Detection of Hepatitis B Virus Antigens in Paraffin-embedded Liver Specimens from the Amazon Region, Brazil

Hepatic viscerotomy of paraffin-preserved old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. The material belongs to the Yellow Fever Collection, Department of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed at that time according to clinical aspects and histopathological findings reporting viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 79 specimens, 69 were collected at the Labrea Region and the other 10 in different other localities in the Amazon Region. The five micra thick histological slices were analyzed for the presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) by immunoperoxidase technique. An immunofluorescence assay was applied to the detection of hepatitis D, yellow fever and dengue virus antigens. Nine (11.4%) histological samples were HBsAg reactive and 5 (6.3%) were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens related to the other pathologies were not detected in this study. Our results confirm that the methodology described may be used to elucidate the aetiology of hepatitis diseases even after a long time of conservation of the specimens.

Are oxygen uptake kinetics modified when using a respiratory snorkel?

PURPOSE: The aim of this study was to compare VO2 kinetics during constant power cycle exercise measured using a conventional facemask (CM) or a respiratory snorkel (RS) designed for breath-by-breath analysis in swimming. METHODS: VO2 kinetics parameters-obtained using CM or RS, in randomized counterbalanced order-were compared in 10 trained triathletes performing two submaximal heavy-intensity cycling square-wave transitions. These VO2 kinetics parameters (ie, time delay: td1, td2; time constant: τ1, τ2; amplitude: A1, A2, for the primary phase and slow component, respectively) were modeled using a double exponential function. In the case of the RS data, this model incorporated an individually determined snorkel delay (ISD). RESULTS: Only td1 (8.9 ± 3.0 vs 13.8 ± 1.8 s, P < .01) differed between CM and RS, whereas all other parameters were not different (τ1 = 24.7 ± 7.6 vs 21.1 ± 6.3 s; A1 = 39.4 ± 5.3 vs 36.8 ± 5.1 mL x min(-1) x kg(-1); td2 = 107.5 ± 87.4 vs 183.5 ± 75.9 s; A2' (relevant slow component amplitude) = 2.6 ± 2.4 vs 3.1 ± 2.6 mL x min(-1) x kg(-1) for CM and RS, respectively). CONCLUSIONS: Although there can be a small mixture of breaths allowed by the volume of the snorkel in the transition to exercise, this does not appear to significantly influence the results. Therefore, given the use of an ISD, the RS is a valid instrument for the determination of VO2 kinetics within submaximal exercise.

Eliminació autotròfica de nitrogen en una pila biològica (microbial fuel cells )

L’aigua i l’energia formen un binomi indissociable. En relació al cicle de l’aigua, des de fa varies dècades s’han desenvolupat diferents formes per recuperar part de l’energia relacionada amb l’aigua, per exemple a partir de centrals hidroelèctriques. No obstant, l’ús d’aquesta aigua també porta associat un gran consum energètic, relacionat sobretot amb el transport, la distribució, la depuració, etc... La depuració d’aigües residuals porta associada una elevada demanda energètica (Obis et al.,2009). En termes energètics, tot i que la despesa elèctrica d’una EDAR varia en funció de diferents paràmetres com la configuració i la capacitat de la planta, la càrrega a tractar, etc... es podria considerar que el rati mig seria d’ aproximadament 0.5 KWh•m-3.Els principals costos d’explotació estan relacionats tant amb la gestió de fangs (28%) com amb el consum elèctric (25%) (50% tractament biològic). Tot i que moltes investigacions relacionades amb el tractament d’aigua residual estan encaminades en disminuir els costos d’operació, des de fa poques dècades s’està investigant la viabilitat de que l’aigua residual fins i tot sigui una font d’energia, canviant la perspectiva, i començant a veure l’aigua residual no com a una problemàtica sinó com a un recurs. Concretament s’estima que l’aigua domèstica conté 9.3 vegades més energia que la necessària per el seu tractament mitjançant processos aerobis (Shizas et al., 2004). Un dels processos més desenvolupats relacionats amb el tractament d’aigües residuals i la producció energètica és la digestió anaeròbia. No obstant, aquesta tecnologia permet el tractament d’altes càrregues de matèria orgànica generant un efluent ric en nitrogen que s’haurà de tractar amb altres tecnologies. Per altre banda, recentment s’està investigant una nova tecnologia relacionada amb el tractament d’aigües residuals i la producció energètica: les piles biològiques (microbial fuel cells, MFC). Aquesta tecnologia permet obtenir directament energia elèctrica a partir de la degradació de substrats biodegradables (Rabaey et al., 2005). Les piles biològiques, més conegudes com a Microbial Fuel Cells (acrònim en anglès, MFC), són una emergent tecnologia que està centrant moltes mirades en el camp de l’ investigació, i que es basa en la producció d’energia elèctrica a partir de substrats biodegradables presents en l’aigua residual (Logan., 2008). Els fonaments de les piles biològiques és molt semblant al funcionament d’una pila Daniell, en la qual es separa en dos compartiments la reacció d’oxidació (compartiment anòdic) i la de reducció (compartiment catòdic) amb l’objectiu de generar un determinat corrent elèctric. En aquest estudi, bàsicament es mostra la posada en marxa d'una pila biològica per a l'eliminació de matèria orgànica i nitrogen de les aigües residuals.

