for microsolid-phase extraction (µ-SPE) in batch mode. Optimum retention of analytes (0.1 to 1.0 mL of plasma/urine) was achieved by fixing plasma/urine pH at 6.5 and assisting the procedure by mechanical shaking (150 rpm, 40 °C, 12 min). Optimum elution conditions implied 2 mL of a 90:10 methanol/acetic acid and ultrasound extraction (35 kHz, 325 W) for 6 min. Good precision was assessed by intra-day and inter-day assays. In addition, the method was found to be accurate after intra-day and inter-day analytical recovery assays and after analyzing control serum and urine control samples. The limits of quantification were in the range of 0.36-0.49 ng L-1 (plasma analysis) and 0.47-0.57 ng L-1 (urine analysis). These values are low enough for confirmative conclusions regarding marijuana abuse through blood and urine analysis. Graphical Abstract ᅟ.


Asunto(s)
Cannabinoides/análisis , Cromatografía Líquida de Alta Presión/métodos , Impresión Molecular , Polímeros/química , Microextracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/métodos , Cannabinoides/sangre , Cannabinoides/orina , Límite de Detección , Reproducibilidad de los Resultados
16.
Proc Natl Acad Sci U S A ; 109(19): 7535-40, 2012 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-22529367

RESUMEN

The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency. Efficient in planta GT was achieved in Arabidopsis thaliana by expression of a site-specific endonuclease that not only cuts within the target but also the chromosomal transgenic donor, leading to an excised targeting vector. Progeny clonal for the targeted allele could be obtained directly by harvesting seeds. Targeted events could be identified up to approximately once per 100 seeds depending on the target donor combination. Molecular analysis demonstrated that, in almost all events, homologous recombination occurred at both ends of the break. No ectopic integration of the GT vector was found.


Asunto(s)
Arabidopsis/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Marcación de Gen/métodos , Plantas/genética , Arabidopsis/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Glucuronidasa/genética , Glucuronidasa/metabolismo , Modelos Genéticos , Plantas/metabolismo , Plantas Modificadas Genéticamente , Recombinación Genética , Plantones/genética , Plantones/metabolismo , Semillas/genética , Semillas/metabolismo , Transformación Genética
18.
Behav Processes ; 220: 105070, 2024 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-38906234

RESUMEN

Instrumental appetitive extinction involves the reduction of a previously reinforced response when its occurrence is no longer rewarded. Two experiments with terrestrial toads (Rhinella arenarum) tested whether the occurrence of a nonreinforced response is necessary for response extinction by varying the time of exposure to nonrewarded goal-box stimuli across groups. In Experiment 1, toads that received the same acquisition training (15 sessions, 1 session/day, 300 s of access to water in the goal box) were randomly assigned to two groups. In Group 600 (n=12), animals spent 600 s in the goal box in 8 daily extinction sessions (water present but inaccessible). In Group 0 (n=11), toads performed the runway response (i.e., walking from the start to the goal box) but were removed as soon as they entered the goal box, thus having minimal exposure to nonrewarded goal-box stimuli. The runway response was weakened in Group 600 across extinction trials, but exhibited little change in Group 0. In Experiment 2, toads were randomly assigned to two groups after the same acquisition training. Group 0 (n=7) was treated the same as Group 0 in the previous experiment. In Group RI (retention interval, n=7), toads remained in their home cage for 13 days. Finally, all animals received 4 extinction sessions with 300 s in the empty goal box. There was little behavioral change in Group 0 during the 13 sessions with minimal exposure to the goal box. In extinction, both groups reduced their runway response at similar rates. Although the procedures were instrumental, extinction of the runway response in toads can be accounted for in terms of a Pavlovian approach response to stimuli paired with reward and nonreward in the goal box.

19.
Physiol Behav ; 278: 114511, 2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38479582

RESUMEN

Successive negative contrast (SNC) has been used to study reward relativity, reward loss, and frustration for decades. In instrumental SNC (iSNC), the anticipatory performance of animals downshifted from a large reward to a small reward is compared to that of animals always reinforced with the small reward. iSNC involves a transient deterioration of anticipatory behavior in downshifted animals compared to unshifted controls. There is scattered information on the optimal parameters to produce this effect and even less information about its neural basis. Five experiments with rats trained in a runway to collect food pellets explored the effects of trial distribution (massed or spaced), amount of preshift training, reward disparity, and reward magnitude on the development of an iSNC effect. Start, run, and goal latencies were measured. Using spaced trials (one trial per day), evidence of the iSNC effect was observed with 24 preshift trials and a 32-to-4 pellet disparity. With massed trials (4 trials per session separated by 30-s intertrial intervals), evidence of iSNC was found with 12 preshift sessions (a total of 48 trials) and a 16-to-2 pellet disparity. The massed-training procedure was then used to assess neural activity in three prefrontal cortex areas using c-Fos expression in animals perfused after the first downshift session. There was evidence of increased activation in the anterior cingulate cortex and a trend toward increased activation in the infralimbic and prelimbic cortices. These procedures open a venue for studying the neural basis of the instrumental behavior of animals that experience reward loss.


Asunto(s)
Condicionamiento Operante , Recompensa , Ratas , Animales , Condicionamiento Operante/fisiología , Motivación , Corteza Prefrontal
20.
J Vis Exp ; (208)2024 Jun 28.
Artículo en Inglés | MEDLINE | ID: mdl-39007624

RESUMEN

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.


Asunto(s)
Fagos de Salmonella , Salmonella enterica , Fagos de Salmonella/fisiología , Salmonella enterica/virología , Lipopolisacáridos , Salmonella/virología , Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología
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