Learning complex texture discrimination.

Higher-order spatial correlations contribute strongly to visual structure and salience, and are common in the natural environment. One method for studying this structure has been through the use of highly controlled texture patterns whose obvious structure is defined entirely by third- and higher-order correlations. Here we examine the effects that longer-term training has on discrimination of 17 such texture types. Training took place in 14 sessions over 42 days. Discrimination performance increased at different rates for different textures. The time required to complete a visit reduced by 25.4% (p=0.0004). Factor analysis was applied to data from the learning and experienced phases of the experiment. This indicated that the gain in speed was accompanied by an increase in the number of mechanisms contributing to discrimination. Learning was not affected by sleep quality but was affected by extreme tiredness (p<0.01). The improved discrimination and speed were retained for 2.5 months. Overall, the effects were consistent with perceptual learning. The observed learning is likely related to the adaptation of innate mechanisms that underlie our ability to identify nonredundant, visually salient structure in natural images. It may involve cortical V2 and appears to involve increased strength, speed, and breadth of connections within our internal representation of this complex perceptual space.

Purification and characterization of the bacteriocin produced by Lactobacillus sakei MBSa1 isolated from Brazilian salami.

AIMS: The study aimed at determining the biochemical characteristics of the bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. METHODS AND RESULTS: Identification of strain MBSa1 was performed by 16S rDNA sequencing. The bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for bacteriocin genes commonly present in Lact. sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2·0 to 6·0 and active against all tested Listeria monocytogenes strains. Maximal production of bacteriocin MBSa1 (1600 AU ml(-1)) in MRS broth occurred after 20 h at 25°C. The molecular mass of produced bacteriocin was 4303·3 Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-α, T-ß, X, P, G and Q). CONCLUSIONS: In the studied conditions, Lact. sakei MBSa1 produced sakacin A, a class II bacteriocin, with anti-Listeria activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study covers the purification and characterization of the bacteriocin produced by a lactic acid bacteria isolated from salami (Lact. sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2·0-6·0) and activity against L. monocytogenes food isolates bring up a potential technological application to improve food safety.

Immunostimulating activity of Uncaria tomentosa in RAW 264.7 macrophages.

Uncaria tomentosa is a plant native to the Amazon that has immunomodulatory and antitumor properties due to the alkaloids found in the plant, being able to modify the immune response by potentiating or suspending the action of cytokines secreted by macrophages that induce the immune response, either by the classical route (M1) or through the alternative route (M2). Macrophages activated by M1 convert L-arginine into L-citrulline and nitric oxide (NO), whereas macrophages activated by the M2 pathway use the enzymatic activity of arginase to convert the same substrate into L-ornithine and urea. The aim of this work was to evaluate the immunostimulating activity of the crude hydroalcoholic extract from the bark of the U. tomentosa stem in RAW 264.7 macrophages. Concentrations of 0.2, 0.1 and 0.05 mg/mL of U. tomentosa extract associated with LPS, INF-γ and IL-4 inducers were tested by determining NO production and arginase enzyme activity. Nitric oxide production was enhanced by the extract when associated with LPS and LPS + INF-γ inducers. In the activity of the arginase enzyme, the extract decreased the stimulation of IL-4 on the enzyme, mainly at 0.2 mg/mL concentration. Therefore, it is concluded that the crude hydroalcoholic extract of the stem bark of U. tomentosa in RAW 264.7 cells, at a concentration of 0.2 mg/mL, showed considerable pro-inflammatory activity.

Increased expression of interleukin-6 gene in gastritis and gastric cancer.

