Morphological and Molecular Analyses of Aponurus laguncula Looss, 1907 (Digenea: Lecithasteridae) Parasitic in Atlantic Spadefish Chaetodipterus faber (Broussonet, 1782) (Acanthuriformes: Ephippidae) from Brazilian Coastal Zone.

OBJECTIVES: An integrative taxonomic description of Aponurus laguncula (Lecithasteridae), a digenean parasitic species of Chaetodipterus faber (Acanthuriformes) from Brazilian Southeast, is provided. Morphological techniques, as whole mounted slides, histology and scanning electron microscopy, and molecular analyses supported that integrative description. METHODS: Fifteen digenean specimens were stained in hydrochloric carmine and mounted on permanent slides. Two specimens were stained in hematoxylin and eosin following histological routine processing. Four parasites were dehydrated through a graded ethanol series, critical point dried with carbon dioxide and coated with gold to scanning electron microscopy analysis. Sequence of the large ribosomal subunit (28S rDNA) gene was generated and used to construct a phylogeny based on maximum likelihood and Bayesian inference analyses. RESULTS: Morphological description and morphometric data obtained in present study were in accordance with previous studies of the species. Use of another morphological techniques, as scanning electron microscopy and histology, corroborated the observed features of whole mounted slides. Also, they provided a better observation of previous reported characteristics and new features reporting, such as an elongated hermaphroditic duct, a smooth tegument and cells that compose the prostatic gland. The molecular sequence obtained in the present study formed a robust clade with available sequences of species of Aponurus. CONCLUSIONS: The integrative taxonomic approach successfully combined morphological observations, including both previously reported features and new descriptions from histological and electron microscopy analyses, with molecular data to identify these specimens as A. laguncula. Moreover, the detailed characterization of structures, such as the gonads in A. laguncula, that would be challenging to analyze using a single technique, was possible. Further molecular studies with less conserved genetic markers should be conducted to understand phylogenetic relationships between Aponurus species.

Integrative Taxonomy of Prosogonotrema bilabiatum Vigueras, 1940 (Digenea: Sclerodistomidae): A Parasite in Atlantic Spadefish Chaetodipterus faber (Broussonet, 1782) (Acanthuriformes: Ephippidae) from Brazil.

OBJECTIVES: The present work aims to expand the knowledge of the digenean species Prosogonotrema bilabiatum (Sclerodistomidae), a parasite of Chaetodipterus faber (Acanthuriformes) from Brazil, with an integrative taxonomic approach, using light microscopy, scanning electron microscopy, histology, and molecular biology. METHODS: Forty-one digenean specimens were stained with hydrochloric carmine for morphological studies. Eleven parasites were dehydrated through a graded ethanol series, critical point dried with carbon dioxide, and coated with gold for scanning electron microscopy analysis. Four specimens were processed following histological routine and stained with hematoxylin and eosin and Gomori trichrome. DNA extracted was amplified using 28S partial primer D1-D3. Maximum likelihood and Bayesian inference were performed for phylogenetic analysis. RESULTS: Morphometric and morphological data of the specimens studied ranged in accordance as observed in previous descriptions of the species. Observations from scanning electron microscopy and histology corroborated with those observed in stained whole mounts. Molecular analysis showed that specimens of P. bilabiatum from Brazil clustered with another two sequences of this species from different hosts and localities, with a high node support value. CONCLUSIONS: The integrative taxonomic approach allowed to record and describe new characteristics of P. bilabiatum related to the tegument, the structure and the arrangement of its tissues. The use of molecular markers confirmed that specimens identified as P. bilabiatum from different hosts and localities are all conspecific. Further studies, mainly molecular with less conserved genetic markers, should be carried out to better understand the phylogenetic relationships of Prosogonotrema with Hemiuroidea.

Integrity of prokaryotic mRNA isolated from complex samples for in vivo bacterial transcriptome analysis.

