Using Redox Proteomics to Gain New Insights into Neurodegenerative Disease and Protein Modification.

Antioxidant defenses in biological systems ensure redox homeostasis, regulating baseline levels of reactive oxygen and nitrogen species (ROS and RNS). Oxidative stress (OS), characterized by a lack of antioxidant defenses or an elevation in ROS and RNS, may cause a modification of biomolecules, ROS being primarily absorbed by proteins. As a result of both genome and environment interactions, proteomics provides complete information about a cell's proteome, which changes continuously. Besides measuring protein expression levels, proteomics can also be used to identify protein modifications, localizations, the effects of added agents, and the interactions between proteins. Several oxidative processes are frequently used to modify proteins post-translationally, including carbonylation, oxidation of amino acid side chains, glycation, or lipid peroxidation, which produces highly reactive alkenals. Reactive alkenals, such as 4-hydroxy-2-nonenal, are added to cysteine (Cys), lysine (Lys), or histidine (His) residues by a Michael addition, and tyrosine (Tyr) residues are nitrated and Cys residues are nitrosylated by a Michael addition. Oxidative and nitrosative stress have been implicated in many neurodegenerative diseases as a result of oxidative damage to the brain, which may be especially vulnerable due to the large consumption of dioxygen. Therefore, the current methods applied for the detection, identification, and quantification in redox proteomics are of great interest. This review describes the main protein modifications classified as chemical reactions. Finally, we discuss the importance of redox proteomics to health and describe the analytical methods used in redox proteomics.

Beneficial Bacteria and Plant Extracts Promote Honey Bee Health and Reduce Nosema ceranae Infection.

The research aims to give new insights on the effect of administering selected bacterial strains, isolated from honey bee gut, and/or a commercial plant extract blend (HiveAlive®) on Nosema ceranae. Analyses were first performed under laboratory conditions such as different infective doses of N. ceranae, the effect of single strains and their mixture and the influence of pollen administration. Daily survival and feed consumption rate were recorded and pathogen development was analysed using qPCR and microscope counts. Biomarkers of immunity and physiological status were also evaluated for the different treatments tested using one bacterial strain, a mixture of all the bacteria and/or a plant extract blend as treatments. The results showed an increase of abaecin transcript levels in the midgut of the honey bees treated with the bacterial mixture and an increased expression of the protein vitellogenin in the haemolymph of honey bees treated with two separate bacterial strains (Bifidobacterium coryneforme and Apilactobacillus kunkeei). A significant effectiveness in reducing N. ceranae was shown by the bacterial mixture and the plant extract blend regardless of the composition of the diet. This bioactivity was seasonally linked. Quantitative PCR and microscope counts showed the reduction of N. ceranae under different experimental conditions. The antiparasitic efficacy of the treatments at field conditions was studied using a semi-field approach which was adapted from research on insecticides for the first time, to analyse antiparasitic activity against N. ceranae. The approach proved to be reliable and effective in validating data obtained in the laboratory. Both the mixture of beneficial bacteria and its association with Hive Alive® are effective in controlling the natural infection of N. ceranae in honey bee colonies.

Interstitial Lung Disease Is Associated with Sleep Disorders in Rheumatoid Arthritis Patients.

OBJECTIVE: To evaluate sleep disorders and associated factors in patients with rheumatoid-arthritis-associated interstitial lung disease (RA-ILD). METHODS: We performed an observational study of 35 patients with RA-ILD (cases) and 35 age- and sex-matched RA patients without ILD (controls). We evaluated sleep disorders (Oviedo Sleep Questionnaire), positive psychological factors (resilience using the Wagnild and Young Resilience Scale, emotional intelligence using the 24-item Trait Meta-Mood Scale), anxiety and depression (Hospital Anxiety and Depression Scale), quality of life (36-item short-form survey), and fatigue (Functional Assessment of Chronic Illness Therapy Questionnaire). Other variables studied included the Charlson Comorbidity Index (CCI) and RA activity according to the DAS28-ESR. RESULTS: Compared to the controls, the cases were characterized by poorer sleep quality with a higher prevalence of insomnia (42% vs. 20%; p = 0.039), greater severity of insomnia (p = 0.001), and lower sleep satisfaction (p = 0.033). They also had poorer resilience and emotional recovery and more severe anxiety and depression. A diagnosis of ILD was the only factor independently associated with the three dimensions of sleep quality. The predictors of poorer sleep satisfaction in patients with RA-ILD were age (ß = -0.379), DAS28-ESR (ß = -0.331), and usual interstitial pneumonia pattern (ß = -0.438). The predictors of insomnia were DAS28-ESR (ß = 0.294), resilience (ß = -0.352), and CCI (ß = 0.377). CONCLUSIONS: RA-ILD is associated with significant sleep disorders. RA-ILD seems to be an independent risk factor for sleep alterations, with a greater impact on insomnia. Age, disease activity, and comorbidity also play a role in sleep disorders in patients with RA-ILD.

