An isolate of the nematophagous fungus Monacrosporium thaumasium for the control of cattle trichostrongyles in south-eastern Brazil.

A mycelial formulation in sodium alginate pellets of the nematophagous fungus Monacrosporium thaumasium (isolate NF34A) was assessed in the biological control of beef cattle trichostrongyles in tropical Brazil. Two groups of ten male Nellore calves aged 6 months, a fungus-treated group and a control group, were fed on a pasture of Brachiaria decumbens naturally infected with larvae of cattle trichostrongyles. The fungus-treated group received doses of sodium alginate mycelial pellets orally (1 g pellets (0.2 g fungus)/10 kg live weight) twice a week for 12 months. At the end of the study there was a significant reduction (P< 0.01) in the number of eggs per gram of faeces and coprocultures of the fungus-treated group--47.8% and 50.2%, respectively--in relation to the control group. There was a 47.3% reduction in herbage samples, collected up to 0-20 cm from faecal pats, between the fungus-treated and control groups, and a 58% reduction when the sampling distance was 20-40 cm from faecal pats (P< 0.01). The treatment with sodium alginate pellets containing the nematode-trapping fungus M. thaumasium reduced trichostrongyles in tropical south-eastern Brazil and could be an effective tool for the biological control of this parasitic nematode in beef cattle. However, in such a tropical climate with low rainfall the fungal viability can be reduced.

Acoustophoresis of monodisperse oil droplets in water: Effect of symmetry breaking and non-resonance operation on oil trapping behavior.

Acoustic manipulation of particles in microchannels has recently gained much attention. Ultrasonic standing wave (USW) separation of oil droplets or particles is an established technology for microscale applications. Acoustofluidic devices are normally operated at optimized conditions, namely, resonant frequency, to minimize power consumption. It has been recently shown that symmetry breaking is needed to obtain efficient conditions for acoustic particle trapping. In this work, we study the acoustophoretic behavior of monodisperse oil droplets (silicone oil and hexadecane) in water in the microfluidic chip operating at a non-resonant frequency and an off-center placement of the transducer. Finite element-based computer simulations are further performed to investigate the influence of these conditions on the acoustic pressure distribution and oil trapping behavior. Via investigating the Gor'kov potential, we obtained an overlap between the trapping patterns obtained in experiments and simulations. We demonstrate that an off-center placement of the transducer and driving the transducer at a non-resonant frequency can still lead to predictable behavior of particles in acoustofluidics. This is relevant to applications in which the theoretical resonant frequency cannot be achieved, e.g., manipulation of biological matter within living tissues.

Acute effect of passive cycle-ergometry and functional electrical stimulation on nitrosative stress and inflammatory cytokines in mechanically ventilated critically ill patients: a randomized controlled trial.

Early mobilization is beneficial for critically ill patients because it reduces muscle weakness acquired in intensive care units. The objective of this study was to assess the effect of functional electrical stimulation (FES) and passive cycle ergometry (PCE) on the nitrous stress and inflammatory cytometry in critically ill patients. This was a controlled, randomized, open clinical trial carried out in a 16-bed intensive care unit. The patients were randomized into four groups: Control group (n=10), did not undergo any therapeutic intervention during the study; PCE group (n=9), lower-limb PCE for 30 cycles/min for 20 min; FES group (n=9), electrical stimulation of quadriceps muscle for 20 min; and FES with PCE group (n=7), patients underwent PCE and FES, with their order determined randomly. The serum levels of nitric oxide, tumor necrosis factor alpha, interferon gamma, and interleukins 6 and 10 were analyzed before and after the intervention. There were no differences in clinical or demographic characteristics between the groups. The results revealed reduced nitric oxide concentrations one hour after using PCE (P<0.001) and FES (P<0.05), thereby indicating that these therapies may reduce cellular nitrosative stress when applied separately. Tumor necrosis factor alpha levels were reduced after the PCE intervention (P=0.049). PCE and FES reduced nitric oxide levels, demonstrating beneficial effects on the reduction of nitrosative stress. PCE was the only treatment that reduced the tumor necrosis factor alpha concentration.

XplorMed: a tool for exploring MEDLINE abstracts.

The most frequent access to the MEDLINE database of scientific abstracts is by keyword search. However, this is often not sufficient because although the user might find all the useful abstracts, these are buried in hundreds that are irrelevant. The exploratory tool XplorMed has been developed to analyse the result of any MEDLINE query. It suggests main groups of related topics and documents, sparing the user the need of reading all abstracts.