Even though several in vitro studies have focused on bacterial biology, the extent of such knowledge is not complete when considering an actual infection. As culture-independent microbiology methods such as high-throughput sequencing became available, important aspects of host-bacterium interactions will be elucidated. Based on microbiological relevance, we considered Bacteroides fragilis in a murine experimental infection as a model system to evaluate the in vivo bacterial transcriptome in host exudates. A disproportionate number of reads belonging to the host genome were retrieved in the first round of pyrosequencing, even after depletion of ribosomal RNA; the average number of reads related to the eukaryotic genome was 71.924-67.7%, whereas prokaryotic reads represented 34.338-32.3% in host exudates. Thus, different treatments were used to improve the prokaryotic RNA yield: i) centrifugation; ii) ultrasonic treatment; and iii) ultrasonic treatment followed by centrifugation. The latter treatment was found to be the most efficient in generating bacterial yields, as it resulted in a higher number of Bacteroides cells. However, the RNA extracted after this treatment was not of sufficient quality to be used in cDNA synthesis. Our results suggest that the methodology routinely used for RNA extraction in transcriptional analysis is not appropriate for in vivo studies in complex samples. Furthermore, the most efficient treatment for generating good bacterial cell yields was not suitable to retrieve high-quality RNA. Therefore, as an alternative methodological approach to enable in vivo studies on host-bacterium interactions, we advise increasing the sequencing depth despite the high costs.

PCR to detect Mycobacterium tuberculosis in respiratory tract samples: evaluation of clinical data.

Tuberculosis (TB) remains as an important public health problem worldwide. Therefore, the rapid detection of M. tuberculosis is of primary importance to effectively reduce transmission in patients. The aims of this study were to evaluate two in-house molecular tests: nested PCR (nPCR) and real-time PCR (rtPCR) to detect M. tuberculosis complex directly from clinical samples. The results were compared to the culture results and to the culture results plus clinical data of patients. The rtPCR and nPCR presented high sensitivity (Se) and specificity (Sp) (rtPCR 97·6% and 91·5%, nPCR 85·7% and 92·7%, respectively) compared to culture. When the results of the molecular tests were compared to the culture plus clinical data the Se and Sp were 90·2% and 97·3% for rtPCR and 80·4% and 98·6% for the nPCR, respectively. The results demonstrated that molecular assays of M. tuberculosis can provide a sensitive and rapid diagnostic of TB, and when used in addition to the clinical data of TB patients will help to improve the Sp of the diagnosis of pulmonary TB.

Survival of Lactobacillus delbrueckii UFV H2b20 in fermented milk under simulated gastric and intestinal conditions.

The survival of Lactobacillus delbrueckii UFV H2b20 was assessed in fermented milk, both during the storage period and after exposure to simulated gastric and intestinal juices, as well the detection of the gene fbpA involved in adherence to human gastrointestinal tract. L. delbrueckii UFV H2b20 remained stable and viable for 28 days under refrigerated storage conditions. After one day of storage, that strain exhibited a one-log population reduction following exposure in tandem to simulated gastric and intestinal juices. After 14 days of storage, a two-log reduction was observed following 90 min of exposure to the simulated gastric conditions. However, the strain did not survive following exposure to the simulated intestinal juice. The observed tolerance to storage conditions and resistance to the simulated gastric and intestinal conditions confirm the potential use of L. delbrueckii UFV H2b20 as a probiotic, which is further reinforced by the detection of fbpA in this strain.

Carbapenem-susceptible Acinetobacter baumannii carrying the ISAba1 upstream blaOXA-51-like gene in Porto Alegre, southern Brazil.

SUMMARY Over the last decade, Acinetobacter baumannii resistant to carbapenems has emerged in many medical centres and is commonly associated with high morbidity and mortality. We investigated potential mechanisms contributing to antimicrobial resistance of 58 clinical isolates of A. baumannii collected during a prolonged city-wide outbreak in five different hospitals in southern Brazil. The integrase gene was detected in 51 (87·9%) isolates of which 36 harboured class 2 integrons alone and 14 had both class 1 and 2 integrons; all carbapenem-resistant isolates displayed class 2 integrons. ISAba1 was found upstream of bla OXA-23-like only in isolates resistant to carbapenems; however, ISAba1 upstream of blaOXA-51-like was present in both susceptible and resistant isolates. This is the first report of a high prevalence of class 2 integrons in A. baumannii in southern Brazil. Moreover, our study suggests that ISAba1/blaOXA-51-like alone is insufficient to confer resistance to carbapenems.

