Early Identification of Infectious Complications in Bariatric Surgery by the Determination of Peritoneal and Systemic Cytokines

Obesity has become a global epidemic and bariatric surgery is one of the therapeutic tools to deal with it. Postoperative complications can occur, such as staple line dehiscence and anastomotic leaks, leading to increased patient mortality. The diagnosis of these complications is frequently difficult. The objective of the present study was to determine whether peritoneal and systemic cytokines could early detect those complications. All patients who underwent open Roux-en-Y gastric bypass from February 2007 to August 2008 were prospectively evaluated. Blood and peritoneal effluent from the drain were collected for the determination of cytokine levels. We also evaluated the clinical signs and the leukograms of the patients. A total of 107 obese patients were studied. Ninety patients had no complications; 17 had at least one infectious complication which include five cases of staple line dehiscence. Until the third postoperative day, the vital signs and the leukogram did not predict the onset of infectious complications, but the cytokines (interleukin-1 beta and interleukin-6) were early markers of these complications. Cytokines are good predictors of poor postoperative evolution in bariatric surgery since peritoneal cytokines diagnose better these infectious complications even before changes in blood count and before the occurrence of clinical manifestations.

Gene expression profile analysis of primary glioblastomas and non-neoplastic brain tissue: identification of potential target genes by oligonucleotide microarray and real-time quantitative PCR

The prognosis of glioblastomas is still extremely poor and the discovery of novel molecular therapeutic targets can be important to optimize treatment strategies. Gene expression analyses comparing normal and neoplastic tissues have been used to identify genes associated with tumorigenesis and potential therapeutic targets. We have used this approach to identify differentially expressed genes between primary glioblastomas and non-neoplastic brain tissues. We selected 20 overexpressed genes related to cell cycle, cellular movement and growth, proliferation and cell-to-cell signaling and analyzed their expression levels by real time quantitative PCR in cDNA obtained from microdissected fresh tumor tissue from 20 patients with primary glioblastomas and from 10 samples of non-neoplastic white matter tissue. The gene expression levels were significantly higher in glioblastomas than in non-neoplastic white matter in 18 out of 20 genes analyzed: P < 0.00001 for CDKN2C, CKS2, EEF1A1, EMP3, PDPN, BNIP2, CA12, CD34, CDC42EP4, PPIE, SNAI2, GDF15 and MMP23b; and NFIA (P: 0.0001), GPS1 (P: 0.0003), LAMA1 (P: 0.002), STIM1 (P: 0.006), and TASP1 (P: 0.01). Five of these genes are located in contiguous loci at 1p31-36 and 2 at 17q24-25 and 8 of them encode surface membrane proteins. PDPN and CD34 protein expression were evaluated by immunohistochemistry and they showed concordance with the PCR results. The present results indicate the presence of 18 overexpressed genes in human primary glioblastomas that may play a significant role in the pathogenesis of these tumors and that deserve further functional investigation as attractive candidates for new therapeutic targets.

Identification and characterization of a new member of snake venom thrombin inhibitors from Bothrops insularis using a proteomic approach

Snake venom C-type lectin-like proteins (CLPs) are ubiquitously found in Viperidae snake venoms and differ from the C-type lectins as they display different biological activities but no carbohydrate-binding activity. Previous analysis of the transcriptome obtained from the Bothrops insularis venom gland showed the presence of two clusters homologous to bothrojaracin (BJC) chains a and P. In an effort to identify a new BJC-like molecule, we used an approach associated with proteomic technologies to identify the presence of the expressed protein and then to purify and characterize a new thrombin inhibitor from B. insularis venom. We also constructed homology models of this protein and BJC, which were compared with other C-type lectin-like family members and revealed several conserved features of this intriguing snake venom toxin family. (C)0 2007 Elsevier Ltd. All rights reserved.