Combined effect of a neonicotinoid insecticide and a fungicide on honeybee gut epithelium and microbiota, adult survival, colony strength and foraging preferences.

Fungicides, insecticides and herbicides are widely used in agriculture to counteract pathogens and pests. Several of these molecules are toxic to non-target organisms such as pollinators and their lethal dose can be lowered if applied as a mixture. They can cause large and unpredictable problems, spanning from behavioural changes to alterations in the gut. The present work aimed at understanding the synergistic effects on honeybees of a combined in-hive exposure to sub-lethal doses of the insecticide thiacloprid and the fungicide penconazole. A multidisciplinary approach was used: honeybee mortality upon exposure was initially tested in cage, and the colonies development monitored. Morphological and ultrastructural analyses via light and transmission electron microscopy were carried out on the gut of larvae and forager honeybees. Moreover, the main pollen foraging sources and the fungal gut microbiota were studied using Next Generation Sequencing; the gut core bacterial taxa were quantified via qPCR. The mortality test showed a negative effect on honeybee survival when exposed to agrochemicals and their mixture in cage but not confirmed at colony level. Microscopy analyses on the gut epithelium indicated no appreciable morphological changes in larvae, newly emerged and forager honeybees exposed in field to the agrochemicals. Nevertheless, the gut microbial profile showed a reduction of Bombilactobacillus and an increase of Lactobacillus and total fungi upon mixture application. Finally, we highlighted for the first time a significant honeybee diet change after pesticide exposure: penconazole, alone or in mixture, significantly altered the pollen foraging preference, with honeybees preferring Hedera pollen. Overall, our in-hive results showed no severe effects upon administration of sublethal doses of thiacloprid and penconazole but indicate a change in honeybees foraging preference. A possible explanation can be that the different nutritional profile of the pollen may offer better recovery chances to honeybees.

Conradi-Hünerman-Happle Syndrome and Obsessive-Compulsive Disorder: a clinical case report.

BACKGROUND: Obsessive-Compulsive Disorder (OCD) is a common and chronic psychiatric disorder with significant morbidity characterized by intrusive, uncontrollable and reoccurring thoughts (i.e., obsessions) and/or ritualistic behaviours (i.e., compulsions). Conradi-Hünerman-Happle Syndrome (CHHS) is a rare inherited X-linked dominant variant of chondrodysplasia punctata, a heterogeneous group of rare bone dysplasias characterized by punctate epiphyseal calcifications of complex etiology and pathophysiology that remain to be defined. Available literature reveals a lacuna in regards to the coexistence of the entities with no clinical reports described. CASE PRESENTATION: A 12 year old female patient with diagnosis of CHHS, presents to psychiatric consultation due to aggravation of her OCD clinical picture, with aggravation of hand-washing frequency during the Covid-19 pandemic with significant functional impact. Psychopharmacological treatment aimed at OCD with Selective Serotonin Reuptake Inhibitor (SSRI) and antipsychotic was instituted with favourable, albeit partial response. CONCLUSIONS: The authors aim to describe a clinical case in which the patient presents with Conradi-Hünerman-Happle Syndrome and Obsessive-Compulsive Disorder. Clinical descriptions of CHHS and OCD are not available in the literature. Through this case description the authors aim to present a rare case as well as discuss an eventual association between etiology and/or pathophysiology of the two disorders.

