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Wolbachia is an obligatory nutritional symbiont of the common bed bug, Cimex lectularius, providing B-vitamins to its host. The biological significance of Wolbachia to bed bugs is investigated primarily by eliminating the symbiont with antibiotics, which is followed by confirmation with molecular assays. In this chapter, we describe a protocol for eliminating Wolbachia in bed bugs using the ansamycin antibiotic rifampicin (also known as rifampin) and three molecular methods to accurately detect and quantify the Wolbachia gene copies in bed bug samples. We describe the digital droplet PCR (ddPCR), a highly sensitive technique for absolute quantification of low abundance target genes, which has proven to be a valuable technique for confirmation of the elimination of Wolbachia.

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Obligate blood feeders, such as Cimex lectularius (common bed bug), have symbiotic associations with nutritional endosymbionts that produce B-vitamins. To quantify the symbiont's contribution to host fitness in these obligate mutualisms, the symbiont must be eliminated and its absence rigorously confirmed. We developed and validated procedures for complete elimination of Wolbachia (Wb) in bed bugs and quantified development and reproduction in bed bugs with and without Wb and with and without B-vitamins supplementation. Aposymbiotic bed bugs had slower nymphal development, reduced adult survivorship, smaller adult size, fewer eggs per female, and lower hatch rate than bed bugs that harbored Wb. In aposymbiotic bed bugs that were fed B-vitamins-supplemented blood, nymph development time, adult survivorship and hatch rate recovered to control levels, but adult size and egg number only partially recovered. These results underscore the nutritional dependence of bed bugs on their Wb symbiont and suggest that Wb may provide additional nutritional benefits beyond the B-vitamin mix that we investigated.

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To support efforts to manage and contain spotted lanternfly (SLF), Lycorma delicatula White (Hemiptera: Fulgoridae), research is being conducted to develop classical biological control methods. To date, two potential biocontrol agents from China have been identified: an egg parasitoid, Anastatus orientalis, and a nymphal parasitoid, Dryinus sinicus Olmi (Hymenoptera: Dryinidae). The research detailed here focuses on investigating the biology and rearing of A. orientalis to assess its potential efficacy in a biocontrol program and optimize its rearing. Female wasps lived significantly longer than male wasps (68 and 23 d, respectively) and females produced an average of 94 total progeny that successfully emerged as adults, with most progeny produced between weeks one and four of the females' lives. The sex ratio of the progeny, with no re-mating, was initially highly female-biased but became progressively more male-biased, likely due to sperm depletion. There was no evidence of additional mortality to SLF eggs from wasp host feeding, but the data were highly variable and the sample size was small. There was high parasitoid emergence when oviposition conditions mimicked mid-September Beijing temperature and photoperiod; however, there was little emergence under 25°C and long-day conditions because most progeny entered a diapause. Storage of parasitized eggs in 5°C chill lowered parasitoid emergence rates. Lastly, there was no evidence that storing field-collected SLF egg masses in 5°C for 10 mo prior to parasitization affected parasitism rates. These findings inform our rearing protocol for A. orientalis and facilitate our testing of this species as a potential biological control agent for SLF.

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Forests make up a large portion of terrestrial plant biomass, and the long-lived woody plants that dominate them possess an array of traits that deter consumption by forest pests. Although often extremely effective against native consumers, invasive species that avoid or overcome these defenses can wreak havoc on trees and surrounding ecosystems. This is especially true when multiple invasive species co-occur, since interactions between invasive herbivores may yield non-additive effects on the host. While the threat posed by invasive forest pests is well known, long-term field experiments are necessary to explore these consumer-host interactions at appropriate spatial and temporal scales. Moreover, it is important to measure multiple variables to get a "whole-plant" picture of their combined impact. We report the results of a 4-yr field experiment addressing the individual and combined impacts of two invasive herbivores, the hemlock woolly adelgid (Adelges tsugae) and elongate hemlock scale (Fiorinia externa), on native eastern hemlock (Tsuga canadensis) in southern New England. In 2011, we planted 200 hemlock saplings into a temperate forest understory and experimentally manipulated the presence/absence of both herbivore species; in 2015, we harvested the 88 remaining saplings and assessed plant physiology, growth, and resource allocation. Adelgids strongly affected hemlock growth: infested saplings had lower above/belowground biomass ratios, more needle loss, and produced fewer new needles than control saplings. Hemlock scale did not alter plant biomass allocation or growth, and its co-occurrence did not alter the impact of adelgid. While both adelgid and scale impacted the concentrations of primary metabolites, adelgid effects were more pronounced. Adelgid feeding simultaneously increased free amino acids local to feeding sites and a ~30% reduction in starch. The cumulative impact of adelgid-induced needle loss, manipulation of nitrogen pools, and the loss of stored resources likely accelerates host decline through disruption of homeostatic source-sink dynamics occurring at the whole-plant level. Our research stresses the importance of considering long-term impacts to predict how plants will cope with contemporary pressures experienced in disturbed forests.