Nymphs of the genus Amblyomma (Acari: Ixodidae) of Brazil: descriptions, redescriptions, and identification key

Together with the larval stage, the nymphal stage of ticks of the genus Amblyomma are the most aggressive ticks for humans entering areas inhabited by wildlife and some domestic animals in Brazil. However, due to the absence of morphological descriptions of the nymphal stage of most Brazilian Amblyomma species, plus the lack of an identification key, little or nothing is known about the life history of Amblyomma spp. nymphs in the country. In the present study, morphological description of the nymphal stage, illustrating important external characters through scanning electron microscopy, is provided for nymphs of 15 Amblyomma species that occur in Brazil, for which the nymphal stage had never been described: A. aureolatum, A. auricularium, A. calcaratum, A. coelebs, A. fuscum, A. humerale, A. incisum, A. latepunctatum, A. naponense, A. nodosum, A. ovate, A. pacae, A. pseudoconcolor, A. scalpturatum, A. varium. In addition, the nymphal stage of 12 Amblyomma species, which had been previously described, are redescribed: A. brasiliense, A. cajennense, A. dissimile, A. dubitatum, A. longirostre, A. oblongoguttatum, A. parked, A. parvum, A. romitii, A. rotundatum, A. tigrinum, A. triste. The descriptions and redescriptions totalized 27 species. Only 2 species (A. geayi, A. goeldii) out of the 29 Amblyomma species established in Brazil are not included in the present study. A dichotomous identification key is included to support taxonomic identification of the nymphal stage of 27 Amblyomma species established in Brazil. (C) 2010 Elsevier GmbH. All rights reserved.

Clinical evaluation and COL2A1 gene analysis in 21 Brazilian families with Stickler syndrome: Identification of novel mutations, further genotype/phenotype correlation, and its implications for the diagnosis

We present clinical and molecular evaluation from a large cohort of patients with Stickler syndrome: 78 individuals from 21 unrelated Brazilian families. The patients were selected in a Hospital with a craniofacial dysmorphology assistance service and clinical diagnosis was based on the presence of cleft palate associated to facial and ocular anomalies of Stickler syndrome. Analysis of COL2A1 gene revealed 9 novel and 4 previously described pathogenic mutations. Except for the mutation c.556G>T (p.Gly186X), all the others were located in the triple helical domain. We did not find genotype/phenotype correlation in relation to type and position of the mutation in the triple helical domain. However, a significantly higher proportion of myopia in patients with mutations located in this domain was observed in relation to those with the mutation in the non-tripe helical domain (c.556G>T; P < 0.04). A trend towards a higher prevalence of glaucoma, although not statistically significant, was observed in the presence of the mutation c.556G>T. It is possible. that this mutation alters the splicing of the mRNA instead of only creating a premature stop codon and therefore it can lead to protein products of different ocular effects. One novel DNA variation (c.1266+7G>C) occurs near a splice site and it was observed to co-segregate with the phenotype in one of the two families with this DNA variation. As in silico analysis predicted that the c.1266+7G>C DNA variation can affect the efficiency of the splicing, we still cannot rule it out as non-pathogenic. Our study also showed that ascertainment through cleft palate associated to other craniofacial signs can be very efficient for identification of Stickler syndrome patients. Still, high frequency of familial cases and high frequency of underdevelopment of distal lateral tibial epiphyses observed in our patients suggested that the inclusion of this information can improve the clinical diagnosis of Stickler syndrome. (C) 2008 Elsevier Masson SAS. All rights reserved.

A Lagrangian identification of major sources of moisture over Central Brazil and La Plata Basin

[1] This work examines the main sources of moisture over Central Brazil and La Plata Basin during the year through a new Lagrangian diagnosis method which identifies the humidity contributions to the moisture budget over a region. This methodology computes budgets of evaporation minus precipitation by calculating changes in the specific humidity along back-trajectories for the previous 10 d. The origin of all air masses residing over each region was tracked during a period of 5 years (2000-2004). These regions were selected because they coincide with two centers of action of a known dipole precipitation variability mode observed in different temporal scales (from intra seasonal up to inter decadal timescales) and are related to the climatic variability of the South American Monsoon System. The results suggested the importance of the tropical south Atlantic as a moisture source for Central Brazil, and of recycling for La Plata basin. It seems that the Tropical South Atlantic plays an important role as a moisture source for Central Brazil and La Plata basin along the year, particularly during the austral summer. The north Atlantic is also an additional source for both regions during the austral summer.

