Neonicotinoids in the agroecosystem: In-field long-term assessment on honeybee colony strength and microbiome.

Bees can be severely affected by various plant protection products (PPP). Among these, neonicotinoid insecticides are of concern as they have been shown to be responsible for extensive honeybee colonies death when released into the environment. Also, sublethal neonicotinoid doses contaminating single honeybees and their colonies (e.g. through contaminated pollen) are responsible for honeybees physiological alterations with probable implication also on microbiome functionality. Honeybees show symbiotic interactions with specific gut bacteria that can enhance the adult host performances. Among the known mechanisms, the modulation of the immune system, the degradation of recalcitrant secondary plant metabolites, pollen digestion, and hormonal signaling, are the most important functional benefits for the host honeybee. To date, few research efforts have aimed at revealing the impact of PPP on the gut microbial community of managed and wild honeybees. The majority of the existing literature relays on cage or semifield tests of short duration for research investigating neonicotinoids-gut microbiome interactions. This research wanted to unravel the impact of two neonicotinoids (i.e. imidacloprid and thiacloprid) in natural field conditions up to 5 weeks of exposure. A long-term impact of neonicotinoids on gut microbial community of honeybees was observed. The alterations affected several microbial genera and species such as Frischella spp., lactobacilli and bifidobacteria, whose shifting is implicated in intestinal dysbiosis. Long-term impact leading to dysbiosis was detected in case of exposure to imidacloprid, whereas thiacloprid exposure stimulated temporary dysbiosis. Moreover, the microbial diversity was significantly reduced in neonicotinoid-treated groups. Overall, the reported results support a compromised functionality of the gut microbial community, that might reflect a lower efficiency in the ecosystemic functionality of honeybees.

Sox2 is not required for melanomagenesis, melanoma growth and melanoma metastasis in vivo.

Melanoma is a dangerous form of skin cancer derived from the malignant transformation of melanocytes. The transcription factor SOX2 is not expressed in melanocytes, however, it has been shown to be differentially expressed between benign nevi and malignant melanomas and to be essential for melanoma stem cell maintenance and expansion in vitro and in xenograft models. By using a mouse model in which BRafV600E mutation cooperates with Pten loss to induce the development of metastatic melanoma, we investigated if Sox2 is required during the process of melanomagenesis, melanoma growth and metastasis and in the acquisition of resistance to BRAF inhibitors (BRAFi) treatments. We found that deletion of Sox2 specifically in Pten null and BRafV600E-expressing melanocytes did not prevent tumor formation and did not modify the temporal kinetics of melanoma occurrence compared to Sox2 wt mice. In addition, tumor growth was similar between Sox2 wt and Sox2 deleted (del) melanomas. By querying publicly available databases, we did not find statistically significant differences in SOX2 expression levels between benign nevi and melanomas, and analysis on two melanoma patient cohorts confirmed that Sox2 levels did not significantly change between primary and metastatic melanomas. Melanoma cell lines derived from both Sox2 genotypes showed a similar sensitivity to vemurafenib treatment and the same ability to develop vemurafenib resistance in long-term cultures. Development of vemurafenib resistance was not dependent on SOX2 expression also in human melanoma cell lines in vitro. Our findings exclude an oncogenic function for Sox2 during melanoma development and do not support a role for this transcription factor in the acquisition of resistance to BRAFi treatments.

Host preference and host colonization of the Asian long-horned beetle, Anoplophora glabripennis (Coleoptera Cerambycidae), in Southern Europe.

The Asian long-horned beetle (ALB), Anoplophora glabripennis (Motschulsky), is a highly polyphagous invasive pest with a broad range of host species, but showing relevant differences between infestation areas. Host preference and host colonization (female fecundity, egg and larval survival) were assessed in a population in Northern Italy by choice and no-choice experiments conducted in both field and laboratory conditions. During 5 years of field observations, ALB was found to infest seven genera of trees: Acer, Aesculus, Betula, Populus, Prunus, Salix and Ulmus. However, Acer, Betula, Ulmus and Salix resulted to be the preferred hosts corresponding to 97.5% (1112) of the 1140 infested trees. In both laboratory and field trials carried out on these four host genera, no-choice experiments recorded the highest host colonization of A. glabripennis on Acer trees, with the highest number of laid eggs and the lowest egg and larval mortality. Ulmus and Salix showed a lower number of laid eggs during laboratory choice test, but egg and larval mortality had mean values similar to Acer. On the contrary, despite the high number of Betula trees felled during the eradication plan carried out in the infestation area, this tree species showed the lowest beetle suitability in terms of number of laid eggs and insect survival. An overestimation of the number of infested Betula occurring during the tree survey may explain the discordance between high number of infested Betula and low beetle suitability. Instead, the large number of infested Acer recorded in the field was probably due to the high abundance of these trees occurring in parks and gardens within the infestation area and to the low adult dispersal of A. glabripennis. Overall, results from this study confirm that host species affects both beetle colonization and breeding performance. The study shows ALB host preference and host suitability varying between tree species, suggesting an ALB acceptance even of sub-optimal hosts.