Helicobacter pylori (H. pylori) induces an intense inflammatory response, mediated by proinflammatory cytokines, including interleukin (IL)-6 and its membrane receptor (IL-6R), which activates important signaling pathways in the development of gastric disease and cancer. We investigated the gene and protein expression of IL-6 and IL-6R and the influence of polymorphisms rs1800795, rs1800796, and rs1800797 on its gene expression together with H. pylori infection. Furthermore, an in-silico analysis was performed to support our results. Gastric biopsies were obtained from patients with gastric symptoms and patients with gastric cancer (GC) and were divided into groups (Control, Gastritis, and Cancer). H. pylori was detected by PCR. Real-time-qPCR was employed to determine gene expression, and western blot assay was used to analyze protein expression levels. PCR-RFLP was used to characterize IL-6 polymorphisms. Bioinformatics analyses were performed using the Gene Expression Omnibus (GEO) database and GEO2R to screen out differentially expressed genes (DEGs). H. pylori was detected in 43.3% of the samples. Statistically significant differences were found for IL-6 (P=0.0001) and IL-6R (P=0.0005) genes among the three groups, regardless of the presence of H. pylori. Among patients with H. pylori infection, the IL-6 and IL-6R gene and protein expressions were significantly increased, highlighting IL-6 gene overexpression in patients with GC. No statistically significant differences were found for the rs1800795, rs1800796, and rs1800797 polymorphisms compared to IL-6 gene expression. The results indicated that the IL-6 polymorphisms do not influence its expression, but IL-6 and IL-6R expression seems to be altered by the presence of H. pylori.

Increased expression of interleukin-6 gene in gastritis and gastric cancer

Helicobacter pylori (H. pylori) induces an intense inflammatory response, mediated by proinflammatory cytokines, including interleukin (IL)-6 and its membrane receptor (IL-6R), which activates important signaling pathways in the development of gastric disease and cancer. We investigated the gene and protein expression of IL-6 and IL-6R and the influence of polymorphisms rs1800795, rs1800796, and rs1800797 on its gene expression together with H. pylori infection. Furthermore, an in-silico analysis was performed to support our results. Gastric biopsies were obtained from patients with gastric symptoms and patients with gastric cancer (GC) and were divided into groups (Control, Gastritis, and Cancer). H. pylori was detected by PCR. Real-time-qPCR was employed to determine gene expression, and western blot assay was used to analyze protein expression levels. PCR-RFLP was used to characterize IL-6 polymorphisms. Bioinformatics analyses were performed using the Gene Expression Omnibus (GEO) database and GEO2R to screen out differentially expressed genes (DEGs). H. pylori was detected in 43.3% of the samples. Statistically significant differences were found for IL-6 (P=0.0001) and IL-6R (P=0.0005) genes among the three groups, regardless of the presence of H. pylori. Among patients with H. pylori infection, the IL-6 and IL-6R gene and protein expressions were significantly increased, highlighting IL-6 gene overexpression in patients with GC. No statistically significant differences were found for the rs1800795, rs1800796, and rs1800797 polymorphisms compared to IL-6 gene expression. The results indicated that the IL-6 polymorphisms do not influence its expression, but IL-6 and IL-6R expression seems to be altered by the presence of H. pylori.

The E6 and E7 genes of HPV-18 are sufficient for inducing two-stage in vitro transformation of human keratinocytes.

Using a recently described keratinocyte assay, we demonstrate that HPV-18 DNA induces two progressive steps in cellular transformation (a large cell and a small cell stage). Both steps of this keratinocyte transformation can be achieved with a subgenomic fragment containing only the HPV-18 regulatory region and E6/E7 genes. Similar to cell lines transformed by the complete HPV-18 genome, keratinocytes transformed by the HPV-18 E6/E7 genes express the major early viral protein (E7) but are non-tumorigenic in nude mice. Interestingly, HPV-18 DNA was noted to be 5 times more efficient than HPV-16 DNA for in vitro keratinocyte transformation, regardless of the method of DNA transfection.

Distinct regions of p53 have a differential role in transcriptional activation and repression functions.