AMP hydrolysis in soluble and microsomal rat cardiac cell fractions: kinetic characterization and molecular identification of 5 `-nucleotidase

The present study describes the enzymatic properties and molecular identification of 5`-nucleotidase in soluble and microsomal fractions from rat cardiac ventricles. Using AMP as a substrate, the results showed that the cation and the concentration required for maximal activity in the two fractions was magnesium at a final concentration of 1 mM. The pH optimum for both fractions was 9.5. The apparent K-m (Michaelis constant) values calculated from the Eadie-Hofstee plot were 59.7 +/- 10.4 mu M and 134.8 +/- 32.1 mu M, with V-max values of 6.7 +/- 0.4 and 143.8 +/- 23.8 nmol P-i/min/mg of protein (means +/- S.D., n = 4) from soluble and microsomal fractions respectively. Western blotting analysis of ecto-5`-nucleotidase revealed a 70 kDa protein in both fractions, with the major proportion present in the microsomal fraction. The presence of these enzymes in the heart probably has a physiological function in adenosine signalling. Furthermore, the presence of ecto-5`-nucleotidase in the microsomal fraction could have a role in the modulation of the excitation-contraction-coupling process through involvement of the Ca2+ influx into the sarcoplasmic reticulum. The measurement of maximal enzyme activities in the two fractions highlights the potential capacity of the different pathways of purine metabolism in the heart.

Screening for endophytic nitrogen-fixing bacteria in Brazilian sugar cane varieties used in organic farming and description of Stenotrophomonas pavanii sp. nov.

A Gram-negative, rod-shaped, non-spore-forming and nitrogen-fixing bacterium, designated ICB 89(T), was isolated from stems of a Brazilian sugar cane variety widely used in organic farming. 16S rRNA gene sequence analysis revealed that strain ICB 89(T) belonged to the genus Stenotrophomonas and was most closely related to Stenotrophomonas maltophilia LMG 958(T), Stenotrophomonas rhizophila LMG 22075(T), Stenotrophomonas nitritireducens L2(T), [Pseudomonas] geniculata ATCC 19374(T), [Pseudomonas] hibiscicola ATCC 19867(T) and [Pseudomonas] beteli ATCC 19861(T). DNA-DNA hybridization together with chemotaxonomic data and biochemical characteristics allowed the differentiation of strain ICB 89(T) from its nearest phylogenetic neighbours. Therefore, strain ICB 89(T) represents a novel species, for which the name Stenotrophomonas pavanii sp. nov. is proposed. The type strain is ICB 89(T) (=CBMAI 564(T) =LMG 25348(T)).

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

Aims: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. Methods and Results: Polyclonal and Mabs against the intimin-conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1 center dot 3 x 10-8 mol l-1, failed in the detection of some of these isolates. Conclusion: All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Identification and characterization of antigenic proteins potentially expressed during the infectious process of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. A P. brasiliensis cDNA expression library was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic-L-amino-acid decarboxylase (Pbddc), lumazine synthase (Pbls) and a homologue of the high affinity copper transporter (Pbctr3) were obtained. Recombinant proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3. The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the in vivo-induced antigen technology (IVIAT), we identified immunogenic proteins expressed at high levels during infection. Quantitative real time RTPCR demonstrated high transcript levels of Pbddc, Pbls and Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of P. brasiliensis. (C) 2009 Elsevier Masson SAS. All rights reserved.

