Evaluation of the efficacy of an antioxidant combination for the modulation of metabolic, endocrine, and clinical parameters in patients with polycystic ovary syndrome.

OBJECTIVE: To evaluate the efficacy of dietary supplementation with a combination of antioxidants (lipoic acid, N-acetylcysteine, vitamin B6, and S-adenosyl-L-methionine) for the modulation of metabolic, endocrine, and clinical parameters in comparison with oral contraception in non-diabetic women newly diagnosed with polycystic ovary syndrome (PCOS). METHODS: This was a prospective, partially randomized, multicenter study in which non-diabetic women with PCOS were recruited under routine clinical practice conditions and distributed in three groups to receive the following regimen for 6 months: 1) antioxidant combination (MN group); 2) oral contraception (OC group); or 3) oral contraception and the antioxidant combination (MN + OC group). General recommendation of healthy diet and regular exercise was given to all patients. Metabolic, endocrine, clinical, and quality of life parameters were recorded at baseline and after 6 months of therapy. RESULTS: A total of 96 women with PCOS were included in the study. After 6 months of treatment, the homeostasis model assessment-estimated insulin resistance (HOMA-IR) level was reduced only in the MN group, with a significant mean reduction of -0.92 points. Androstenedione was significantly reduced in all groups. Clinical parameters that significantly improved in all groups were hirsutism, acne, irregular menstruation, and quality of life, with no statistical differences between the groups. CONCLUSIONS: This study showed that the antioxidant combination might be a suitable therapy for patients with PCOS when oral contraceptive is not indicated, because in all groups clinical parameters, irregular menstruation as well as androstenedione and quality of life were significantly improved with no statistical difference between groups.

Antimicrobial Susceptibility Profiles Among Neonatal Early-onset Sepsis Pathogens.

BACKGROUND: Empiric administration of ampicillin and gentamicin is recommended for newborns at risk of early-onset sepsis (EOS). There are limited data on antimicrobial susceptibility of all EOS pathogens. METHODS: Retrospective review of antimicrobial susceptibility data from a prospective EOS surveillance study of infants born ≥22 weeks' gestation and cared for in Neonatal Research Network centers April 2015-March 2017. Nonsusceptible was defined as intermediate or resistant on final result. RESULTS: We identified 239 pathogens (235 bacteria, 4 fungi) in 235 EOS cases among 217,480 live-born infants. Antimicrobial susceptibility data were available for 189/239 (79.1%) isolates. Among 81 Gram-positive isolates with ampicillin and gentamicin susceptibility data, all were susceptible in vitro to either ampicillin or gentamicin. Among Gram-negative isolates with ampicillin and gentamicin susceptibility data, 72/94 (76.6%) isolates were nonsusceptible to ampicillin, 8/94 (8.5%) were nonsusceptible to gentamicin, and 7/96 (7.3%) isolates were nonsusceptible to both. Five percent or less of tested Gram-negative isolates were nonsusceptible to each of third or fourth generation cephalosporins, piperacillin-tazobactam, and carbapenems. Overall, we estimated that 8% of EOS cases were caused by isolates nonsusceptible to both ampicillin and gentamicin; these were most likely to occur among preterm, very-low birth weight infants. CONCLUSIONS: The vast majority of contemporary EOS pathogens are susceptible to the combination of ampicillin and gentamicin. Clinicians may consider the addition of broader-spectrum therapy among newborns at highest risk of EOS, but we caution that neither the substitution nor the addition of 1 single antimicrobial agent is likely to provide adequate empiric therapy in all cases.

Effects of Dietary Calcium Propionate Supplementation on Hypothalamic Neuropeptide Messenger RNA Expression and Growth Performance in Finishing Rambouillet Lambs.