Thyroid hormone reduces inflammatory cytokines improving glycaemia control in alloxan-induced diabetic wistar rats.

AIM: This study aimed at evaluating whether thyroid hormone treatment could improve glycaemia and insulin response in alloxan-induced diabetic rats by altering cytokine expression in the skeletal muscle and epididymal white adipose tissue (eWAT) as well as altering inflammatory cell infiltration in eWAT. METHODS: Diabetes mellitus (DM) was induced in male Wistar rats by alloxan injection, and a subset of the diabetic rats was treated with T3 (1.5 µg per 100 g body weight) for a 28-day period (DT3 ). Cytokines were measured in serum (MILIplex assay kit) as well as in soleus and EDL skeletal muscles and eWAT by Western blotting. Thyroid function was evaluated by morphological, molecular and biochemical parameters. Cardiac function was assessed by measuring heart rate, blood pressure, maximal rate of pressure development (dp/dtmax ) and decline (dp/dtmin ) as well as the contractility index (CI). Sixty rats were used in the study. RESULTS: Diabetic rats exhibited decreased thyroid function and increased inflammatory cytokines in serum, soleus muscle and eWAT. T3 treatment decreased glycaemia and improved insulin sensitivity in diabetic animals. These alterations were accompanied by decreased TNF-alpha and IL-6 content in soleus muscle and eWAT, and inflammatory cell infiltration in eWAT. T3 treatment did not affect cardiac function of diabetic rats. CONCLUSIONS: The present data provide evidence that T3 treatment reduces glycaemia and improves insulin sensitivity in diabetic rats, and that at least part of this effect could result from its negative modulation of inflammatory cytokine expression.

Long-term type 1 diabetes impairs decidualization and extracellular matrix remodeling during early embryonic development in mice.

INTRODUCTION: Endometrial decidualization and associated extracellular matrix (ECM) remodeling are critical events to the establishment of the maternal-fetal interface and successful pregnancy. Here, we investigated the impact of type 1 diabetes on these processes during early embryonic development, in order to contribute to the understanding of the maternal factors associated to diabetic embryopathies. METHODS: Alloxan-induced diabetic Swiss female mice were bred after different periods of time to determine the effects of diabetes progression on the development of gestational complications. Furthermore, the analyses focused on decidual development as well as mRNA expression, protein deposition and ultrastructural organization of decidual ECM. RESULTS: Decreased number of implantation sites and decidual dimensions were observed in the group mated 90-110 days after diabetes induction (D), but not in the 50-70D group. Picrosirius staining showed augmentation in the fibrillar collagen network in the 90-110D group and, following immunohistochemical examination, that this was associated with increase in types I and V collagens and decrease in type III collagen and collagen-associated proteoglycans biglycan and lumican. qPCR, however, demonstrated that only type I collagen mRNA levels were increased in the diabetic group. Alterations in the molecular ratio among distinct collagen types and proteoglycans were associated with abnormal collagen fibrillogenesis, analyzed by transmission electron microscopy. CONCLUSIONS: Our results support the concept that the development of pregnancy complications is directly related with duration of diabetes (progression of the disease), and that this is a consequence of both systemic factors (i.e. disturbed maternal endocrine-metabolic profile) and uterine factors, including impaired decidualization and ECM remodeling.

Emx2 is a dose-dependent negative regulator of Sox2 telencephalic enhancers.