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We investigated the insertion of small potassium (K+) channel proteins (KcvMA-1D and KcvNTS) into model membranes and the lipid-protein structural interference, combining neutron reflectometry and electrophysiology. Neutron reflectometry experiments showed how the transverse structure and mechanical properties of the bilayer were modified, upon insertion of the proteins in single model-membranes, either supported on solid substrate or floating. Parallel electrophysiology experiments were performed on the same channels reconstituted in free-standing planar lipid bilayers, of both typical composition and matched to the neutron reflectometry experiment, assessing their electrical features. Functional and structural results converge in detecting that the proteins, conical in shape, insert with a directionality, cytosolic side first. Our work addresses the powerful combination of the two experimental approaches. We show here that membrane structure spectroscopy and ion channel electrophysiology can become synergistic tools in the analysis of structural-functional properties of biomimetic complex environment.

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INTRODUCTION: An elevation of cardiac troponins has been described in healthy athletes after endurance exercises. The clinical significance of this increase is unclear and the lack of awareness of this phenomenon may lead to inappropriate management of these subjects. AIM: We sought to determine wether an intensive cycling training could determine a biomarkers elevation. METHODS: We evaluated serum high sensitivity cardiac troponin T, NH(2)-terminal pro-brain natriuretic peptide, CK-MB and CK in 21 healthy male preadolescent athletes (age 9.2 ± 1.7 years) after an intensive cycling training prolonged until muscular exhaustion (mean duration 16'41″). During exercise heart rhythm and rate were monitored with Holter. RESULTS: 62% of the group had an elevation of cardiac biomarkers: specifically, 6 children had an increase in troponin levels; 3 of them had an elevation of pro-brain natriuretic peptides as well. Pro-brain natriuretic peptides resulted increased in 9 subjects. There was no relation between troponin elevation and heart rate, age or exercise duration; subjects with increased pro-brain natriuretic peptides had mean and maximal heart rates lower than children with normal natriuretic peptides levels. Other sports were performed in 50% of subjects with normal troponins and only in 17% of those with increased values. CONCLUSIONS: A short, high-intensity exercise caused an elevation of cardiac biomarkers in 62% of our subjects. The grade of training may influence the release of troponin and this increase is probably related to a temporary discrepancy between O2 delivery and consumption. Increases in natriuretic peptides levels are possibly expression of different adaptations to exercise.

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We report the identification of a novel mutation in the fork-head box C1 (FOXC1) gene which occurred de novo in an Italian patient with unrecognized Axenfeld-Rieger syndrome. He was previously diagnosed as having late recognized primary congenital glaucoma at the age of 14 years and was subsequently subjected to multiple surgical interventions due to uncontrolled intraocular pressure and progressive visual field loss. After exclusion of mutations in CYP1B1 and MYOC, trio-whole-exome sequencing revealed de novo in frame deletion in the coding region of the FOXC1 gene (c.407_409delGTC, p.V137del) leading to a deletion of the evolutionary conserved amino acid Valine at position 137 of the protein. Molecular modeling predicted that Val137 deletion impairs FOXC1 DNA-binding capacity and transcriptional activation. Since loss-of-function mutations in FOXC1 are associated with Axenfeld-Rieger syndrome, the genetic findings in combination with re-evaluation of the patient's clinical data resulted in a corrected diagnosis of Axenfeld-Rieger syndrome with developmental glaucoma. We therefore suggest that in addition to CYP1B1 and MYOC, FOXC1 should be included in the genetic analysis of cases with unclear glaucomatous phenotypes to ensure proper diagnosis, adequate treatment and appropriate genetic counseling.