Protection of glioblastoma cells from cisplatin cytotoxicity via protein kinase Ciota-mediated attenuation of p38 MAP kinase signaling.

Glioblastoma multiforme is an aggressive form of brain cancer that responds poorly to chemotherapy and is generally incurable. The basis for the poor response of this cancer to chemotherapy is not well understood. The atypical protein kinases C (PKCiota and PKCzeta) have previously been implicated in leukaemia cell chemoresistance. To assess the role of atypical PKC in glioblastoma cell chemoresistance, RNA interference was used to deplete human glioblastoma cells of PKCiota. Transfection of cells with either of two different RNA duplexes specific for PKCiota caused a partial sensitisation to cell death induced by the chemotherapy agent cisplatin. To screen for possible mechanisms for PKCiota-mediated chemoresistance, microarray analysis of gene expression was performed on RNA from glioblastoma cells that were either untreated or depleted of PKCiota. This identified sets of genes that were regulated either positively or negatively by PKCiota. Within the set of genes that were negatively regulated by PKCiota, the function of the gene coding for GMFbeta, an enhancer of p38 mitogen-activated protein kinase (MAP kinase) signaling, was investigated further, as the p38 MAP kinase pathway has been previously identified as a key mediator of cisplatin cytotoxicity. The expression of both GMFbeta mRNA and protein increased upon PKCiota depletion, and this was accompanied by an increase in cisplatin-activated p38 MAP kinase signaling. Transient overexpression of GMFbeta increased cisplatin-activated p38 MAP kinase signaling and also sensitised cells to cisplatin cytotoxicity. The increase in cisplatin cytotoxicity seen with PKCiota depletion was blocked by the p38 MAP kinase inhibitor SKF86002. These data show that PKCiota can confer partial resistance to cisplatin in glioblastoma cells by suppressing GMFbeta-mediated enhancement of p38 MAP kinase signaling.

Effect of the presence of cholesterol in the interfacial microenvironment on the modulation of the alkaline phosphatase activity during in vitro mineralization.

Mineralization of the skeleton starts within cell-derived matrix vesicles (MVs); then, minerals propagate to the extracellular collagenous matrix. Tissue-nonspecific alkaline phosphatase (TNAP) degrades inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and contributes Pi (Phosphate) from ATP to initiate mineralization. Compared to the plasma membrane, MVs are rich in Cholesterol (Chol) (∼32%) and TNAP, but how Chol influences TNAP activity remains unclear. We have reconstituted TNAP in liposomes of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) combined with Chol or its derivatives Cholestenone (Achol) and Ergosterol (Ergo). DPPC plus 36% sterols in liposome increased the catalytic activity of TNAP toward ATP. The presence of Chol also increased the propagation of minerals by 3.4-fold. The catalytic efficiency of TNAP toward ATP was fourfold lower in DOPC proteoliposomes as compared to DPPC proteoliposomes. DOPC proteoliposomes also increased biomineralization by 2.8-fold as compared to DPPC proteoliposomes. TNAP catalyzed the hydrolysis of ATP more efficiently in the case of the proteoliposome consisting of DOPC with 36% Chol. The same behavior emerged with Achol and Ergo. The organization of the lipid and the structure of the sterol influenced the surface tension (γ), the TNAP phosphohydrolytic activity in the monolayer, and the TNAP catalytic efficiency in the bilayers. Membranes in the Lα phase (Achol) provided better kinetic parameters as compared to membranes in the Lo phase (Chol and Ergo). In conclusion, the physical properties and the lateral organization of lipids in proteoliposomes are crucial to control mineral propagation mediated by TNAP activity during mineralization.

Re-annotating the Mycoplasma pneumoniae genome sequence: adding value, function and reading frames.

Four years after the original sequence submission, we have re-annotated the genome of Mycoplasma pneumoniae to incorporate novel data. The total number of ORFss has been increased from 677 to 688 (10 new proteins were predicted in intergenic regions, two further were newly identified by mass spectrometry and one protein ORF was dismissed) and the number of RNAs from 39 to 42 genes. For 19 of the now 35 tRNAs and for six other functional RNAs the exact genome positions were re-annotated and two new tRNA(Leu) and a small 200 nt RNA were identified. Sixteen protein reading frames were extended and eight shortened. For each ORF a consistent annotation vocabulary has been introduced. Annotation reasoning, annotation categories and comparisons to other published data on M.pneumoniae functional assignments are given. Experimental evidence includes 2-dimensional gel electrophoresis in combination with mass spectrometry as well as gene expression data from this study. Compared to the original annotation, we increased the number of proteins with predicted functional features from 349 to 458. The increase includes 36 new predictions and 73 protein assignments confirmed by the published literature. Furthermore, there are 23 reductions and 30 additions with respect to the previous annotation. mRNA expression data support transcription of 184 of the functionally unassigned reading frames.