The wild type p53 tumor suppressor protein transactivates genes carrying p53 responsive elements and represses several TATA containing promoters. We report in vivo gene regulation assays where deletion of the N-terminal 75 residues (delta N75) results in loss of transactivation of p53CON and repression of an HPV 6 reporter. In contrast, removal of the C-terminal 75 (delta C75) amino acids resulted in a truncated protein capable of trans-activating p53CON but not able to repress the HPV 6 reporter. In vitro protein association assays revealed that the delta N75 protein, but not the delta C75 truncated protein, could oligomerize with the wild type p53 protein. Co-transfection assays with wild type p53 showed that the delta N75 mutant protein has a dominant negative effect on trans-activation function. However, it does not affect the ability of wild type p53 to repress transcription from the HPV 6 receptor. The delta C75 protein had no effect on the ability of the wild type p53 to activate p53CON or repress the HPV 6 reporter. These results suggest that distinct regions of p53 have a differential role in transcriptional activation and repression functions.

Multidrug-resistant Escherichia coli isolated from a dog with a history of urolithiasis: case report / Escherichia coli multirresistente isolada de cão com histórico de urolitíase: relato de caso

Bacterial resistance is a reality in both human and veterinary health, it limits the therapeutic arsenal and raises the costs of the patient's treatment. A dog with signs of cystitis received treatment with 5mg/kg enrofloxacin at three consecutive times, with low effectiveness. The presence of urethral uroliths was identified and urohydropulsion was done. The animal presented a new obstruction, for which a cystotomy was performed, but continued with signs of infection. Uroculture and antimicrobial susceptibility test were then performed. Escherichia coli was identified, which was resistant to 13 antibiotics, being sensitive only to piperacillin-tazobactam and amikacin. In the screening test for ß-lactamase, the production of ESßL was detected. The qPCR indicated the presence of the bla CTXm, bla DHA, bla OXA, bla IMP, bla TEM, bla GIM, bla SIM, bla SPM and bla SME genes, which may lead to a phenotypic resistance profile for ampicillin, amoxicillin-clavulanate, aztreonam, cefepime cefoxitin, cefuroxime, ceftazidime, ceftriaxone, imipenem, and piperacillin-tazobactam. This case reaffirms the value that laboratory analysis adds to the diagnosis and treatment of cystitis and urolithiasis, which can define the direction of evolution of the prognosis and the speed at which the patient's health will be restored.(AU)

A resistência bacteriana aos antibióticos é uma realidade, tanto na saúde humana quanto veterinária, limita o arsenal terapêutico e eleva os custos relacionados ao tratamento do paciente. Um cão, com sinais de cistite, recebeu tratamento com enrofloxacina, na dose de 5mg/kg, em três momentos seguidos, com baixa efetividade. Identificou-se presença de urólitos uretrais e foi feita uro-hidropropulsão. O animal apresentou nova obstrução, para a qual foi realizada uma cistotomia, mas continuou com sinais de infecção. Realizou-se, então, urocultura e teste de antibiograma. Foi identificada Escherichia coli, que se mostrou resistente a 13 antibióticos, sendo sensível somente à piperacilina-tazobactam e amicacina. No teste de triagem para ß-lactamase, detectou-se a produção de ESßL. A qPCR indicou presença dos genes blaCTXm, blaDHA, blaOXA, blaIMP, blaTEM, blaGIM, blaSIM, blaSPM e blaSME, que podem conduzir um perfil fenotípico de resistência para ampicilina, amoxicilina-ácido clavulânico, aztreonam, cefepima, cefoxitina, cefuroxima, ceftazidima, ceftriaxona, imipenem, piperacilina-tazobactam. Este caso reafirma o valor que a análise laboratorial agrega ao diagnóstico e tratamento da cistite e da urolitíase, podendo definir o sentido de evolução do prognóstico e a velocidade em que a saúde do paciente será restabelecia.(AU)

Atividade biológica in vitro de própolis e óleos essenciais sobre o fungo Colletotrichum musae isolado de bananeira (Musa spp. ) / In vitro biological activity of propolis and essential oils on the fungus Colletotrichum musae isolated from banana Musa spp