The transcription factor Sox2 is essential for neural stem cells (NSC) maintenance in the hippocampus and in vitro. The transcription factor Emx2 is also critical for hippocampal development and NSC self-renewal. Searching for 'modifier' genes affecting the Sox2 deficiency phenotype in mouse, we observed that loss of one Emx2 allele substantially increased the telencephalic ß-geo (LacZ) expression of a transgene driven by the 5' or 3' Sox2 enhancer. Reciprocally, Emx2 overexpression in NSC cultures inhibited the activity of the same transgene. In vivo, loss of one Emx2 allele increased Sox2 levels in the medial telencephalic wall, including the hippocampal primordium. In hypomorphic Sox2 mutants, retaining a single 'weak' Sox2 allele, Emx2 deficiency substantially rescued hippocampal radial glia stem cells and neurogenesis, indicating that Emx2 functionally interacts with Sox2 at the stem cell level. Electrophoresis mobility shift assays and transfection indicated that Emx2 represses the activities of both Sox2 enhancers. Emx2 bound to overlapping Emx2/POU-binding sites, preventing binding of the POU transcriptional activator Brn2. Additionally, Emx2 directly interacted with Brn2 without binding to DNA. These data imply that Emx2 may perform part of its functions by negatively modulating Sox2 in specific brain areas, thus controlling important aspects of NSC function in development.

The transcription factor Sox2 is required for osteoblast self-renewal.

The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2-3 passages failed to yield any Sox2-null cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2-null cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

Early postoperative cognitive recovery after remifentanil-propofol or sufentanil-propofol anaesthesia for supratentorial craniotomy: a randomized trial.

BACKGROUND AND OBJECTIVE: This study was designed to evaluate early postoperative cognitive recovery after total intravenous anaesthesia with remifentanil-propofol or sufentanil-propofol in patients undergoing craniotomy for supratentorial expanding lesions. METHODS: Sixty patients were consecutively enrolled, and randomly assigned to one of two study groups: remifentanil-propofol or sufentanil-propofol anaesthesia. To evaluate cognitive function the Short Orientation Memory Concentration Test (SOMCT) and Rancho Los Amigos Scale (RLAS) were administered to all patients in a double-blind procedure before surgery at 15, 45 min and 3 h after extubation. RESULTS: Mean extubation time was similar in the two groups (13 +/- 5 min vs. 19 +/- 6 min). A significantly larger number of patients in the remifentanil-propofol group than in the sufentanil-propofol group required antihypertensive medication postoperatively to maintain mean arterial pressure within 20% of baseline (18/30 vs. 4/29; P = 0.0004). Intergroup analysis showed no differences in baseline SOMCT scores (28 +/- 1 vs. 28 +/- 1) whereas mean SOMCT scores at 15, 45 min and 3 h after extubation were significantly higher in the remifentanil-propofol group (30 patients) than in the sufentanil-propofol group (29 patients) (22 +/- 3 vs. 16 +/- 3; P < 0.0001 and 27 +/- 1 vs. 22 +/- 3; P < 0.0001; 28 +/- 1 vs. 26 +/- 2; P = 0.0126). CONCLUSIONS: In conclusion, propofol-remifentanil and propofol-sufentanil are both suitable for fast-track neuroanaesthesia and provide similar intraoperative haemodynamics, awakening and extubation times. Despite a higher risk of treatable postoperative hypertension propofol-remifentanil allows earlier cognitive recovery.

Occurrence of aflatoxins M(1) and M(2) in milk commercialized in Ribeirão Preto-SP, Brazil.

Aflatoxins are toxic metabolites found in foods and feeds. When ruminants eat foodstuffs containing aflatoxins B(1) and B(2), these toxins are metabolized and excreted as aflatoxin M(1) and M(2) in milk. The aim was to determine the incidence of these aflatoxins in commercial milk collected from supermarkets in Ribeirão Preto-SP, Brazil, and consisting of 60 ultrahigh temperature (UHT) milk samples and 79 pasteurized milk samples. The milk samples were analysed according to method 986.16 of AOAC International. None of the milk samples analysed were contaminated with aflatoxin M(2), and aflatoxin M(1) was detected in 29 (20.9%) of samples in the range 50-240 ng l(-1). The results show that despite a high occurrence of aflatoxin M(1) in commercial pasteurized and UHT milk sold in Ribeirão Preto in 1999 and 2000, the contamination level of these toxins could not be considered a serious public health problem according to MERCOSUR Technical Regulations. However, levels in 20.9% of the milk samples exceeded the concentration of 50 ng l(-1) permitted by the European Union. Although it is not necessary to continue monitoring the incidence and levels of aflatoxins M(1) and M(2) in milk samples, surveillance could be appropriate.