The Bacillus subtilis Gne (GneA, GalE) protein can catalyse UDP-glucose as well as UDP-N-acetylglucosamine 4-epimerisation.

Mutations in the Bacillus subtilis gene that affect the activity of the uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 4-epimerase (EC 5.1.3.7) were shown to map to galE, the structural gene of the UDP-glucose (Glc) 4-epimerase (EC 5.1.3.2). This genetic evidence that the same enzyme can catalyse the epimerisation of hexoses as well as of their N-acetylated forms is confirmed by in vitro assays with purified enzyme. It appears that in B. subtilis, Gne (GneA, GalE) is involved in two distinct and essential functions, i.e., cell detoxification under certain growth conditions and the biosynthesis of anionic cell wall polymers. We discuss the evidence that such enzymes capable of utilizing both UDP-hexoses and UDP-N-acetylhexosamines are present in other organisms.

Sequencing of simple sequence repeat anchored polymerase chain reaction amplification products of Biomphalaria glabrata

Simple sequence repeat anchored polymerase chain reaction amplification (SSR-PCR) is a genetic typing technique based on primers anchored at the 5' or 3' ends of microsatellites, at high primer annealing temperatures. This technique has already been used in studies of genetic variability of several organisms, using different primer designs. In order to conduct a detailed study of the SSR-PCR genomic targets, we cloned and sequenced 20 unique amplification products of two commonly used primers, CAA(CT)6 and (CA)8RY, using Biomphalaria glabrata genomic DNA as template. The sequences obtained were novel B. glabrata genomic sequences. It was observed that 15 clones contained microsatellites between priming sites. Out of 40 clones, seven contained complex sequence repetitions. One of the repeats that appeared in six of the amplified fragments generated a single band in Southern analysis, indicating that the sequence was not widespread in the genome. Most of the annealing sites for the CAA(CT)6 primer contained only the six repeats found within the primer sequence. In conclusion, SSR-PCR is a useful genotyping technique. However, the premise of the SSR-PCR technique, verified with the CAA(CT)6 primer, could not be supported since the amplification products did not result necessarily from microsatellite loci amplification.

Should Hyperglycemia in Critically Ill Children Be Treated?

Introduction: In adults, strict control of hyperglycemia reduces mortality and morbidity. There is controversy in medical patients and neurological patients who can suffer of neuroglucopenia. Objectives: To determine prevalence and prognostic significance of hyperglycemia among critically ill non-diabetic children. To evaluate which patients will best benefit of insulin treatment. Methods: Retrospective study using blood glucose levels (GLUC: 9015 values, 923 patients) in our PICU from 01.2003 to 12.2005. 11 Patients with DKA were excluded. Overall PICU mortality was 3.7%. Hyperglycemia was defined at 6.1 mmol/L and different cutoff values (6.1, 8.3 and 11.1 mmol/l) were analyzed for glycemia at admission (GLUC). Sustained hyperglycemia was evaluated with the area under the curve normalized per hour (48h-AUC/h) for the first 48 h. The prevalence of hypo (_3mmol/L), hyperglycemia and PICU death were analyzed. Results: Trough the use of different cutoff values (_6.1, _8.3 and _11.1 mmol/l), prevalence of hyperglycemia at admission was 31.8 %, 16.8% and 10.3%; associated mortality was 2.8%, 4.0% and 15.2% respectively, significantly correlated to cutoff values (r_0.95, p_0.05). Prevalence of hypoglycemia at admission was low (0.9% with no death). 48h-AUC(mmol/L/h) was computed in 747 children (30 deaths). Prevalence of hyperglycemic 48h-AUC values was 47.5%, 17.3% and 4.0% with a respective mortality of 3.4%, 6.3% and 20.7% (r_0.97, p_0.03). For those with high GLUC and high 48h-AUC (_ 11.1 mmol/L) mortality was high (31.5%), but it decrease dramatically to 5.5% when 48h-AUC decrease spontaneously to values _8.3 mmol/L/h. Finally, when patients with severe neurological lesions (GCS_3, n_22) where excluded, increased mortality was observed only for GLUC (n_ 86) and 48h-AUC (n_26) higher than 11.1 mmol/L. Conclusions: Hyperglycemia at admission and even more sustained hyperglycemia (AUC) are highly correlated to mortality in PICU. But children who will have benefit of insulin therapy represent only 3% of our population, much lower than for adults.