The objective of this experiment was to evaluate the effects of feeding different levels concentrations of dietary calcium propionate (CaPr) on lambs' growth performance; ruminal fermentation parameters; glucose-insulin concentration; and hypothalamic mRNA expression for neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin (POMC). Thirty-two individually fed lambs were randomly assigned to four treatments: (1) control diet (0 g/kg of CaPr), (2) low CaPr, (30 g/kg dry matter (DM)), (3) medium CaPr, (35 g/kg DM), and (4) high CaPr (40 g/kg DM). After 42 days of feeding, lambs were slaughtered for collecting samples of the hypothalamus. Data were analyzed as a complete randomized design, and means were separated using linear and quadratic polynomial contrast. Growth performance was not affected (p ≥ 0.11) by dietary CaPr inclusion. The ruminal concentration of total volatile fatty acids (VFA) increased linearly (p = 0.04) as dietary CaPr increased. Likewise, a linear increase in plasma insulin concentration (p = 0.03) as dietary CaPr concentration increased. The relative mRNA expression of NPY exhibited a quadratic effect (p < 0.01), but there were significant differences in the mRNA expression of AgRP and POMC (p ≥ 0.10). Dietary calcium propionate did not improve lamb growth performance in lambs feed with only forage diets. Intake was not correlated with feed intake with mRNA expression of neuropeptides.

Pomegranate variety and pomegranate plant part, relevance from bioactive point of view: a review.

Pomegranate (Punica granatum L.) belongs to the Punicaceae plant family. It is an important fruit due to its nutritional and medicinal properties. Pomegranates are widely distributed around the world and, therefore, have a broad genetic diversity, resulting in differences in their phytochemical composition. The scientific community has focused on the positive health effects of pomegranate as a whole, but the different varieties have rarely been compared according to their bioactive compounds and bioactivity. This review aims to provide a holistic overview of the current knowledge on the bioactivity of pomegranate trees, with an emphasis on differentiating both the varieties and the different plant parts. This review intends to provide a general and organized overview of the accumulated knowledge on pomegranates, the identification of the most bioactive varieties, their potential consumption pathways and seeks to provide knowledge on the present gaps to guide future research.

A close neuroanatomical relationship between the enkephalinergic (methionine-enkephalin) and tachykininergic (substance P) systems in the alpaca diencephalon.

INTRODUCTION: In the alpaca diencephalon, the distribution of immunoreactive cell bodies and fibers containing methionine-enkephalin (MET) or substance P (SP) has been studied. MATERIAL AND METHODS: The immunohistochemical study was performed by standard method on the diencephalon of four male alpacas that lived at sea level. RESULTS: Nerve fibers containing MET or SP were widely distributed in the thalamus and hypothalamus. METand SP-immunoreactive fibers showed a similar distribution in the whole diencephalon. Immunoreactive cell bodies containing MET or SP were only observed in the hypothalamus. The distribution of MET-immunoreactive cell bodies was more widespread than that observed for cell bodies containing SP. CONCLUSIONS: A close neuroanatomical relationship between the tachykininergic (SP) and enkephalinergic (MET) systems was observed in the whole diencephalon suggestive of the existence of multiple physiological interactions between both systems.

In vitro and computational studies of natural products related to perezone as anti-neoplastic agents.

Many natural phyto-products as perezone (Per) exhibit anti-cancer activities. Using experimental and computational studies, it was described that Poly ADP-ribose polymerase 1(PARP-1) inhibition and the induction of oxidative stress state explain the pro-apoptotic activity of Per. The aim of this study was to evaluate two phyto-products related to Per as anti-cancer agents: hydroxyperezone (OHPer) and its monoangelate (OHPer-MAng). These molecules were structurally characterized employing thermal analysis, IR spectrophotometry and X-ray diffraction techniques. The phyto-compounds evaluated in vitro in six cancer cell lines (K562, MCF-7, MDA-MB-231, HeLa, U373, A549) and non-malignant cells determinate their cytotoxicity, type of induced cell death, ability to avoid cell migration and changes at the redox status of the cell. Using, in vitro and computational studies provided the inhibition of PARP-1 and its potential binding mode. Cell proliferation assays demonstrated that OHPer-MAng treatment significantly induces apoptosis in triple negative breast cancer (TNBC) cell line (MDA-MB-231 IC50 = 3.53 µM), being particularly less cytotoxic to Vero cells (IC50 = 313.92 µM), human lymphocytes (IC50 = 221.46 µM) and rat endothelial cells (IC50=> 400 µM). The treatment of MDA-MB-231 cells with OHPer-MAng showed inhibition of migration by cancer cells. The induction of an oxidative stress state, similar to other quinones and PARP-1 inhibition explains the pro-apoptotic activity of OHPer-MAng. Docking studies showed that OHPer-MAng establishes great non-bonding interactions with the lateral chains of Tyr235, Hys201, Tyr246, Ser203, Asn207, and Gly233 located at the catalytic site of PARP-1, also demonstrating the anti-cancer activity of OHPer-MAng in TNBC cell line.