RESUMO: No Brasil existem várias doenças fúngicas que acometem a bananeira. Destas, pode-se citar a antracnose, responsável por grandes prejuízos à cultura, cujo agente causal é o fungo Colletotrichum musae. A principal forma de controle dessa enfermidade é através da aplicação de fungicidas a base de tiabendazol ou tiofanato metílico. Esse manejo, embora eficiente, favorece o desenvolvimento de resistência do patógeno, causa danos ao ambiente e ao produtor, deixando ainda resíduos nos frutos. Esses fatores têm favorecido a busca por substâncias alternativas com capacidade de controlar o fungo e que não sejam nocivas ao ambiente e, principalmente, que sejam seguras ao consumidor final. Dentre as opções, surge o interesse pelo uso de certos óleos essenciais e da própolis, ambos conhecidos por possuírem propriedades fungicidas. O presente trabalho foi desenvolvido com o objetivo de determinar o potencial fungitóxico "in vitro" da própolis e dos óleos essenciais de palmarosa (Cymbopogon martinii), de teatree (Melaleuca alternifolia), de cravo (Eugenia caryophyllata), e de eucalipto (Corymbia citriodora), sobre Colletotrichum musae. O desenvolvimento experimental consistiu em adicionar inóculos fúngicos de 5 mm, obtidos a partir de colônias puras, ao meio de cultura BDA (batata-dextrose-ágar) acrescido das referidas substâncias em diferentes concentrações (0, 25, 50, 75, 100 e 125 µL/L). Paralelo aos tratamentos realizou-se teste com o fungicida padrão para comparações das médias. A eficiência das substâncias sobre o fungo foi determinada através das avaliações do crescimento micelial das colônias (média de duas medidas diametralmente opostas). Os valores de crescimento micelial obtidos foram utilizados também para o cálculo do índice de velocidade de crescimento micelial. O delineamento experimental utilizado foi inteiramente casualizado em esquema fatorial 5 x 6 + 1, (cinco substâncias em seis concentrações + fungicida), com cinco repetições. Os óleos de tea tree, cravo e palmarosa foram eficientes no controle do fungo Colletotrichum musae não diferindo do fungicida a partir da dose de 50 µL/L em todas as avaliações, apresentando potencial para controle em cultivos orgânicos ou em sistemas de manejo integrado.

ABSTRACT: In Brazil there are several fungi that cause diseases on banana plants. These include the "anthracnose", which is responsible for major crop losses and whose causative agent is the fungus Colletotrichum musae. The main way to control this disease is through the application of fungicides based on thiabendazole and thiophanate-methyl. Although this management is effective, it favors the development of pathogen resistance, which causes damage to the environment and producer and also leaves residues in fruits. These factors have encouraged the search for alternative substances to control the fungus and that are not harmful to the environment and particularly to the final consumer. Among the options, there is interest in the use of essential oils and propolis, both known to have antifungal activity. The present work was developed with the objective of determining the potential of propolis and essential oils of palmarosa (Cymbopogon martinii), tea tree (Melaleuca alternifolia), clove (Eugenia caryophyllata) and eucalyptus (Corymbia citriodora) in the in vitro control of the fungus Colletotrichum musae. The experimental development consisted in adding 5 mm fungal inoculants, obtained from pure colonies, in PDA culture (potato-dextrose-agar) plus the aforementioned substances in different concentrations (0, 25, 50, 75, 100 and 125 µL/L). At the same time as these treatments, we carried out a test with the fungicide to compare the averages. The efficiency of the substances on the fungus was determined through evaluations of the mycelial growth of the colonies (average of two diametrically opposed measures). The values of mycelial growth obtained were also used for the calculation of the speed index of the mycelial growth. The experimental design was completely randomized in 5 x 6 + 1 (5 substances in 6 concentrations + fungicide) factorial design, with 5 repetitions. The tea tree, clove and palmarosa oils were efficient in the control of the fungus Colletotrichum musae, which can be used as a control option in organic crops or in integrated management systems.