Homology-based method for identification of protein repeats using statistical significance estimates.

Short protein repeats, frequently with a length between 20 and 40 residues, represent a significant fraction of known proteins. Many repeats appear to possess high amino acid substitution rates and thus recognition of repeat homologues is highly problematic. Even if the presence of a certain repeat family is known, the exact locations and the number of repetitive units often cannot be determined using current methods. We have devised an iterative algorithm based on optimal and sub-optimal score distributions from profile analysis that estimates the significance of all repeats that are detected in a single sequence. This procedure allows the identification of homologues at alignment scores lower than the highest optimal alignment score for non-homologous sequences. The method has been used to investigate the occurrence of eleven families of repeats in Saccharomyces cerevisiae, Caenorhabditis elegans and Homo sapiens accounting for 1055, 2205 and 2320 repeats, respectively. For these examples, the method is both more sensitive and more selective than conventional homology search procedures. The method allowed the detection in the SwissProt database of more than 2000 previously unrecognised repeats belonging to the 11 families. In addition, the method was used to merge several repeat families that previously were supposed to be distinct, indicating common phylogenetic origins for these families.

Adaptation of protein surfaces to subcellular location.

In vivo, proteins occur in widely different physio-chemical environments, and, from in vitro studies, we know that protein structure can be very sensitive to environment. However, theoretical studies of protein structure have tended to ignore this complexity. In this paper, we have approached this problem by grouping proteins by their subcellular location and looking at structural properties that are characteristic to each location. We hypothesize that, throughout evolution, each subcellular location has maintained a characteristic physio-chemical environment, and that proteins in each location have adapted to these environments. If so, we would expect that protein structures from different locations will show characteristic differences, particularly at the surface, which is directly exposed to the environment. To test this hypothesis, we have examined all eukaryotic proteins with known three-dimensional structure and for which the subcellular location is known to be either nuclear, cytoplasmic, or extracellular. In agreement with previous studies, we find that the total amino acid composition carries a signal that identifies the subcellular location. This signal was due almost entirely to the surface residues. The surface residue signal was often strong enough to accurately predict subcellular location, given only a knowledge of which residues are at the protein surface. The results suggest how the accuracy of prediction of location from sequence can be improved. We concluded that protein surfaces show adaptation to their subcellular location. The nature of these adaptations suggests several principles that proteins may have used in adapting to particular physio-chemical environments; these principles may be useful for protein design.

Comparison of ARM and HEAT protein repeats.

ARM and HEAT motifs are tandemly repeated sequences of approximately 50 amino acid residues that occur in a wide variety of eukaryotic proteins. An exhaustive search of sequence databases detected new family members and revealed that at least 1 in 500 eukaryotic protein sequences contain such repeats. It also rendered the similarity between ARM and HEAT repeats, believed to be evolutionarily related, readily apparent. All the proteins identified in the database searches could be clustered by sequence similarity into four groups: canonical ARM-repeat proteins and three groups of the more divergent HEAT-repeat proteins. This allowed us to build improved sequence profiles for the automatic detection of repeat motifs. Inspection of these profiles indicated that the individual repeat motifs of all four classes share a common set of seven highly conserved hydrophobic residues, which in proteins of known three-dimensional structure are buried within or between repeats. However, the motifs differ at several specific residue positions, suggesting important structural or functional differences among the classes. Our results illustrate that ARM and HEAT-repeat proteins, while having a common phylogenetic origin, have since diverged significantly. We discuss evolutionary scenarios that could account for the great diversity of repeats observed.

Bioinformatics: from genome data to biological knowledge.

Recently, molecular biologists have sequenced about a dozen bacterial genomes and the first eukaryotic genome. We can now obtain answers to detailed questions about the complete set of genes of an organism. Bioinformatics methods are increasingly used for attaching biological knowledge to long lists of genes, assigning genes to biological pathways, comparing the gene sets of different species, identifying specificity factors, and describing sets of highly conserved proteins common to all domains of life. Substantial progress has recently been made in the availability of primary and added-value databases, in the development of algorithms and of network information services for genome analysis. The pharmaceutical industry has greatly benefited from the accumulation of sequence data through the identification of targets and candidates for the development of drugs, vaccines, diagnostic markers and therapeutic proteins.