Sensibilidad del test de diagnóstico rápido SARS-CoV-2 Panbio en Atención Primaria / Sensitivity of the Panbio COVID-19 rapid antigen detection test in primary care

Introducción y objetivos: los pediatras de Atención Primaria necesitamos técnicas de diagnóstico rápido (TDR) fiables para prevenir la propagación de la enfermedad COVID-19 mediante un cribado temprano y eficaz a la espera de una vacuna. El objetivo de este trabajo fue evaluar como novedad en Atención Primaria, tanto en adultos como niños, sintomáticos y contactos asintomáticos, la sensibilidad (S) de los test de antígeno SARS-CoV-2 Panbio del laboratorio Abbott respecto a la reacción en cadena de la polimerasa (PCR).Pacientes y métodos: se incluyeron 591 pacientes (222 menores de 14 años) (249 sintomáticos y 342 contactos). Se calculó la sensibilidad (S) y la especificidad (E) junto con sus intervalos de confianza (IC) del 95%. La independencia de los dos resultados ha sido analizada mediante el test de McNemar.Resultados: la S del test en adultos fue del 81% (IC 95%: 66,16-96,34) y en niños del 80% (IC 95%: 34,94-100) dentro de los 5 primeros días. En contactos se evaluó la S en los cinco primeros días, en adultos (68%; IC 95%: 51,13-86,37), del 5.º al 9.º día (85%) y en niños (66%; IC 95%: 30,31-100). El tipo de contacto más frecuente fue domiciliario en un 52% de los casos. La E fue 100% en todos los casos.Conclusiones: el test rápido de antígeno SARS-CoV-2 Panbio puede ser útil para diagnóstico de adultos y niños los primeros cinco días de inicio de síntomas, así como entre el 5.º y 9.º día tras el contacto con positivo COVID-19 confirmado, pendiente de interpretar en futuros estudios. (AU)

Introduction and objectives: primary care paediatricians need reliable rapid diagnostic techniques (RDTs) to prevent the spread of coronavirus disease 19 (COVID-19) through early and effective screening while awaiting a vaccine. The objective of this study was to evaluate the sensitivity (Sen) of the Abbott laboratory SARS-CoV-2 Panbio antigen test, newly introduced in primary care, in both adults and children (symptomatic and asymptomatic contacts) in comparison to the polymerase chain reaction (PCR) test.Sample and methods: the study included 591 patients (222 aged less than 14 years) from 7 primary care centres; of who 249 were symptomatic and 342 asymptomatic contacts. We calculated the Sen and specificity (Spe) with their 95% confidence intervals (CIs). We assessed the independence of the two results with the McNemar test.Results: the Sen of the test within 5 days from onset was 81% in adults (95% CI, 66.16-96.34) and 80% in children (95% CI: 34.94-100). In contacts, we assessed the Sen within 5 days, in adults (68%; 95% CI: 51.13- 86.37), in 5 to 9 days (85%) and in children (66%; 95% CI: 30.31-100). The most frequent source of exposure were household contacts (52% of the cases). The Spe was 100% in every case.Conclusions: the Panbio SARS-CoV-2 rapid antigen test can be useful for diagnosis in adults and children within 5 days of onset, and from days 5 to 9 in contacts of confirmed COVID 19 cases. Further studies are required for adequate interpretation of the latter result. (AU)

Guía atrapada durante cambio de electrodo del seno coronario en terapia de resincronización cardiaca / Trapped guidewire during coronary sinus electrode exchange in cardiac resynchronization therapy

Resumen Se presenta el caso de una paciente con atrapamiento de guía al interior del seno coronario durante el procedimiento de cambio de un electrodo (Sentus ProMRI OTW BP L-85) por desalojo asociado a disfunción de la terapia de resincronización cardiaca. Durante el implante del nuevo electrodo se presentó atrapamiento y retención intravascular de la guía utilizada para su posicionamiento a nivel del seno coronario, lo cual hizo imposible su remoción. La paciente no aceptó tratamiento quirúrgico, se encuentra en vigilancia médica y permanece asintomática desde hace 3 años.