The oncogenic role of human papillomavirus proteins.

Each human papillomavirus (HPV) type is genotypically distinct and infects epithelial cells at unique anatomic sites. Among the HPV types described, a subgroup is associated with genital disease and a subset of these is found in 90% of genital cancers. Although in benign infections the viral genome is present as an episome, in cancers it is integrated. The integration event invariably results in the expression of two viral proteins, E6 and E7. These two proteins are capable of transforming cells individually and cooperate to immortalize primary human epithelial cells. Molecular analysis has revealed that the E6 protein encoded by the HPV "high risk" types prevalent in cancers forms a tripartite complex with the p53 tumor suppressor protein and a cellular protein termed E6-AP, resulting in the degradation of p53. The E7 protein encoded by "high-risk" HPV types shows high-affinity association with the retinoblastoma tumor suppressor, pRb. The E7 protein associates also with other cellular factors known to play a role in cell cycle regulation. This review discusses the evidence, molecular and biological, in vitro and in vivo, supporting a direct role for the "high-risk" HPV type encoded E6 and E7 proteins in cervical carcinogenesis.

E2 of cottontail rabbit papillomavirus is a nuclear phosphoprotein translated from an mRNA encoding multiple open reading frames.

The papillomavirus E2 protein is a transcription trans-activator and as such as a paramount effect on viral functions. We have identified and characterized the cottontail rabbit papillomavirus E2 protein expressed from the late simian virus 40 promoter in COS-7 cells. E2 was shown to be a highly phosphorylated 49-kilodalton protein. Subcellular fractionation and indirect immunofluorescent staining indicated that E2 was located in the nuclei. E2 was expressed from a vector which contained just the open reading frame. ORF E2 and also from vectors which extended farther upstream and also expressed E7 or the short E6 protein. However, the level of E2 was very low in cells transfected with a vector expressing the long E6 protein. Mapping of transcripts with nuclease S1 and exonuclease ExoVII in cells expressing the short E6 and E2 proteins showed that E2 was translated from an RNA which encoded the short E6, E7, and E2 proteins but served as mRNA for only the short E6 and E2 proteins.

Identification and characterization of the CRPV E7 protein expressed in COS-7 cells.

The papillomavirus E7 protein may play an important role in oncogenesis as it is the major viral protein present in human cervical cancer-derived cell lines. Because of the relevance of the cottontail rabbit papillomavirus (CRPV) system for the study of viral-induced malignancies, we characterized its E7 protein expressed by a heterologous strong promoter. An E7-specific antiserum was obtained by immunizing rabbits with a Trp E-E7 fusion protein containing the 88 carboxyl-terminal amino acids of E7. This antiserum specifically precipitated from [35S]cysteine but not from 32PO4-labeled transiently transfected COS-7 cells a 14-kDa protein. The protein was present only in the soluble cytoplasmic fraction and exhibited a heterogeneous sedimentation rate in nondenaturing glycerol gradients.

Single amino acid substitutions in "low-risk" human papillomavirus (HPV) type 6 E7 protein enhance features characteristic of the "high-risk" HPV E7 oncoproteins.

HPV types associated with genital disease are termed "high-risk" or "low-risk" viruses according to their prevalence in cancers. Two viral genes, E6 and E7, are invariably expressed in cervical carcinomas. The E7 gene product has been found to bind the retinoblastoma tumor suppressor protein and to be phosphorylated by casein kinase II. Although present in both high- and low-risk E7 proteins, these activities are diminished in the low-risk HPV-6 E7 polypeptide. To better understand the oncogenic potential of the HPV-6 E7 protein, we replaced four of its amino acids with HPV-16 E7 residues present in the analogous region of the N-terminal half of the protein. Replacement of the arginine at position 4 of the HPV-6 E7 protein with an aspartate present in HPV-16 E7 slowed the mobility of the protein when expressed in vivo. Replacement of the glycine at position 22 with an aspartate resulted in higher affinity for retinoblastoma protein binding. Replacement of valine residues at positions 30 and 37 with asparagine and aspartate, respectively, resulted in higher levels of casein kinase II phosphorylation. The substitution at position 22 was the only mutation that exhibited increased transforming activity, suggesting a correlation between the HPV E7 protein affinity for the retinoblastoma tumor suppressor protein and its ability to transform established cells. Our results show that subtle changes in sequence may result in marked differences in biological activity of HPV oncogenes.