Reverse transcription-polymerase chain reaction construction of plasmid-based, full-length cDNA libraries from Leishmania infantum for in vitro expression screening

We describe a streamlined reverse transcription-polymerase chain reaction methodology for constructing full-length cDNA libraries of trypanosomatids on the basis of conserved sequences located at the 5' and 3'ends of trans-spliced mRNAs. The amplified cDNA corresponded to full-length messengers and was amenable to in vitro expression. Fractionated libraries could be rapidly constructed in a plasmid vector by the TA cloning method (Invitrogen). We believe this is useful when there are concerns over the use of restriction enzymes and phage technology as well as in cases where expression of proteins in their native conformation is desired.

Mammographie-Screening in der Schweiz: eine retrospektive Analyse zur Umsetzung

[Table des matières] 1. Problemstellung. 1.1. Programme in anderen Ländern. 1.2. Situation in der Schweiz. 2. Methodisches Vorgehen. 2.1. Dokumentationsanalyse. 2.2. Experteninterviews. 3. Resultate : Beschreibung der wesentlichen Aktivitäten zur Programmentwicklung. 3.1. Wesentliche Aktivitäten in der Schweiz vor der Einführung des KVG. 3.2. Grundeinstellung zum organisierten Mammographie-Screening. 3.3. Gesetzliche Grundlagen. 3.4. Strukturen im schweizerischen Gesundheitswesen. 3.5. Finanzierung. 3.6. Rollen und Kompetenzen. 3.7. Wirksamkeit. 3.8. Internationale Expertise. 4. Lösungsansätze.

Resistance of Aedes aegypti from the state of São Paulo, Brazil, to organophosphates insecticides

Since the reintroduction of Aedes aegypti in the state of São Paulo, in the middle of the 1980-decade, organophosphate insecticides are being used to control the dengue vector. In 1996, an annual program for monitoring the susceptibility of Ae. aegypti to the insecticides was implemented. Some of the results of this monitoring program are presented. Ae. aegypti populations from ten localities have been submitted to bioassays with the diagnostic dose of temephos and fenitrothion. Only two (Marília and Presidente Prudente) remain susceptible to both insecticides and one (Santos) exhibits true resistance. Ae. aegypti from the remaining localities showed an incipient altered susceptibility. Resistance ratios varied from 1.2 to 2.9 for temephos and from 1.5 to 3.2 to fenitrothion, indicating moderate levels of resistance. Biochemical assays did not detect alterations in the enzyme acetilcholinesterase, but indicated that resistance is associated with esterases.

Eleutherinone, a novel fungitoxic naphthoquinone from Eleutherine bulbosa (Iridaceae)

The dichloromethane extract prepared from the underground parts of Eleutherine bulbosa (Miller) Urban (Iridaceae) showed strong activity in the direct bioautography assay with the phytopathogenic fungus Cladosporium sphaerospermum. This assay was used to guide the fractionation of this extract and allowed the isolation of four compounds: the new naphthoquinone eleutherinone[8-methoxy-1-methyl-1,3-dihydro-naphtho(2,3-c)furan-4,9 -dione] and the known compounds, previously isolated from this species, eleutherin [9-methoxy-1(R),3(S)-dimethyl-3,4-dihydro-1H-benzo(g)isochromene-5,10-dione], isoeleutherin [9-methoxy-1(R),3(R)-dimethyl-3,4-dihydro-1H-benzo(g)isochromene-5,10-dione], and eleutherol [4-hydroxy-5-methoxy-3(R)-methyl-3H-naphtho(2,3-c)furan-1 -one]. All quinonoid compounds showed strong antifungal activity in the bioautography assay at 100 µg/spot, while eleutherol was inactive.

Hepatic intra-arterial versus intravenous fotemustine in patients with liver metastases from uveal melanoma (EORTC 18021): a multicentric randomized trial.