Acute effect of passive cycle-ergometry and functional electrical stimulation on nitrosative stress and inflammatory cytokines in mechanically ventilated critically ill patients: a randomized controlled trial

Early mobilization is beneficial for critically ill patients because it reduces muscle weakness acquired in intensive care units. The objective of this study was to assess the effect of functional electrical stimulation (FES) and passive cycle ergometry (PCE) on the nitrous stress and inflammatory cytometry in critically ill patients. This was a controlled, randomized, open clinical trial carried out in a 16-bed intensive care unit. The patients were randomized into four groups: Control group (n=10), did not undergo any therapeutic intervention during the study; PCE group (n=9), lower-limb PCE for 30 cycles/min for 20 min; FES group (n=9), electrical stimulation of quadriceps muscle for 20 min; and FES with PCE group (n=7), patients underwent PCE and FES, with their order determined randomly. The serum levels of nitric oxide, tumor necrosis factor alpha, interferon gamma, and interleukins 6 and 10 were analyzed before and after the intervention. There were no differences in clinical or demographic characteristics between the groups. The results revealed reduced nitric oxide concentrations one hour after using PCE (P<0.001) and FES (P<0.05), thereby indicating that these therapies may reduce cellular nitrosative stress when applied separately. Tumor necrosis factor alpha levels were reduced after the PCE intervention (P=0.049). PCE and FES reduced nitric oxide levels, demonstrating beneficial effects on the reduction of nitrosative stress. PCE was the only treatment that reduced the tumor necrosis factor alpha concentration.

Serum leptin levels in normal children: relationship to age, gender, body mass index, pituitary-gonadal hormones, and pubertal stage.

UNLABELLED: It is commonly accepted that at least in girls puberty starts when a minimum level of body mass or a certain amount of body fat are present. However the precise signal by which adipose stores inform the hypothalamus of the degree of energetic reserves is unknown. Leptin is a hormone produced by the adipocytes to regulate food intake and energy expenditure at the hypothalamic level. To understand whether leptin is the adipose tissue signal that allows puberty, 789 normal children of both sexes, age 5-15 yr, were transversally studied. Leptin levels, as well as gonadal and gonadotropins, levels, were analyzed in addition to the determination of auxological parameters. In an age-related analysis, leptin levels in girls rose from 5-15 yr (from 4.3 +/- 0.4 to 8.5 +/- 0.9 micrograms/L) in parallel with body weight. Boys always had lower leptin levels than girls (3.3 +/- 0.3 micrograms/L at 5 yr), but they rose in parallel with weight until 10 yr (5.3 +/- 0.7 micrograms/L), when a striking decrease was observed until 15 yr (3.0 +/- 0.3 micrograms/L). In girls, leptin was the first hormone to rise followed by FSH and later by LH and estradiol. A similar pattern occurred in boys, despite the fact that leptin dropped after 10 yr when testosterone rises. Divided into three pubertal stages, i.e. P1 = prepuberty, P2 = early puberty, and P3 = overt puberty, in girls the four hormones rose progressively from P1 to P3, but from P2 to P3 the present increment was greater for LH and estradiol. In boys, leptin decreased from P1 to P3, whereas FSH, LH, and testosterone rose. The age-related changes were not caused by adiposity variations, because data did not change when subtracting values of children over 97% of standard deviation score of body mass index. IN CONCLUSION: 1) leptin appears to increase in both boys and girls before the appearance of other reproductive hormones related to puberty; 2) leptin levels in boys are always lower than in girls, although they increase with age until the age 10 yr; 3) leptin in boys declines about the time testosterone increases. Leptin may well be a permissive factor for the initiation of pubertal events.

Growth hormone (GH) response to GH-releasing peptide-6 in type 1 diabetic patients with exaggerated GH-releasing hormone-stimulated GH secretion.