Overexpression of a modified eIF4E regulates potato virus Y resistance at the transcriptional level in potato.

BACKGROUND: Potato virus Y (PVY) is a major pathogen of potatoes with major impact on global agricultural production. Resistance to PVY can be achieved by engineering potatoes to express a recessive, resistant allele of eukaryotic translation initiation factor eIF4E, a host dependency factor essential to PVY replication. Here we analyzed transcriptome changes in eIF4E over-expressing potatoes to shed light on the mechanism underpinning eIF4E-mediated recessive PVY resistance. RESULTS: As anticipated, modified eIF4E-expressing potatoes demonstrated a high level of resistance, eIF4E expression, and an unexpected suppression of the susceptible allele transcript, likely explaining the bulk of the potent antiviral phenotype. In resistant plants, we also detected marked upregulation of genes involved in cell stress responses. CONCLUSIONS: Our results reveal a previously unanticipated second layer of signaling attributable to eIF4E regulatory control, and potentially relevant to establishment of a broader, more systematic antiviral host defense.

Green Approach Extraction of Perezone from the Roots of Acourtia platyphilla (A. Grey): A Comparison of Four Activating Modes and Supercritical Carbon Dioxide.

Perezone, a sesquiterpene quinone, is a very important molecule due to its pharmacological activities in addition to the fact that it is considered to be the first secondary metabolite isolated in the new world (America-Mexico, 1852). This study aims to offer a green comparative study about the extraction of the target molecule from the roots of the vegetable specimen Acourtia platyphilla (A. Grey). The study was performed comparing five different modes of extraction: supercritical CO2, electromagnetic infrared and microwave irradiations, mechanical-wave ultrasound versus typical mantle heating procedure. An exhaustive comparative-discussion of the obtained results is provided. It is worth noting that the corresponding quantifications were established using 1H NMR, correlating appropriately the integrals of the vinylic proton H-6 of perezone with the aromatic singlet of p-dinitrobenzene employed as an internal reference. It is also important to highlight that the four presented procedures are novel modes to extract perezone. Finally, a complementary study about the solubility of the target sesquiterpene quinone related to the use of supercritical CO2 is also reported.

Impact of selenium co-administration on methylmercury exposed eleutheroembryos and adult zebrafish (Danio rerio): Changes in bioaccumulation and gene expression.

Mercury still represents one of the most hazardous threats for the aquatic ecosystem due to its high toxicity, and the fact that it can be easily incorporated into the food chain by accumulation in fish as MeHg. On the other hand, selenium is a micronutrient that is part of different antioxidant enzymes that regulate the cellular redox state, and whose complex interaction with Hg has been extensively studied from a toxicological point of view. In order to evaluate the protective effect of Se(IV) co-administration against MeHg accumulation and toxicity, we have selected an in-vivo model at two developmental stages: zebrafish eleutheroembryos and adult fish. Embryos were exposed during 48 h to MeHg (5 or 25 µg/l) and a concentration of Se (IV) representing a molar ratio close to one (2.5 or 12.5 µg/l), while adult zebrafish were exposed during 72 h to either 25 µg/l of MeHg alone or co-exposed with 12.5 µg/l of Se (IV). A significant decrease in MeHg bioaccumulation factor was observed in eleutheroembryos co-exposed to Se(IV). A time-dependent accumulation of MeHg was observed in all the analyzed organs and tissues of adult fish, which was significantly reduced in the muscular tissue and the intestine by Se(IV) co-administration. However, such protection against MeHg bioaccumulation was not maintained in the brain and liver. The data derived from the gene expression analysis also demonstrated the protective effect of Se(IV) against MeHg-induced oxidative stress and the activation of different defense mechanisms by Se(IV) co-administration.

Effect of an anxiolytic botanical containing Souroubea sympetala and Platanus occidentalis on in-vitro diazepam human cytochrome P450-mediated metabolism.