Abstract It is reported the case of a patient with guidewire trapping inside the coronary sinus during an electrode exchange procedure (Sentus ProMRI OTW BP L-85) due to dislocation associated with dysfunction of cardiac re-synchronization therapy. During the implantation of the new electrode, entrapment of the guidewire used for its positioning at the level of the coronary sinus and intravascular retention were presented, making it impossible to remove it. The patient did not accept surgical treatment and has been under medical surveillance, asymptomatic for three years.

Screening of Dietary Ingredients against the Honey Bee Parasite Nosema ceranae.

Nosema ceranae is a major pathogen in the beekeeping sector, responsible for nosemosis. This disease is hard to manage since its symptomatology is masked until a strong collapse of the colony population occurs. Conversely, no medicaments are available in the market to counteract nosemosis, and only a few feed additives, with claimed antifungal action, are available. New solutions are strongly required, especially based on natural methods alternative to veterinary drugs that might develop resistance or strongly pollute honey bees and the environment. This study aims at investigating the nosemosis antiparasitic potential of some plant extracts, microbial fermentation products, organic acids, food chain waste products, bacteriocins, and fungi. Honey bees were singularly infected with 5 × 104 freshly prepared N. ceranae spores, reared in cages and fed ad libitum with sugar syrup solution containing the active ingredient. N. ceranae in the gut of honey bees was estimated using qPCR. The results showed that some of the ingredients administered, such as acetic acid at high concentration, p-coumaric acid, and Saccharomyces sp. strain KIA1, were effective in the control of nosemosis. On the other hand, wine acetic acid strongly increased the N. ceranae amount. This study investigates the possibility of using compounds such as organic acids or biological agents including those at the base of the circular economy, i.e., wine waste production, in order to improve honeybee health.

Effects of Synthetic Acaricides and Nosema ceranae (Microsporidia: Nosematidae) on Molecules Associated with Chemical Communication and Recognition in Honey Bees.

Acaricides and the gut parasite Nosema ceranae are commonly present in most productive hives. Those stressors could be affecting key semiochemicals, which act as homeostasis regulators in Apis mellifera colonies, such as cuticular hydrocarbons (CHC) involved in social recognition and ethyl oleate (EO) which plays a role as primer pheromone in honey bees. Here we test the effect of amitraz, coumaphos, tau-fluvalinate and flumethrin, commonly applied to treat varroosis, on honey bee survival time, rate of food consumption, CHC profiles and EO production on N. ceranae-infected and non-infected honey bees. Different sublethal concentrations of amitraz, coumaphos, tau-fluvalinate and flumethrin were administered chronically in a syrup-based diet. After treatment, purified hole-body extracts were analyzed by gas chromatography coupled to mass spectrometry. While N. ceranae infection was also shown to decrease EO production affecting survival rates, acaricides showed no significant effect on this pheromone. As for the CHC, we found no changes in relation to the health status or consumption of acaricides. This absence of alteration in EO or CHC as response to acaricides ingestion or in combination with N. ceranae, suggests that worker honey bees exposed to those highly ubiquitous drugs are hardly differentiated by nest-mates. Having determined a synergic effect on mortality in worker bees exposed to coumaphos and Nosema infection but also, alterations in EO production as a response to N. ceranae infection it is an interesting clue to deeper understand the effects of parasite-host-pesticide interaction on colony functioning.

Sub-lethal effects of the consumption of Eupatorium buniifolium essential oil in honeybees.

When developing new products to be used in honeybee colonies, further than acute toxicity, it is imperative to perform an assessment of risks, including various sublethal effects. The long-term sublethal effects of xenobiotics on honeybees, more specifically of acaricides used in honeybee hives, have been scarcely studied, particularly so in the case of essential oils and their components. In this work, chronic effects of the ingestion of Eupatorium buniifolium (Asteraceae) essential oil were studied on nurse honeybees using laboratory assays. Survival, food consumption, and the effect on the composition of cuticular hydrocarbons (CHC) were assessed. CHC were chosen due to their key role as pheromones involved in honeybee social recognition. While food consumption and survival were not affected by the consumption of the essential oil, CHC amounts and profiles showed dose-dependent changes. All groups of CHC (linear and branched alkanes, alkenes and alkadienes) were altered when honeybees were fed with the highest essential oil dose tested (6000 ppm). The compounds that significantly varied include n-docosane, n-tricosane, n-tetracosane, n-triacontane, n-tritriacontane, 9-tricosene, 7-pentacosene, 9-pentacosene, 9-heptacosene, tritriacontene, pentacosadiene, hentriacontadiene, tritriacontadiene and all methyl alkanes. All of them but pentacosadiene were up-regulated. On the other hand, CHC profiles were similar in healthy and Nosema-infected honeybees when diets included the essential oil at 300 and 3000 ppm. Our results show that the ingestion of an essential oil can impact CHC and that the effect is dose-dependent. Changes in CHC could affect the signaling process mediated by these pheromonal compounds. To our knowledge this is the first report of changes in honeybee cuticular hydrocarbons as a result of essential oil ingestion.