Increased E6/E7 transcription in HPV 18-immortalized human keratinocytes results from inactivation of E2 and additional cellular events.

We characterized the state of the viral genome and transcription of human papillomavirus 18 oncogenes, E6 and E7, in immortalized human keratinocytes. At passage 9 after transfection with HPV 18 a homogeneous population of immortal clones was present. These cells have the viral DNA integrated within the E2 orf, accompanied by its partial deletion, similarly to what has been found in cervical carcinoma specimens. Transcription of the E6 and E7 oncogenes is mediated by the major viral early promoter (P105). Interestingly, transcriptional activity from this promoter increased upon continued in vitro passage of the cells. This event is concomitant with an increase in the proliferation rate of the cells. Reintroduction of the HPV 18 E2 gene into these cells resulted in repression of P105. However, the amount of E2 was limited in the HPV 18-immortalized cells. These data suggest that both viral and cellular factors play a role in increasing levels of E6 and E7 transcription providing the host cell with a proliferation advantage necessary for tumor growth.

The two proteins encoded by the cottontail rabbit papillomavirus E6 open reading frame differ with respect to localization and phosphorylation.

Cottontail rabbit papillomavirus-induced tumors contain two E6-coding transcripts. A major transcript can code for a short E6 protein initiated at AUG codon 2, and a minor one could code for a long E6 initiated at AUG 1. We have identified the two proteins expressed in COS-7 cells (M. Barbosa and F. O. Wettstein, J. Virol. 61:2938-2942, 1987). The properties of the two proteins are distinctly different. The long E6 is predominantly present in the nucleus, in which it appears to be associated with the nuclear matrix. Minor portions of the long E6 are located in equal amounts in both the soluble cytoplasmic and the membrane fractions. The short E6 is a soluble cytoplasmic protein phosphorylated at serine residues.

Transcription of the cottontail rabbit papillomavirus early region and identification of two E6 polypeptides in COS-7 cells.

Cottontail rabbit papillomavirus (CRPV) early proteins are present at very low levels in virus-induced tumors and cannot be detected by immunological methods. Furthermore, cells in culture are not readily transformed by the virus. To overcome these difficulties in identifying and characterizing the putative transforming protein(s) coded by the E6 open reading frame, the early cottontail rabbit papillomavirus region was expressed under the control of the late simian virus 40 promoter. Mapping of the transcripts in transiently transfected COS-7 cells indicated that transcription was initiated in the late region of simian virus 40. Two E6-coded polypeptides were identified, representing translation products initiated at the first and second AUG codons.

Identification of the major cottontail rabbit papillomavirus late RNA cap site and mapping and quantitation of an E2 and minor E6 coding mRNA in papillomas and carcinomas.

The capsite of the 2.6- and 4.8-kb major late transcripts of cottontail rabbit papillomavirus (CRPV) has been mapped by primer extension. A leader exon of about 300 nucleotides common to both RNAs is located in the untranslated region of the genome upstream of the capsites for early transcripts. In contrast to the early capsites which are all preceded by TATA boxes, no such sequence is present 30 nucleotides upstream of the late capsite. These data indicate that the switch from early to late transcription involves recognition of a new promoter and suppression of transcription termination at the early polyadenylation site. We have also identified a minor exon with a coding potential for a putative E2 transactivating protein. Quantitation by S1 mapping of the E2 coding exon and a minor exon coding for a full-sized E6 protein unique in size to the highly oncogenic CRPV did not reveal differences in the level of transcription between papillomas and carcinomas.