OBJECTIVES: A novel anxiolytic natural health product (NHP) containing Souroubea sympetala and Platanus occidentalis is available for the companion animal market and is currently being developed for clinical evaluation. Addressing the risk of potential NHP-drug interactions, this study investigated S. sympetala and P. occidentalis plant extracts, and their identified bioactive compounds, for effects on the activity of cytochrome P450 (CYP) isozymes and the metabolism of the conventional anti-anxiety medication diazepam. METHODS: Souroubea sympetala and P. occidentalis extracts, a 1 : 1 blend of the two extracts, and five triterpenes were tested for inhibitory effects on human recombinant CYP3A4, CYP2D6, CYP2C9 and CYP2C19 activity using a fluorometric plate assay. Direct effects on the metabolism of diazepam were evaluated using human liver microsomes with drug and metabolite quantification by ultra-high-pressure liquid chromatography and mass spectroscopy. KEY FINDINGS: The active substances betulinic acid (BA) and ursolic acid (UA) strongly inhibited CYP3A4 activity while UA and lupeol moderately inhibited CYP2C19. All extracts exhibited strong activity against the tested isozymes at 50-100 µg/ml. BA and all plant extracts blocked the formation of major diazepam metabolites. CONCLUSIONS: Betulinic acid, UA and both the extracts and blended product are expected to affect the metabolism of diazepam when given in high dose.

Safety evaluation of a new anxiolytic product containing botanicals Souroubea spp. and Platanus spp. in dogs.

Separation anxiety and noise aversion are common behavioral problems in dogs. They elicit fear responses such as cowering, seeking out the owner, and attempting to escape. This can result in property damage, injury to the dog, and disruption of the owner-pet bond, possibly leading to pet abandonment or euthanasia. A novel botanical anxiolytic product was evaluated for safety in dogs as the target animal species. Its intended use is for the treatment and prevention of anxiety and noise aversion in dogs. It contains a defined mixture of Souroubea spp. vine and Platanus spp. bark, delivering the active principle, betulinic acid, at a recommended dose of 1 mg/kg body weight (BW). In the current target animal safety study, 16 healthy male beagle dogs were administered either a placebo or the newly formulated botanical tablets at 0.5×, 2.5×, or 5× the recommended dose (1 mg/kg BW) over 28 d. The dogs were monitored for occurrence of any systemic or local adverse events. In the investigation presented here, there were no clinically significant adverse effects following treatment, as determined by clinical observations, physical examinations, BW, hematology, clinical biochemistry, and urinalysis. Pharmacokinetic analysis demonstrated that the concentration of betulinic acid in serum was below 0.020 µg/mL in treated animals. Under the conditions of these studies, the formulated blend of S. sympetala and P. occidentalis, when administered up to 5× the intended dose for 28 consecutive d, showed no adverse effects on the health of dogs.

L'anxiété de séparation et une aversion au bruit sont des problèmes de comportement fréquents chez les chiens. Elles élicitent des réponses de peur telles que des tremblements, la recherche du propriétaire, et une tentative de fuite. Elles peuvent résulter en des dommages à la propriété, des blessures au chien, et un bris du lien propriétaire-animal, pouvant potentiellement mener à l'abandon de l'animal ou l'euthanasie. Un nouveau produit anxiolytique botanique a été évalué pour sa sécurité chez les chiens, l'espèce animale cible. Son utilisation visée est pour le traitement et la prévention de l'anxiété et de l'aversion au bruit chez les chiens. Le produit contient un mélange défini de vigne de Souroubea spp. et d'écorce de Platanus spp., fournissant le principe actif, l'acide bétulinique, à un dosage recommandé de 1 mg/kg de poids corporel (PC). Dans l'étude de sécurité chez l'espèce animale cible, 16 chiens mâles de race beagle en santé ont reçu soit un placebo ou les nouvelles tablettes botaniques à 0,5×, 2,5×, ou 5× la dose recommandée (1 mg/kg PC) pendant 28 jours. Les chiens ont été observés pour l'apparition de manifestions adverses systémiques ou locales. Dans l'étude présentée ici, il n'y eut aucun effet clinique adverse significatif suivant le traitement, tel que déterminé par les observations cliniques, les examens physiques, le PC, et les résultats des analyses hématologiques, de biochimie clinique et urinaires. L'analyse pharmacocinétique a démontré que la concentration d'acide bétulinique dans le sérum était moins de 0,020 µg/mL chez les animaux traités. Dans les conditions des présentes études, le mélange de S. sympetala et de P. occidentalis, lorsqu'administré jusqu'à 5× le dosage prévu pendant 28 jours consécutifs, n'a démontré aucun effet adverse sur la santé des chiens.(Traduit par Docteur Serge Messier).