Complications related to cardiac implantable electronic devices / Complicaciones asociadas al implante de dispositivos cardiacos electrónicos

Abstract Introduction: complications due to cardiac implantable electronic devices have been sparsely studied despite the increased number and complexity of these procedures in a population with multiple comorbidities. Objective: to determine the complication rate and associated risk factors at a reference center in Colombia. Methods: retrospective cohort study, which included patients who had a cardiac electronic device implanted between 2012 and 2015. Clinical records were reviewed to determine if patients developed complications during the year after the procedure, and, if so, which type and which clinical variables could be related to. Results: a total of 897 patients were included, 620 with pacemaker implants and 277 with other devices. The average age was 71.4 years, 63.9% were men, almost all the patients had a chronic disease, and 70% were de novo implants. The global complication rate was 10.9%; Lead displacement (3.6%) and pocket hematoma (3.3%) were the most frequent complications; 7.5% were major complications, and 73.5% occurred in the first month after procedure. The hospitalization rate associated with complications was 9.5%, and the median hospital stay was seven days, with 66.3% of these patients requiring new interventions. The mortality rate was 0.2% Conclusions: complications associated with cardiac implantable electronic devices occur red mainly in the first trimester after the initial intervention, were more frequent in patients under 80 years old, increased according to device complexity, and were not related to with the studied comorbidities.

Resumen Introducción: las complicaciones secundarias al implante de dispositivos cardiacos electrónicos han sido poco estudiadas a pesar del aumento en número y complejidad de estos procedimientos en población con múltiples comorbilidades. Objetivo: determinar la tasa de complicaciones del implante de dispositivos y los factores de riesgo asociados, en un centro de referencia en Colombia. Métodos: estudio de cohorte retrospectiva, que incluyó pacientes a quienes se les implantó dispositivo electrónico cardiaco entre 2012 y 2015. Se revisó la historia clínica para determinar si durante un año posterior al procedimiento, presentaron complicaciones, de qué tipo y con qué variables clínicas podría asociarse. Resultados: se incluyeron 897 pacientes, 620 con implante de marcapaso y 277 otros dispositivos. La edad promedio fue 71.4 años, 63.9% hombres, con múltiples enfermedades crónicas, 70% fueron implantes de novo. Se observó una tasa de complicaciones del 10.9%, la cual varía de acuerdo con el tipo de dispositivo. El desalojo del electrodo (3.6%) y el hematoma del bolsillo (3.3%) fueron las complicaciones más frecuentes, 7.5% fueron complicaciones mayores y 73.5% se presentaron en el primer mes postoperatorio. La tasa de hospitalización asociada a complicación fue 9.5%, mediana de estancia de 7 días, con un 66.3% de los pacientes en requerimiento de reintervención. La tasa de mortalidad fue del 0.2%. Conclusiones: las complicaciones asociadas al implante de dispositivos eléctricos cardiacos se presentaron principalmente en el primer trimestre, fueron más frecuentes en menores de 80 años, aumentaron con la complejidad del dispositivo y no se relacionaron con las comorbilidades estudiadas.