A combined enrichment/polymerase chain reaction based method for the routine screening of Streptococcus agalactiae in pregnant women

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates. Guidelines from CDC recommend universal screening of pregnant women for rectovaginal GBS colonization. The objective of this study was to compare the performance of a combined enrichment/PCR based method targeting the atr gene in relation to culture using enrichment with selective broth medium (standard method) to identify the presence of GBS in pregnant women. Rectovaginal GBS samples from women at ¡Ý36 weeks of pregnancy were obtained with a swab and analyzed by the two methods. A total of 89 samples were evaluated. The prevalence of positive results for GBS detection was considerable higher when assessed by the combined enrichment/PCR method than with the standard method (35.9% versus 22.5%, respectively). The results demonstrated that the use of selective enrichment broth followed by PCR targeting the atr gene is a highly sensitive, specific and accurate test for GBS screening in pregnant women, allowing the detection of the bacteria even in lightly colonized patients. This PCR methodology may provide a useful diagnostic tool for GBS detection and contributes for a more accurate and effective intrapartum antibiotic and lower newborn mortality and morbidity.

A combined enrichment/polymerase chain reaction based method for the routine screening of Streptococcus agalactiae in pregnant women.

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates. Guidelines from CDC recommend universal screening of pregnant women for rectovaginal GBS colonization. The objective of this study was to compare the performance of a combined enrichment/PCR based method targeting the atr gene in relation to culture using enrichment with selective broth medium (standard method) to identify the presence of GBS in pregnant women. Rectovaginal GBS samples from women at ≥36 weeks of pregnancy were obtained with a swab and analyzed by the two methods. A total of 89 samples were evaluated. The prevalence of positive results for GBS detection was considerable higher when assessed by the combined enrichment/PCR method than with the standard method (35.9% versus 22.5%, respectively). The results demonstrated that the use of selective enrichment broth followed by PCR targeting the atr gene is a highly sensitive, specific and accurate test for GBS screening in pregnant women, allowing the detection of the bacteria even in lightly colonized patients. This PCR methodology may provide a useful diagnostic tool for GBS detection and contributes for a more accurate and effective intrapartum antibiotic and lower newborn mortality and morbidity.

A combined enrichment/polymerase chain reaction based method for the routine screening of Streptococcus agalactiae in pregnant women

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates. Guidelines from CDC recommend universal screening of pregnant women for rectovaginal GBS colonization. The objective of this study was to compare the performance of a combined enrichment/PCR based method targeting the atr gene in relation to culture using enrichment with selective broth medium (standard method) to identify the presence of GBS in pregnant women. Rectovaginal GBS samples from women at 36 weeks of pregnancy were obtained with a swab and analyzed by the two methods. A total of 89 samples were evaluated. The prevalence of positive results for GBS detection was considerable higher when assessed by the combined enrichment/PCR method than with the standard method (35.9% versus 22.5%, respectively). The results demonstrated that the use of selective enrichment broth followed by PCR targeting the atr gene is a highly sensitive, specific and accurate test for GBS screening in pregnant women, allowing the detection of the bacteria even in lightly colonized patients. This PCR methodology may provide a useful diagnostic tool for GBS detection and contributes for a more accurate and effective intrapartum antibiotic and lower newborn mortality and morbidity.

Evaluation of liquid-liquid extraction process for separating acrylic acid produced from renewable sugars.

In this article, the separation and the purification of the acrylic acid produced from renewable sugars were studied using the liquid-liquid extraction process. Nonrandom two-liquids and universal quasi-chemical models and the prediction method universal quasi-chemical functional activity coefficients were used for generating liquid-liquid equilibrium diagrams for systems made up of acrylic acid, water, and solvents (diisopropyl ether, isopropyl acetate, 2-ethyl hexanol, and methyl isobutyl ketone) and the results were compared with available liquid-liquid equilibrium experimental data. Aspen Plus (Aspen Technology, Inc., version 2004.1) software was used for equilibrium and process calculations. High concentration of acrylic acid was obtained in this article using diisopropyl ether as solvent.

Sera from chronic chagasic patients depress cardiac electrogenesis and conduction.

We report results obtained with sera from 58 chronic chagasic patients that were evaluated for effects on heart rate and atrioventricular (AV) conduction in isolated rabbit hearts and screened for the presence of muscarinic and beta-adrenergic activity. We show that sera from 26 patients decreased heart rate, while 10 increased it and 22 had no effect. Additionally, sera from 20 of the 58 patients blocked AV conduction. Muscarinic activation seems to be involved in both effects, but is not the only mechanism, since atropine did not antagonize the decrease in heart rate in 23% of sera or AV block in 40%. Sera from patients with complex arrhythmias were significantly more effective in depressing both heart rate and AV conduction. Sera that induce increases in heart rate seem to operate exclusively through beta-adrenergic activation. Two of these sera, evaluated with respect to intercellular communication in primary cultures of embryonic cardiomyocytes were able to block gap junction conductance evaluated by a dye injection technique after 24-h exposure. The mechanisms underlying this uncoupling effect are currently being investigated.