New Botanical Anxiolytics for Use in Companion Animals and Humans.

As part of our ongoing research into botanical therapies for anxiety disorders, the neotropical vine Souroubea sympetala was chosen for study as a phytochemical discovery strategy focusing on rare Central American plant families. When orally administered to male Sprague-Dawley rats, the crude plant extract, its ethyl acetate fraction, supercritical carbon dioxide fraction, or its isolated triterpenes reduced anxiety and/or fear-related behavior in standardized behavioral models. Pharmacological studies showed that the extracts acted at the benzodiazepine GABAA receptor and reduced corticosterone levels. A preparation containing Souroubea fortified with a second triterpene containing plant, Platanus occidentalis, was shown to be safe in a 28-day feeding trial with beagles at 5 times the intended dose. Subsequent trials with beagles in a thunderstorm model of noise aversion showed that the material reduced anxiety behaviors and cortisol levels in dogs. The formulation has been released for the companion animal market in Canada and the USA under the Trademark "Zentrol." Ongoing research is exploring the use of the material in treatment of anxiety and post-traumatic stress in humans.

Lipid remodelling is a widespread strategy in marine heterotrophic bacteria upon phosphorus deficiency.

Upon phosphorus (P) deficiency, marine phytoplankton reduce their requirements for P by replacing membrane phospholipids with alternative non-phosphorus lipids. It was very recently demonstrated that a SAR11 isolate also shares this capability when phosphate starved in culture. Yet, the extent to which this process occurs in other marine heterotrophic bacteria and in the natural environment is unknown. Here, we demonstrate that the substitution of membrane phospholipids for a variety of non-phosphorus lipids is a conserved response to P deficiency among phylogenetically diverse marine heterotrophic bacteria, including members of the Alphaproteobacteria and Flavobacteria. By deletion mutagenesis and complementation in the model marine bacterium Phaeobacter sp. MED193 and heterologous expression in recombinant Escherichia coli, we confirm the roles of a phospholipase C (PlcP) and a glycosyltransferase in lipid remodelling. Analyses of the Global Ocean Sampling and Tara Oceans metagenome data sets demonstrate that PlcP is particularly abundant in areas characterized by low phosphate concentrations. Furthermore, we show that lipid remodelling occurs seasonally and responds to changing nutrient conditions in natural microbial communities from the Mediterranean Sea. Together, our results point to the key role of lipid substitution as an adaptive strategy enabling heterotrophic bacteria to thrive in the vast P-depleted areas of the ocean.

Predictive value of neonatal MRI showing no or minor degrees of brain injury after hypothermia.

BACKGROUND: Magnetic resonance imaging is a surrogate biomarker for major neurodevelopmental disabilities in survivors of perinatal hypoxic-ischemic encephalopathy because injury to the basal ganglia/thalami is highly predictive of major neuromotor and cognitive problems. Major disabilities and the appearance of neonatal magnetic resonance imaging are improved with therapeutic hypothermia. We evaluated neurodevelopmental outcomes when conventional magnetic resonance imaging showed minimal or no brain injury. METHODS: Institutional review board-approved series of 62 infants (≥36 weeks; ≥1800 g; 34 boys/28 girls) cooled for hypoxic-ischemic encephalopathy between 2005 and 2011 who underwent neonatal magnetic resonance imaging and Bayley Scales of Infant and Toddler Development-III at 22 ± 7 months of age. Magnetic resonance imaging at 5-14 (mean 8) days was scored as normal (score = 0), showing focal gray or white matter injury only (score = 1), or basal ganglia/thalamic and/or watershed lesions with or without more extensive hemispheric injury (score = 2). Sensitivity, specificity, and positive and negative predictive values for magnetic resonance scores 0 and 1 and statistical interaction between magnetic resonance imaging score and age at magnetic resonance imaging were determined. RESULTS: Magnetic resonance score = 0 was seen in 35/62 patients; 26/35 (74%) were typically developing, seven (20%) had moderate and two (6%) had severe delay. Magnetic resonance score = 1 was seen in 17/62 (27%) patients; 5/17 (29%) were normal, 11/17 (65%) had moderate delay, and 1/17 (6%) had severe neurodevelopmental delay. Of the 52 patients with magnetic resonance scores of 0 and 1, 40% were abnormal. The negative predictive value of a normal magnetic resonance imaging was 74%. For score 1, sensitivity was 95% (confidence interval 63%-83%), specificity 84% (confidence interval 70%-90%), positive predictive value 84% (confidence interval 71%-93%), and negative predictive value 74% (confidence interval 62%-82%). CONCLUSIONS: Caution is warranted when prognosticating about neurodevelopmental status in early childhood after hypoxic ischemic encephalopathy with cooling, and longer follow-up studies are needed to determine the prognostic significance of a neonatal magnetic resonance imaging showing no or minor degrees of brain injury.