In type 1 diabetes mellitus (DM 1), high GH basal levels and exaggerated GH responses to several stimuli, including GHRH, have been described. GH-releasing peptide-6 (GHRP-6) is a synthetic hexapeptide that specifically stimulates GH release, both in vitro and in vivo. The aim of this study was to evaluate the effects of GHRP-6 alone or in combination with GHRH on GH secretion in DM 1. Six type 1 diabetic males and six age-, sex-, and body mass index-matched control volunteers were studied. Each subject received GHRH (100 microg iv), GHRP-6 (90 microg iv), and GHRH plus GHRP-6 on three separate days. GH peak values were higher in DM 1 patients than in control volunteers, after GHRH (52.2+/-9.8 vs. 19.3+/-6.0 microg/L; P = 0.016), GHRP-6 (66.2+/-9.6 vs. 39.9+/-6.3 microg/L; P = 0.05), and GHRH plus GHRP-6 (81.8+/-4.4 vs. 53.7+/-8.2 microg/L; P = 0.01). An additive GH response to combined administration of these two peptides was observed in diabetic patients. Serum insulin-like growth factor (IGF)-1 levels were diminished in DM 1, with respect to normal subjects (145.2+/-21.5 vs. 269.7+/-42.0 microg/L; P = 0.01), whereas IGF-binding protein-3 levels were not significantly different between DM-1 and controls. In summary, GHRP-6 is a potent stimulus for GH secretion in DM 1. The combined administration of GHRP-6 plus GHRH constitutes the most powerful stimulus for GH secretion in DM 1. These patients exhibit a greater GH secretory capacity than normal subjects, probably caused by a diminished tone in the IGF-1 sustained negative feedback control exerted upon somatotroph responsiveness.

Automated extraction of information in molecular biology.

We review data mining techniques in molecular biology, specifically those that extract information from the scientific literature itself. As more of the biological literature is published electronically, there is an opportunity, and even a need, to automatically summarize the literature in a customized way, for example by associating keywords to a topic. These keywords can be extracted from relevant publications. The process of keyword extraction can be automated and optimized to keep literature pointers automatically up-to-date or to filter relevant information from the literature. To illustrate these points, OMIM (Online Mendelian Inheritance in Man), a database of human inherited diseases, was linked to the literature and keywords were derived that covered distinct aspects such as genetic information on the one hand and disease-specific protein and phenotypic information on the other. They were used to extract information that is helpful for keeping entries about disease up-to-date.

Updated catalogue of homologues to human disease-related proteins in the yeast genome.

The recent availability of the full Saccharomyces cerevisiae genome offers a perfect opportunity for revising the number of homologues to human disease-related proteins. We carried out automatic analysis of the complete S. cerevisiae genome and of the set of human disease-related proteins as identified in the SwissProt sequence data base. We identified 285 yeast proteins similar to 155 human disease-related proteins, including 239 possible cases of human-yeast direct functional equivalence (orthology). Of these, 40 cases are suggested as new, previously undiscovered relationships. Four of them are particularly interesting, since the yeast sequence is the most phylogenetically distant member of the protein family, including proteins related to diseases such as phenylketonuria, lupus erythematosus, Norum and fish eye disease and Wiskott-Aldrich syndrome.

Acute otitis media in children with bronchiolitis.

OBJECTIVE: We investigated the prevalence and the etiology of acute otitis media (AOM) in children with bronchiolitis to determine whether AOM in such children is due entirely or mainly to respiratory syncytial virus (RSV), in which case routine antimicrobial treatment would not be appropriate. METHODS: The study group consisted of children aged 2 to 24 months with bronchiolitis. In patients with AOM at entry, nasal washings for RSV enzyme-linked immunosorbent assay were obtained, and Gram-stained smear, bacterial culture, and reverse transcriptase polymerase chain reaction to detect the presence of RSV were performed on middle-ear aspirates. Patients without AOM were reevaluated at 48 to 72 hours, 8 to 10 days, and 18 to 22 days. RESULTS: Forty-two children with bronchiolitis were enrolled. Sixty-two percent had AOM at entry or developed AOM within 10 days. An additional 24% had or eventually developed otitis media with effusion. Only 14% remained free of both AOM and otitis media with effusion throughout the 3-week observation period. All patients with AOM had 1 or more bacterial pathogens isolated from one or both middle-ear aspirates. Of 33 middle-ear aspirates, Streptococcus pneumoniae was isolated in 15, Haemophilus influenzae in 8, Moraxella catarrhalis in 8, and Staphylococcus aureus in 2. Two middle-ear aspirates yielded 2 pathogens each; 2 aspirates had no growth. RSV was identified in 17 (71%) of 24 patients with AOM. CONCLUSION: Bacterial AOM is a complication in most children with bronchiolitis. Accordingly, in patients with bronchiolitis and associated AOM, antimicrobial treatment is indicated.

Local utilization of metacresolsulfonic acid combined with streptomycin in the treatment of actinomycosis.

The effectiveness of combining metacresolsufonic acid with streptomycin in the treatment of actinomycosis, diagnosed either clinically or in the laboratory, was evaluated in 12 bovines and 2 equines. Eighty-seven percent of treated animals were considered clinically cured and did not show any signs of relapse after a six-month follow-up period. Therapeutic diagnosis by clinical observation was the procedure of choice when it was not possible to obtain laboratory diagnosis.