Ovule-specific MADS-box proteins have conserved protein-protein interactions in monocot and dicot plants.

OsMADS13 is a rice MADS-box gene that is specifically expressed in developing ovules. The amino acid sequence of OsMADS13 shows 74% similarity to those of FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, the products of two MADS-box genes that are necessary and sufficient to determine ovule identity in Petunia. To assess whether OsMADS13, the putative rice ortholog of FBP7 and FBP11, has an equivalent function, several analyses were performed. Ectopic expression of FBP7 and FBP11 in Petunia results in ectopic ovule formation on sepals and petals. Here we show that ectopic expression of OsMADS13 in rice and Arabidopsis does not result in the formation of such structures. Furthermore, ectopic expression of FBP7 and FBP11 in Arabidopsis also fails to induce ectopic ovule formation. To determine whether protein-protein interactions involving putative class D MADS-box proteins have been conserved, yeast two-hybrid assays were performed. These experiments resulted in the identification of three putative partners of OsMADS13, all of them encoded by AGL2-like genes. Interestingly the Petunia FBP7 protein also interacts with AGL2-like proteins. The evolutionary conservation of the MADS-box protein partners of these ovule-specific factors was confirmed by exchange experiments which showed that the protein partners of OsMADS13 interact with FBP7 and vice versa.

Intubación nasotracheal con la guía de trasiluminación a un paciente con enfermedad de Paget y dificultad de intubación traqueal prevista / Nasotracheal intubation with a lightwand device in a patient with Paget´s disease and expected intubation difficulty

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Photometric assay for polynucleotide phosphorylase.

Polynucleotide phosphorylase (PNPase) is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of NDPs. It is also believed to play a key role in turnover of prokaryotic transcripts, thus regulating gene expression. At the moment, only radioisotopic methods are available for assaying PNPase in crude extracts; these involve incubating [32P]phosphate and poly(A) in the presence of the enzyme, separating [32P]phosphate from [32P]ADP, and quantifying ADP by scintillation counting. Photometric assay using pyruvate kinase and lactate dehydrogenase as auxiliary enzymes is not feasible in crude extracts because of endogenous ATPase activities, which regenerate ADP from the ATP released by pyruvate kinase. Here, we present a simple photometric assay that uses a cyclic detection system which, due to the sequential action of pyruvate kinase and hexokinase, results in an exponential increase of ADP and glucose 6-phosphate. Glucose 6-phosphate is then revealed by a glucose-6-phosphate dehydrogenase reaction. Based on the theoretical model, a linear increase in absorbance is predicted as a function of the square of the reaction time, with a slope proportional to PNPase activity. Experimental data confirmed the theoretical predictions and showed that the assay was quantitative and unquestionably specific. We also devised a simple procedure for determining absolute enzyme activities (expressed in micromoles of product formed per minute) using exact amounts of pure PNPase as internal standards.

Enrichment of the diet with synthetic and natural sources of provitamin A.

The use of available food rich in provitamin A and retinol as well as fortification of local food are known to result in adequate vitamin A status. In Brazil, several regional foods are known to be good sources of provitamin A such as buriti, several palm oils, mango and others. Improving the consumption of these locally available natural sources of provitamin and vitamin A would cover the needs of the vulnerable population. At the same time fortification of industrialized foods with natural and/or synthetic forms of provitamin A could speed up and fill the gap between requirement and low intake of this vitamin in many parts of the country. This approach has been considered by many as the most effective intervention program to prevent micronutrient deficiencies in developing countries. Our previous studies on the subject have shown that cooking vegetable oil, mainly soybean oil, is a very good alternative vehicle to be fortified and supply vitamin A to the population. Lately we have also enriched the same soybean oil with beta-carotene. Addition of this provitamin A to the oil showed it to be stable when heated at cooking and frying temperatures (retention of 92.4 +/- 6.7% and 65.4 +/- 8.6%, respectively). When rat or human food was prepared with carotene-enriched cooking oil, its bioavailability in experimental animals and absorption in humans were shown to be adequate. An alternative for Brazil, besides adding chemical forms of the vitamin to the cooking oil, would be to mix available carotene-rich palm oil to the soybean oil. There are already regional uses of carotenoid-rich palm oils in the preparation of local dishes in some parts of Brazil and this would facilitate its acceptance by the population. Enrichment of common foods in Brazil, such as soybean oil, with chemical forms of beta-carotene or mixing rich sources of provitamin A can be a good alternative to improve the intake of vitamin A by the Brazilian population.