Analytical and bioanalytical approaches to unravel the selenium-mercury antagonism: a review.

Mercury is a well-known risk to ecosystems and human health. Considering that no effective treatment is available to counteract mercury toxicity, the effectiveness of different trace elements, agents and nutrients with antioxidant properties to protect or reverse mercury toxicity is crucial. In this article we present the main analytical and bioanalytical strategies that have been used to study the potential of selenium as a protective agent against mercury-induced toxicity. We review the different seleno-species and routes of administration tested, and consider the analytical approaches used to evaluate the influence of selenium on the uptake and assimilation of mercury by model animals. In addition, we describe a variety of methods for toxicity assessment that have been used to elucidate the antagonism mercury-selenium, and critically review the main results obtained to date. Future potential research interests that could provide a clearer picture of this phenomenon are also presented.

Cloning and biochemical characterization of an endo-1,4-ß-mannanase from the coffee berry borer Hypothenemus hampei.

BACKGROUND: The study of coffee polysaccharides-degrading enzymes from the coffee berry borer Hypothenemus hampei, has become an important alternative in the identification for enzymatic inhibitors that can be used as an alternative control of this dangerous insect. We report the cloning, expression and biochemical characterization of a mannanase gene that was identified in the midgut of the coffee berry borer and is responsible for the degradation of the most abundant polysaccharide in the coffee bean. METHODS: The amino acid sequence of HhMan was analyzed by multiple sequence alignment comparisons with BLAST (Basic Local Alignment Search Tool) and CLUSTALW. A Pichia pastoris expression system was used to express the recombinant form of the enzyme. The mannanase activity was quantified by the 3,5-dinitrosalicylic (DNS) and the hydrolitic properties were detected by TLC. RESULTS: An endo-1,4-ß-mannanase from the digestive tract of the insect Hypothenemus hampei was cloned and expressed as a recombinant protein in the Pichia pastoris system. This enzyme is 56% identical to the sequence of an endo-ß-mannanase from Bacillus circulans that belongs to the glycosyl hydrolase 5 (GH5) family. The purified recombinant protein (rHhMan) exhibited a single band (35.5 kDa) by SDS-PAGE, and its activity was confirmed by zymography. rHhMan displays optimal activity levels at pH 5.5 and 30°C and can hydrolyze galactomannans of varying mannose:galactose ratios, suggesting that the enzymatic activity is independent of the presence of side chains such as galactose residues. The enzyme cannot hydrolyze manno-oligosaccharides such as mannobiose and mannotriose; however, it can degrade mannotetraose, likely through a transglycosylation reaction. The K(m) and k(cat) values of this enzyme on guar gum were 2.074 mg ml(-1) and 50.87 s(-1), respectively, which is similar to other mannanases. CONCLUSION: This work is the first study of an endo-1,4-ß-mannanase from an insect using this expression system. Due to this enzyme's importance in the digestive processes of the coffee berry borer, this study may enable the design of inhibitors against endo-1,4-ß-mannanase to decrease the economic losses stemming from this insect.

Generación de anticuerpos policlonales para la detección de la variante genotípica GIII de PVY, en cultivos de tomate de Árbol y papa de Colombia / Generation of policlonal antibodies for detection of the PVY genotypic variant GIII in tamarillo and potato crops from Colombia