BACKGROUND: In uveal melanoma (UM) with metastatic disease limited to the liver, the effect of an intrahepatic treatment on survival is unknown. We investigated prospectively the efficacy and toxicity of hepatic intra-arterial (HIA) versus systemic (IV) fotemustine in patients with liver metastases from UM. PATIENTS AND METHODS: Patients were randomly assigned to receive either IV or HIA fotemustine at 100 mg/m(2) on days 1, 8, 15 (and 22 in HIA arm only) as induction, and after a 5-week rest period every 3 weeks as maintenance. Primary end point was overall survival (OS). Response rate (RR), progression-free survival (PFS) and safety were secondary end points. RESULTS: Accrual was stopped after randomization of 171 patients based on the results of a futility OS analysis. A total of 155 patients died and 16 were still alive [median follow-up 1.6 years (range 0.25-6 years)]. HIA did not improve OS (median 14.6 months) when compared with the IV arm (median 13.8 months), hazard ratio (HR) 1.09; 95% confidence interval (CI) 0.79-1.50, log-rank P = 0.59. However, there was a significant benefit on PFS for HIA compared with IV with a median of 4.5 versus 3.5 months, respectively (HR 0.62; 95% CI 0.45-0.84, log-rank P = 0.002). The 1-year PFS rate was 24% in the HIA arm versus 8% in the IV arm. An improved RR was seen in the HIA (10.5%) compared with IV treatment (2.4%). In the IV arm, the most frequent grade ≥3 toxicity was thrombocytopenia (42.1%) and neutropenia (62.6%), compared with 21.2% and 28.7% in the HIA arm. The main grade ≥3 toxicity related to HIA was catheter complications (12%) and liver toxicity (4.5%) apart from two toxic deaths. CONCLUSION: HIA treatment with fotemustine did not translate into an improved OS compared with IV treatment, despite better RR and PFS. Intrahepatic treatment should still be considered as experimental. EUDRACT NUMBER AND CLINICALTRIALSGOV IDENTIFIER: 2004-002245-12 and NCT00110123.

Nineteen additional unpredicted transcripts from human chromosome 21.

The identification of all human chromosome 21 (HC21) genes is a necessary step in understanding the molecular pathogenesis of trisomy 21 (Down syndrome). The first analysis of the sequence of 21q included 127 previously characterized genes and predicted an additional 98 novel anonymous genes. Recently we evaluated the quality of this annotation by characterizing a set of HC21 open reading frames (C21orfs) identified by mapping spliced expressed sequence tags (ESTs) and predicted genes (PREDs), identified only in silico. This study underscored the limitations of in silico-only gene prediction, as many PREDs were incorrectly predicted. To refine the HC21 annotation, we have developed a reliable algorithm to extract and stringently map sequences that contain bona fide 3' transcript ends to the genome. We then created a specific 21q graphical display allowing an integrated view of the data that incorporates new ESTs as well as features such as CpG islands, repeats, and gene predictions. Using these tools we identified 27 new putative genes. To validate these, we sequenced previously cloned cDNAs and carried out RT-PCR, 5'- and 3'-RACE procedures, and comparative mapping. These approaches substantiated 19 new transcripts, thus increasing the HC21 gene count by 9.5%. These transcripts were likely not previously identified because they are small and encode small proteins. We also identified four transcriptional units that are spliced but contain no obvious open reading frame. The HC21 data presented here further emphasize that current gene prediction algorithms miss a substantial number of transcripts that nevertheless can be identified using a combination of experimental approaches and multiple refined algorithms.

BOLD responses to trigeminal nerve stimulation.

The current study investigates a new model of barrel cortex activation using stimulation of the infraorbital branch of the trigeminal nerve. A robust and reproducible activation of the rat barrel cortex was obtained following trigeminal nerve stimulation. Blood oxygen level-dependent (BOLD) effects were obtained in the primary somatosensory barrel cortex (S1BF), the secondary somatosensory cortex (S2) and the motor cortex. These cortical areas were reached from afferent pathways from the trigeminal ganglion, the trigeminal nuclei and thalamic nuclei from which neurons project their axons upon whisker stimulation. The maximum BOLD responses were obtained for a stimulus frequency of 1 Hz, a stimulus pulse width of 100 μs and for current intensities between 1.5 and 3 mA. The BOLD response was nonlinear as a function of frequency and current intensity. Additionally, modeling BOLD responses in the rat barrel cortex from separate cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO(2)) measurements showed good agreement with the shape and amplitude of measured BOLD responses as a function of stimulus frequency and will potentially allow to identify the sources of BOLD nonlinearities. Activation of the rat barrel cortex using trigeminal nerve stimulation will contribute to the interpretation of the BOLD signals from functional magnetic resonance imaging studies.

Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extractdisplayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 µg/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 µg/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 µg/ml) and B. subtilis (MIC at 3.9 and 7.8 µg/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 µg/ml and 3.12 µg/ml, respectively. Both compounds presented MIC of 3.12 µg/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 µg/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.