Obstructive sleep apnea diagnosed by peripheral arterial tonometry in patients with cardiovascular disease / Apnea obstructiva del sueño diagnosticada por tonometría arterial periférica en pacientes con enfermedad cardiovascular

Abstract Introduction: Obstructive sleep apnea is an independent cardiovascular risk factor which can be diagnosed by means of a portable device (WatchPAT®) capable to perform automatic analysis of the peripheral artery tonometry, its amplitude and variability, with a high grade of correlation to polysomnography. Objective: To describe the use of WatchPAT® for diagnosing obstructive sleep apnea in patients with cardiovascular disease. Methods: A case series study of patients evaluated in cardiovascular consultation who underwent to home sleep monitoring using the WatchPAT®200U system between February 1, 2017 to February 1, 2018, due to suspected obstructive sleep apnea. Results: 37 patients were included, with an average age of 57.6 years, most of whom were male. Cardiovascular diseases were: uncontrolled or predominantly nocturnal hypertension (37.8%), recurrent palpitations and/or chronic fatigue (21.6%); sleep disturbances were informed only in 27% of the patients. Obstructive sleep apnea was identified in 97.3% of the patients (29.7% moderate and 56.8% severe), 37.8% had high blood pressure, 49.5% had established heart disease, and 18.9% were diabetic. Only, fifty percent of the patients with severe obstructive sleep apnea and 45% with moderate obstructive sleep apnea snored. Conclusion: Cardiovascular disease specialists should participate more actively in the search and diagnosis of obstructive sleep apnea; it is highly prevalent in patients with nocturnal or resistant high blood pressure, heart rhythm disorders or myocardial structural damage. Obstructive sleep apnea can be diagnosed using portable devices such as WatchPAT®.

Resumen Introducción: La apnea obstructiva del sueño es un factor independiente de riesgo cardiovascular. Para su diagnóstico existen dispositivos portátiles (WatchPAT®) que utilizan el análisis automático de la amplitud y variabilidad del tono arterial periférico logrando una alta correlación con la polisomnografía. Objetivo: Describir el uso del WatchPAT® para el diagnóstico de apnea obstructiva del sueño en pacientes con patología cardiovascular. Métodos: Serie de casos que incluyó pacientes valorados en consulta cardiovascular a quienes se les realizó monitorización del sueño en casa mediante WatchPAT®200U por sospecha de apnea obstructiva del sueño entre el 1( de febrero de 2017 y el 1( de febrero de 2018. Resultados: Se incluyeron 37 pacientes, edad promedio 57,6 años, la mayoría hombres. Las enfermedades cardiovasculares principales fueron: hipertensión arterial no controlada o de predominio nocturno (37,8%), palpitaciones recurrentes y/o fatiga crónica (21,6%); se informaron alteraciones del sueño solo en 27% de los pacientes. Se identificó apnea obstructiva del sueño en 97,3% de los pacientes (29,7% moderado y 56,8% severo); 3,8% eran hipertensos, 45,9% tenían enfermedad cardíaca establecida y 18,9% eran diabéticos. Solo 50% de los pacientes con apnea obstructiva del sueño severa y 45% moderada roncaban. Conclusión: Los especialistas en patología cardiovascular deben tener una participación más activa en la búsqueda y diagnóstico de apnea obstructiva del sueño, altamente prevalente en pacientes con hipertensión arterial nocturna o resistente, en trastornos del ritmo cardiaco o en aquellos con daño estructural del miocardio. Su diagnóstico puede realizarse con dispositivos portátiles como WatchPAT®.

Nosema ceranae in South American Native Stingless Bees and Social Wasp.

Besides the incipient research effort, the role of parasites as drivers of the reduction affecting pollinator populations is mostly unknown. Given the worldwide extension of the beekeeping practice and the diversity of pathogens affecting Apis mellifera populations, honey bee colonies are a certain source of parasite dispersion to other species. Here, we communicate the detection of the microsporidium Nosema ceranae, a relatively new parasite of honey bees, in stingless bees (Meliponini) and the social wasp Polybia scutellaris (Vespidae) samples from Argentina and Brazil by means of duplex PCR. Beyond the geographic location of the nests, N. ceranae was detected in seven from the eight Meliponini species analyzed, while Nosema apis, another common parasite of A. mellifera, was absent in all samples tested. Further research is necessary to determine if the presence of the parasite is also associated with established infection in host tissues. The obtained information enriches the current knowledge about pathologies that can infect or, at least, be vectored by native wild pollinators from South America.