El potyvirus PVY es uno de los agentes causales más frecuentemente asociados a problemas virales en cultivos de papa y tomate de árbol en Colombia. Dada la importancia económica de las enfermedades causadas por PVY y a la necesidad de generar material de siembra certificado por su sanidad viral, es fundamental la generación de herramientas de diagnóstico que permitan la detección temprana de este virus. En este trabajo se reporta la obtención de anticuerpos policlonales específicos, útiles para la detección del genotipo III de PVY (GIII), una de las tres variantes que recientemente han sido reportadas en cultivos de papa y tomate de árbol de la región Andina de Colombia. Como antígeno, se utilizó un péptido sintético diseñado a partir de la región variable del extremo N-terminal del gen de la cápside viral. La sensibilidad de los anticuerpos fue evaluada mediante pruebas de ELISA y dot-blot utilizando péptidos sintéticos. Se realizó una prueba piloto para validar el uso de los anticuerpos a partir de plantas sintomáticas y asintomáticas obtenidas de una región donde confluyen cultivos de ambas solanéceas, encontrándose que los anticuerpos generados ofrecen mayores niveles de detección que los anticuerpos comerciales comúnmente utilizados para detectar los serotipos PVY-O,C y PVY-N de este virus.

PVY is one of the potyvirus more frequently associated with viral infections in tomato and tamarillo crops in Colombia. Due to the economic impact of PVY and the need to certify seeds as virus-free it is important to develop diagnostic tools that allow its premature detection. In this work, the obtention of antibodies detecting the genotype III of PVY is reported. This genotype is one of the three PVY variants infecting tamarillo and potato in the Andean region of Colombia. The N-terminal variable region of the coat protein was chosen as antigen for antibody production. The sensibility of these antibodies was tested by ELISA and dot-blot using synthetic peptides. A pilot test was performed on symptomatic and non-symptomatic plants from a mixed orchard of tamarillo and potato. The generated antibodies showed higher detection levels than the commercial antibodies commonly used to detect the PVY-O,C and PVY-N serotypes.

Anxiolytic activity of a supercritical carbon dioxide extract of Souroubea sympetala (Marcgraviaceae).

The purpose of this work was to develop an extraction technique to yield a betulinic acid-(BA) enriched extract of the traditional anti-anxiety plant Souroubea sympetala Gilg (Marcgraviaceae). Five extraction techniques were compared: supercritical carbon dioxide extraction (SCE), conventional solvent extraction with ethyl acetate (EtOAc), accelerated solvent extraction (ASE), ultrasonic assisted extraction (UAE) and soxhlet extraction (Sox). The EtOAc and SCE extraction methods resulted in BA-enriched extracts, with BA concentrations of 6.78 ± 0.2 and 5.54 ± 0.2 mg/g extract, respectively, as determined by HPLC-APCI-MS. The bioactivity of the BA-enriched extracts was compared in the elevated plus maze (EPM), a validated rodent anxiety behaviour assay. Rats orally administered a 75 mg/kg dose of SCE extract exhibited anxiolysis as compared with vehicle controls, with a 50% increase in the percent time spent in the open arms, a 73% increase in unprotected head dips and a 42% decrease in percent time spent in the closed arms. No significant differences were observed between the SCE and EtOAc extracts for these measures, but the animals dosed with SCE extract had significantly more unprotected head dips than those dosed with the EtOAc extract. The SCE extract demonstrated a dose-response in the EPM, with a trend toward decreased anxiety at 25 mg/kg, and significant anxiolysis was only observed at 75 mg/kg dose. This study demonstrates that SCE can be used to generate a betulinic acid-enriched extract with significant anxiolysis in vivo. Further, the study provides a scientific basis for the ethnobotanical use of this traditional medicine and a promising lead for a natural health product to treat anxiety.

Towards a target label-free suboptimum oligonucleotide displacement-based detection system.

A novel method for the future development of label-free DNA sensors is proposed here. The approach is based on the displacement of a labelled suboptimum mutated oligonucleotide hybridised with the immobilised biotin-capture probe. The target fully complementary to the biotin-capture probe can displace the labelled oligonucleotide causing a subsequent decrease of the signal that verifies the presence of the target. The decrease of signal was demonstrated to be proportional to the target concentration. A study of the hybridisation of mutated and complementary labelled oligonucleotides with an immobilised biotin-capture probe was carried out. Different kinetic and thermodynamic behaviour was observed for heterogeneous hybridisation of biotin-capture probe with complementary or suboptimum oligonucleotides. The displacement method evaluated colourimetrically achieved the objective of decreasing the response time from 1 h for direct hybridisation of 19-mer oligonucleotides in the direct enzyme-linked oligonucleotide assay (ELONA) to 5 min in the case of displacement detection in the micromolar concentration range.