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MAIN CONCLUSION: Multiplicity of protease inhibitors induced by predators may increase the understanding of a plant's intelligent behavior toward environmental challenges. Information about defense mechanisms of non-genomic model plant passion fruit (Passiflora edulis Sims) in response to predator attack is still limited. Here, via biochemical approaches, we showed its flexibility to build-up a broad repertoire of potent Kunitz-type trypsin inhibitors (KTIs) in response to methyl jasmonate. Seven inhibitors (20-25 kDa) were purified from exposed leaves by chromatographic techniques. Interestingly, the KTIs possessed truncated Kunitz motif in their N-terminus and some of them also presented non-consensus residues. Gelatin-Native-PAGE established multiple isoforms for each inhibitor. Significant differences regarding inhibitors' activity toward trypsin and chymotrypsin were observed, indicating functional polymorphism. Despite its rarity, two of them also inhibited papain, and such bifunctionality suggests a recruiting process onto another mechanistic class of target protease (cysteine-type). All inhibitors acted strongly on midgut proteases from sugarcane borer, Diatraea saccharalis (a lepidopteran insect) while in vivo assays supported their insecticide properties. Moreover, the bifunctional inhibitors displayed activity toward midgut proteases from cowpea weevil, Callosobruchus maculatus (a coleopteran insect). Unexpectedly, all inhibitors were highly effective against midgut proteases from Aedes aegypti a dipteran insect (vector of neglected tropical diseases) opening new avenues for plant-derived PIs for vector control-oriented research. Our results reflect the KTIs' complexities in passion fruit which could be wisely exploited by influencing plant defense conditions. Therefore, the potential of passion fruit as source of bioactive compounds with diversified biotechnological application was strengthened.

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Aims: The NEVO™ sirolimus eluting coronary stent is designed to improvelong-term PCI safety by combining sirolimus release from reservoirs withbioabsorbable polymer to reduce spatial and temporal polymer exposure.Absorption of drug and polymer within approximately three months limits theduration of vessel wall exposure to the polymer. Thereafter, only a biologically inertbare-metal platform remains. The NEVO™ stent was first evaluated in the multicenterrandomised NEVO RES-Elution I trial which demonstrated its superiority tothe TAXUS Liberté Paclitaxel - eluting stent.

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Introducción: Las malformaciones congénitas del corazón son el defecto de nacimiento más común y la primera causa de muerte por malformaciones neonatales, incluido el primer año de la vida. Objetivo: Determinar los factores de riesgo asociados a las cardiopatías congénitas en el niño. Métodos: Se realizó un estudio analítico retrospectivo de casos y controles sobre los factores de riesgo asociados a las cardiopatías congénitas, en un grupo de 48 niños cardiópatas pertenecientes a los grupos básicos de trabajo del Policlínico Capitán Roberto Fleites de Santa Clara, Villa Clara, desde enero de 2000 hasta diciembre de 2008; se aplicó un formulario y se revisaron historias clínicas individuales de los niños y las progenitoras. Se utilizó el examen físico, la ecocardiografía bidimensional y el electrocardiograma para verificar el diagnóstico. Para el análisis e interpretación de los resultados, se utilizó el análisis porcentual y la estimación de riesgo; con el propósito de evaluar el efecto de todos los factores asociados, se realizó un análisis multivariado para calcular la razón de posibilidades bajo estos presupuestos, para lo cual se utilizó la regresión logística. Resultados: Los factores que más se relacionaron con las cardiopatías congénitas en su efecto combinado fueron la ingestión de medicamentos (OR = 17,8); le siguen en orden, la hipertensión arterial (OR = 6,04) y las cardiopatías congénitas (OR = 6,6), así como la ingestión de alcohol (OR = 6,67). Conclusiones: Se identificaron los antecedentes familiares de diabetes mellitus, así como la anemia, diabetes mellitus gestacional y el hábito de fumar, como factores de riesgo(AU)