A real-time polymerase chain reaction assay for the identification and quantification of American Leishmania species on the basis of glucose-6-phosphate dehydrogenase

A real-time polymerase chain reaction (PCR) test was developed on the basis of the Leishmania glucose-6-phosphate dehydrogenase locus that enables identification and quantification of parasites. Using two independent pairs of primers in SYBR-Green assays, the test identified etiologic agents of cutaneous leishmaniasis belonging to both subgenera, Leishmania (Viannia) and Leishmania (Leishmania) in the Americas. Furthermore, use of TaqMan probes enables distinction between L. (V.) braziliensis or L. (V.) peruviania from the other L. (Viannia) species. All assays were negative with DNA of related trypanosomatids, humans, and mice. The parasite burden was estimated by normalizing the number of organisms per total amount of DNA in the sample or per host glyceraldehyde-3-phosphate dehydrogenase copies. The real-time PCR assay for L. (Leishmania) subgenus showed a good linear correlation with quantification on the basis of a limiting dilution assay in experimentally infected mice. The test successfully identifies and quantifies Leishmania in human biopsy specimens and represents a new tool to study leishmaniasis.

Identification of geodesic chirping Alfven modes and q-factor estimation in hot core tokamak plasmas in ASDEX Upgrade

Alfven eigenmodes (AE) driven by ion cyclotron resonance heating are usually registered by different diagnostic channels in the hot core plasmas of large tokamaks like JET and ASDEX Upgrade. These AE appear very near to the extremum points of Alfven wave continuum, which is modified by the geodesic effect due to poloidal mode coupling. It is shown that the AE spectrum may be explored as the magnetic spectroscopy (like Alfven cascades by Sharapov et al 2001 Phys. Lett. A 289 127) to determine the q-factor minimum and geodesic frequency at the magnetic axis in standard sawtoothed discharges without reversed shear.

Identification of genes associated with local aggressiveness and metastatic behavior in soft tissue tumors

Soft tissue tumors represent a group of neoplasia with different histologic and biological presentations varying from benign, locally confined to very aggressive and metastatic tumors. The molecular mechanisms responsible for such differences are still unknown. The understanding of these molecular alterations mechanism will be critical to discriminate patients who need systemic treatment from those that can be treated only locally and could also guide the development of new drugs` against this tumors. Using 102 tumor samples representing a large spectrum of these tumors, we performed expression profiling and defined differentially expression genes that are likely to be involved in tumors that are locally aggressive and in tumors with metastatic potential. We described a set of 12 genes (SNRPD3, MEGF9, SPTAN-1, AFAP1L2, ENDOD1, SERPIN5, ZWINTAS, TOP2A, UBE2C, ABCF1, MCM2, and ARL6IP5) showing opposite expression when these two conditions were compared. These genes are mainly related to cell-cell and cell-extracellular matrix interactions and cell proliferation and might represent helpful tools for a more precise classification and diagnosis as well as potential drug targets.

Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma

The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue were obtained from six patients. A custom-built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down-regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave-one-out patient cross-validation. Among the down-regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein-coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (P<0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (P<0.05), were confirmed in clear cell RCC by Real-time RT-PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells. (C) 2008 Wiley-Liss, Inc.

Identification of species formed after pyridine adsorption on iron, cobalt, nickel and silver electrodes by SERS and theoretical calculations

The adsorption of pyridine (py) on Fe, Co, Ni and Ag electrodes was studied using surface-enhanced Raman scattering (SERS) to gain insight into the nature of the adsorbed species. The wavenumber values and relative intensities of the SERS bands were compared to the normal Raman spectrum of the chemically prepared transition metal complexes. Raman spectra of model clusters M(4)(py) (four metal atoms bonded to one py moiety) and M(4)(alpha-pyridil) where M = Ag, Fe, Co or Ni were calculated by density functional theory (DFT) and used to interpret the experimental SERS results. The similarity of the calculated M(4)(py) spectra with the experimental SERS spectra confirm the molecular adsorption of py on the surface of the metallic electrodes. All these results exclude the formation of adsorbed alpha-pyridil species, as suggested previously. Copyright (C) 2009 John Wiley & Sons, Ltd.