Sublethal effects of acaricides and Nosema ceranae infection on immune related gene expression in honeybees.

Nosema ceranae is an obligate intracellular parasite and the etiologic agent of Nosemosis that affects honeybees. Beside the stress caused by this pathogen, honeybee colonies are exposed to pesticides under beekeeper intervention, such as acaricides to control Varroa mites. These compounds can accumulate at high concentrations in apicultural matrices. In this work, the effects of parasitosis/acaricide on genes involved in honeybee immunity and survival were evaluated. Nurse bees were infected with N. ceranae and/or were chronically treated with sublethal doses of coumaphos or tau-fluvalinate, the two most abundant pesticides recorded in productive hives. Our results demonstrate the following: (1) honeybee survival was not affected by any of the treatments; (2) parasite development was not altered by acaricide treatments; (3) coumaphos exposure decreased lysozyme expression; (4) N. ceranae reduced levels of vitellogenin transcripts independently of the presence of acaricides. However, combined effects among stressors on imagoes were not recorded. Sublethal doses of acaricides and their interaction with other ubiquitous parasites in colonies, extending the experimental time, are of particular interest in further research work.

Immune-related gene expression in nurse honey bees (Apis mellifera) exposed to synthetic acaricides.

The mite Varroa destructor is an ectoparasite affecting honey bees worldwide. Synthetic acaricides have been among the principal tools available to beekeepers for its control, although several studies have shown its negative effects on honey bee physiology. Recent research suggests that those molecules strongly impact on immune signaling cascades and cellular immunity. In the present work, LC(50) in six-day-old bees were determined for the following acaricides: tau-fluvalinate, flumethrin, amitraz and coumaphos. According to this obtained value, a group of individuals was treated with each acaricide and then processed for qPCR analysis. Transcript levels for genes encoding antimicrobial peptides and immune-related proteins were assessed. Flumethrin increased the expression of hymenoptaecin when comparing treated and control bees. Significant differences were recorded between coumaphos and flumethrin treatments, while the first one reduced the expression of hymenoptaecin and abaecin, the last one up-regulated their expressions. No significant statistically changes were recorded in the expression levels of vitellogenin, lysozyme or glucose dehydrogenase among bees treated with acaricides and control bees. This work constitutes the first report, under laboratory conditions, about induction of immune related genes in response to synthetic miticides.

Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity.

The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). The constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. In the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function.

Phagocytic amoebocyte sub populations in the perivisceral coelom of the sea urchin Lytechinus variegatus (Lamarck, 1816).

The echinoderms are deuterostomic animals with a nonspecific immune system similar to that of vertebrates. Among coelomocytes, phagocytic amoebocytes have a key role in the nonspecific immune response in sea urchin, being responsible for microorganisms elimination through phagocytosis and also for humoral secretions of a wide spectrum. Sub-populations of phagocytic amoebocytes (PA) have been previously described and two distinct sub populations in the oral (OR) and aboral (AB) regions of the perivisceral coelom of L.variegatus in the present study were found. In the OR there is a higher number of PA with higher phagocytic capacity after 30 minutes of incubation with yeast and higher percentage of intranuclear iron crystalloids. The germicide capacity under the fluorescence technique did not show any difference. SDS-PAGE analysis showed different protein patterns between coelomocytes of OR and AB. Gravitational force had no effect in PA distribution and no physical barrier was found in the perivisceral coelom. The other coelomocyte (vibratile cells, red spherulocytes and white spherulocytes) populations were not different in OR compared with AB in their distribution. Some aspects of the possible causes of the differences found for PA are discussed in the paper.

The intracellular distribution and secretion of endopeptidases 24.15 (EC 3.4.24.15) and 24.16 (EC 3.4.24.16).

Endopeptidase 24.15 (EC 3.4.24.15; EP24.15) and endopeptidase 24.16 (EC 3.4.24.16; EP24.16) are enzymes involved in general peptide metabolism in mammalian cells and tissues. This review will focus on morphological and biochemical aspects related to the subcellular distribution and secretion of these homologous enzymes in the central nervous system. These are important issues for a better understanding of the functions of EP24.15 and EP24.16 within neuroendocrine systems.