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BACKGROUND: The impact of incomplete stent apposition (ISA) after drug-eluting stent implantation determined by intravascular ultrasound (IVUS) on late clinical events is not well defined. OBJECTIVE: To evaluate the clinical impact of ISA after sirolimus-eluting stent (SES) placement during a follow-up period of 4 years. DESIGN: Pooled analysis from the RAVEL, E-SIRIUS and SIRIUS trials, three randomised, multicentre studies comparing SES and bare-metal stents (BMS). METHODS: IVUS at angiographic follow-up was available in 325 patients (SES: n = 180, BMS: n = 145). IVUS images were reviewed for the presence of ISA defined as one or more unapposed stent struts. Clinical follow-up was available for a 4-year period in all patients. Frequency, predictors and clinical sequel of ISA at follow-up after SES and BMS implantation were determined. RESULTS: ISA at follow-up was more common after SES (n = 45 (25%)) than after BMS (n = 12 (8.3%), p<0.001). Canadian Cardiology Society class III or IV angina at stent implantation (odds ratio (OR) = 4.69, 95% CI 2.15 to 10.23, p<0.001) and absence of diabetes (OR = 3.42, 95% CI 1.05 to 11.1, p = 0.041) were predictors of ISA at follow-up after SES placement. Rate of myocardial infarction tended to be slightly higher for ISA than for non-ISA patients. When SES patients only were considered, major adverse cardiac event free survival at 4 years was identical for those with and without ISA at follow-up (11.1% vs 16.3%, p = 0.48). CONCLUSIONS: ISA at follow-up is more common after SES implantation than after BMS implantation. Considering the current very sensitive IVUS definition, ISA appears to be an IVUS finding without significant impact on the incidence of major adverse cardiac events even during long-term follow-up.

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El tratamiento ambulatorio es una de las medidas que pueden imponer los jueces a los menores con conductas delictivas. Este artículo propone una reflexión sobre la aplicación de estos tratamientos, partiendo de la experiencia de un programa de salud mental cuya finalidad es atender a adolescentes y jóvenes denunciados a la justicia. Se analiza como abordar cuestiones como la motivación y al vinculación frente a la imposición de la medida, su focalización y desarrollo, así como la necesidad del trabajo interdisciplinar con los equipos de Justicia Juvenil para realizarlos (AU)

One of the measures which can be imposed by Spanish juvenile courts on delinquent minors is an outpatient treatment. This paper is a reflection on the application of this kind of treatmens in a mental health service for adolescents vhoy have been reported to the juvenile justice sustem. Several relevant issues involved in theses treatments such as personal motivation and bondung facing the imposition of the mesasure, psychodynamic focus and develpment, and the need for an interdisciplinary approach, ate all examined in this paper (AU)

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Three methods (using GM3 quantities ranging from a few milligrams to grams) have been developed to prepare, in high yield, the three derivatives of ganglioside GM3 [alpha-Neu5Ac-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-ceramide]: deacetyl-GM3 [alpha-Neu-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-ceramide], lyso-GM3 [alpha-Neu5Ac-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-sphingosine], and deacetyl-lyso-GM3 [alpha-Neu-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-sphingosine]. This is the first report of the preparation of lyso-GM3 by a one-pot reaction. We can now define the optimal conditions for the different preparations. Preparation of deacetyl-GM3: alkaline reagent, 2 M KOH in water; GM3 concentration, 33 mg/ml; reaction temperature, 90 degrees C; reaction time, 3.5 h; nitrogen atmosphere. Preparation of deacetyl-lyso-GM3: alkaline reagent, 8 M KOH in water; GM3 concentration, 10 mg/ml; reaction temperature, 90 degrees C; reaction time, 18 h; nitrogen atmosphere. Preparation of lyso-GM(3): alkaline reagent, 1 M sodium tert-butoxide in methanol; GM3 concentration, 10 mg/ml; reaction temperature, 80 degrees C; reaction time, 18 h; anhydrous conditions. The percentage yield of deacetyl-GM3 was 70;-75%, that of deacetyl-lyso-GM3 100%, and of lyso-GM3 36;-40%.Deacetyl-GM3, deacetyl-lyso-GM3, and lyso-GM3 were purified by column chromatography, and chemical structures were confirmed by electron spray-mass spectrometry.