Campus Bio-Medico technique for nasolaryngeal ventilation with reinforced laryngeal mask in dental surgery: a patient report.

The authors report the usefulness of a prototype nasal laryngeal mask airway (LMA) used successfully in a disabled 20-year-old woman with severe psychomotor retardation and a compromised airway with predictable indexes of impossible tracheal intubation in direct laryngoscopy. A 16-ch Foley catheter was inserted through the patient's left nostril and guided through her mouth. A size-3 reinforced LMA was positioned and connected to the distal end of the catheter. The LMA-reinforced tube was removed in a retrograde fashion by pulling the catheter up with the patient breathing spontaneously. The duration of the entire operation was 3 hours 20 minutes, and the patient was able to breathe spontaneously and at a 98% saturation average. Nasal reinforced LMA seems to be an interesting solution in patients undergoing 1-day dental or maxillofacial surgery, but is especially appropriate when nasotracheal intubation is too invasive or technically impossible.

Absorption, by humans, of beta-carotene from fortified soybean oil added to rice: effect of heat treatment.

OBJECTIVE: This study was carried out to evaluate the absorption of beta-carotene in humans when rice is prepared with refined cooking soybean oil fortified with beta-carotene and to assess the effect of heat treatment on its bioavailability. METHODS: Sixteen healthy adults subjects participated in two experimental trials. Studies were carried out during two experimental periods of 11 days with a 12-day interval between them. Beta carotene was added to the soybean cooking oil and rice was cooked with it or it was added to the rice after cooking. Experimental diets included these two kinds of rice during the first day and fasting blood samples were collected on different days. All of the test diets were low in carotenoids. Plasma carotenoids were measured by HPLC method. beta-carotene absorption was calculated through postabsorptive peak rise in plasma beta-carotene and the total area under the absorption curve was determined by the trapezoidal method for the 11-day period. RESULTS: Absorption of carotene from heated or unheated fortified soybean oil were similar. Peak plasma carotene rise was different in men and women, p < 0.05 (0.66 +/- 0.097 vs. 1.04 +/- 0.117 mumol/l, respectively). Plasma alpha-carotene and retinol showed no variation. CONCLUSIONS: Results demonstrate that beta-carotene added to soybean oil used in the preparation of rice is absorbed, heated or not, and could be a practical source of provitamin A. Developing countries looking for strategies to increase vitamin A intake could use fortification of vegetable oils with synthetic beta-carotene as a simple method.

Effect of heat treatment on the biological value of beta-carotene added to soybean cooking oil in rats.

Vitamin A deficiency is one of the major nutritional problems in the world, most common in developing countries. Food fortification is a recognised approach to supply vitamins and minerals to needed populations. Vegetable cooking oils were previously suggested by us as a carrier for vitamin A fortification. Fortification of cooking oil with beta-carotene could also be a strategy to prevent vitamin A deficiency. The objective of this article is to start studies on the use of cooking soya oil as a vehicle for synthetic carotene, to evaluate its stability to heat treatment, and to test its bioavailability and bioconversion to vitamin A in rats. Batches of carotene-fortified soybean oil were prepared, containing 2, 4 and 8 RE/g of diet. Some of them were heated to test its stability. At 100 degrees C there was no loss of carotene, at higher temperature carotene retention was 65%. The bioavailability and bioconversion of beta-carotene added to soybean oil was measured through feeding nursing rats and their pups method. Weight gain was good and plasma vitamin A increased significantly in all groups. Liver vitamin A values of rats fed diets with fortified soybean oil heated at 100 degrees C was similar to the 4 RE non-heated fortified oil group (0.72 +/- 0.06 and 0.64 +/- 0.08 mumol/g, respectively). Heated at 170 degrees C the liver total vitamin A value was reduced (0.45 +/- 0.04 mumol/g), but kept bioavailable vitamin A equivalent to 2 RE (0.47 +/- 0.09 mumol/g). Bioconversion of beta-carotene to vitamin A was validated by the plasma and liver findings. beta-carotene added to soybean oil showed good stability to heat and its bioconversion to vitamin A was shown in rat assays. beta-carotene mixed well with edible soybean oil and the fortified cooking oil showed potential as a carrier to be used for the prevention of vitamin A deficiency.