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Anticoagulation for the pregnant patient with valve disease is problematic: both the underlying thrombotic disorder and the pharmacologic agents available for its treatment pose significant risks to the mother and fetus. There are no randomized controlled trial data available to guide decision-making for this patient population. Clinical treatment algorithms usually are derived from patient registries or case series with the obvious limitations of retrospective review, selection bias, historical controls, and small patient numbers. Prospective trials clearly are needed, but clinical research in the pregnant patient presents a myriad of ethical and legal challenges. Warfarin and unfractionated heparin, the mainstays of anticoagulant therapy, fall quite short in any analysis of efficacy and safety. There is an increasing use of low molecular weight heparins (LMWHs) in clinical practice but without evidence-based validation. Anticoagulant management of the pregnant patient must begin with full disclosure of the hazards and limitations of all forms of available treatments, preferably prior to conception. Treatment should be predicated on an assessment of the relative risks of thrombosis and hemorrhage. Careful monitoring and dosage adjustment are required throughout gestation, labor, delivery, and the puerperium.

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There is less certainty about the recommendations for radio-frequency ablation as a therapeutic option for Wolff-Parkinson-White syndrome in children as opposed to adults because of the different natural history and the age-related risks of ablation. To help decision-making, we evaluated the long term clinical evolution and electrophysiologic characteristics of pre-excitation in our series of children and young adults. We reviewed the clinical course of 109 patients below the age of 18 years who had been followed up over a period of 9+4 years, with a range from one to 27 years. This corresponded to 986 patient-years. We examined also the electrophysiologic data from 98 of those patients who underwent a transesophageal study. At the discovery of pre-excitation, 59% of patients were asymptomatic, while 29% developed supraventricular tachycardia during follow-up. The peak incidence of the onset of supraventricular tachycardia occurred during infancy. These patients had the highest incidence of subsequent spontaneous disappearance of the tachycardia (53%), such a favourable evolution being encountered in only 12% of patients in whom the abnormal rhythm first appeared after 12 years of age. Chronic medical treatment was required in 47% of patients, and proved completely effective in 45% of cases. The potential to induce atrial fibrillation, and the incidence pre-excitation considered to be high risk, with the shortest pre-excited RR interval equal to or less than 220 msec, was lowest in the group of patients aged less than 6 years of age, and highest in those older than 12 years of age (p <0.001). Pathways producing arrhythmia with high risk were more common in symptomatic (29%) than in asymptomatic patients (7%) (p<0.001). No mortality occurred. On the basis of our findings, we suggest that ablation should be avoided before the age of 5 or 6 years. Thereafter, the procedure should become the first line of treatment for symptomatic patients older than 12 years of age.

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Src family kinases play a relevant role in the development and differentiation of neuronal cells. They are abundant in sphingolipid-enriched membrane domains of many cell types, and these domains are hypothesized to function in bringing together molecules important to signal transduction. We studied the association of Src family tyrosine kinases and their negative regulatory kinase, Csk, with sphingolipids in sphingolipid-enriched domains of rat cerebellar granule cells differentiated in culture. We find that c-Src, Lyn and Csk are enriched in the sphingolipid-enriched fraction prepared from these cells. Coimmunoprecipitation experiments show that these and sphingolipids are part of the same domain. Cross-linking experiments with a photoactivable, radioactive GD1b derivative show that c-Src and Lyn, which are anchored to the membrane via a myristoyl chain, associate directly with GD1b. Csk, which is not inserted in the hydrophobic core of the membrane, is not photolabeled by this ganglioside. These results suggest that lipid-lipid, lipid-protein, and protein-protein interactions cooperate to maintain domain structure. We hypothesize that such interactions might play a role in the process of neuronal differentiation.

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A simple procedure is described for preparing GM3 ganglioside, from a few milligrams to grams, from GM1-lactone (Sonnino et al., (1985) Glycoconjugate J 2: 343-54) [1]. The synthesis was carried out under the following optimal conditions: 30 mM GM1-lactone in 0.25 M H2SO4 in DMSO, 30 min, 70 degrees C, nitrogen atmosphere, strong stirring. The yield of GM3 was 55%. The procedure applied to milligram amounts of GD1b-dilactone gave GD3 ganglioside.

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The purpose of this study was to assess the feasibility of stent implantation without predilation in patients with a single, noncalcified coronary artery lesion. A total of 122 patients were randomized to receive a stent with or without predilation; direct stent placement was possible in 59 of the 61 patients (97%) with an immediate and long-term clinical follow-up similar to that observed in the group of patients treated conventionally.

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