Sera from chronic chagasic patients depress cardiac electrogenesis and conduction

We report results obtained with sera from 58 chronic chagasic patients that were evaluated for effects on heart rate and atrioventricular (AV) conduction in isolated rabbit hearts and screened for the presence of muscarinic and beta-adrenergic activity. We show that sera from 26 patients decreased heart rate, while 10 increased it and 22 had no effect. Additionally, sera from 20 of the 58 patients blocked AV conduction. Muscarinic activation seems to be involved in both effects, but is not the only mechanism, since atropine did not antagonize the decrease in heart rate in 23 percent of sera or AV block in 40 percent. Sera from patients with complex arrhythmias were significantly more effective in depressing both heart rate and AV conduction. Sera that induce increases in heart rate seem to operate exclusively through beta-adrenergic activation. Two of these sera, evaluated with respect to intercellular communication in primary cultures of embryonic cardiomyocytes were able to block gap junction conductance evaluated by a dye injection technique after 24-h exposure. The mechanisms underlying this uncoupling effect are currently being investigated

Central nervous system metastases from breast carcinoma. A clinical and laboratorial study in 47 patients.

In this retrospective study, 47 patients with clinical diagnosis of central nervous system metastases of breast cancer were evaluated by computerized tomography (CT), magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) examination. The patients were divided in 2 groups: 1, without leptomeningeal neoplasm and 2, with leptomeningeal neoplasm. In the group 2, the time interval between the primary disease and the central nervous system metastasis as well as the survival time were shorter than in group 1 (40 and 4.3 months in group 2 versus 57 and 10 months respectively, in group 1). In both groups the most common neurological symptoms and signs were intracranial hypertension and motor deficits. The most sensitive diagnostic methods were CT and MRI in group 1, and the CSF examination in group 2. The use of the tumor markers CEA and CA-15.3 in the routine examination of CSF showed promising results, mainly in leptomeningeal forms.

Autonomic innervation of the salivary glands in cebid monkeys: a histochemical study.

The autonomic innervation of the major and minor salivary glands was studied in five species of cebid monkeys using acetylcholinesterase (AChE) and catecholamine histochemistry. Catecholamine-containing and AChE-positive nerve fibres were observed in the vessels and secretory endpieces of all glands, with no apparent predominance of one type over the other. In the intralobular ducts, however, the cholinergic innervation predominates. In the major salivary and minor sublingual glands the density of the nervous supply was higher, whereas in the secondary mandibular and posterior lingual glands it was less dense. The morphological patterns of salivary gland innervation found in Cebidae are compared with those of the related family Callitrichidae.

The pineal recess of the opossum: a scanning and transmission electron microscope study.

Scanning and transmission electron microscope studies were performed in the pineal recess region of eighteen adult Didelphis albiventris opossums. With the scanning electron microscope, three concentric regions could be observed in the pineal recess: a central zone lined by cells free of cilia or microvilli, or by a meshwork of interdigitating cell processes; an intermediate zone lined by microvillous cells; and a peripheral zone, lined by ciliated cells. The transmission electron microscope revealed that the central zone is lined mainly by pinealocytes. The lining cells are frequently overlaid by a meshwork of pinealocytic and glial processes. A "subsurface labyrinth" (a meshwork of cellular processes underneath the lining cells) is constantly seen in the central zone. The intermediate zone is lined by elongated microvillous ependymal cells, and the peripheral zone by typical ependyma. The large liquor-contacting area in the pineal recess region, as well as the peculiar organization of its surface, suggest a complex interrelationship between the liquor and the pineal gland of the opossum.

The pineal recess of the opossum: a scanning and transmission electron microcospe study

Scanning and transmission electron microscope studies were performed in the pineal recess region of eighteen adult Didelphis albiventris opossums. With the scanning electron microscope, three concentric regions could be observed in the pineal recess: a central zone lined by cells free of cilia or microvilli, or by a meshwork of interdigitating cell processes; an intermediate zone lined by microvillous cells; and a peripheral zone, lined by ciliated cells. The transmission electron microscope revealed that the central zone is lined mainly by pinealocytes. The lining cells are frequently overlaid by a meshwork of pinealocytic and gliak oricesses. A <> (a meshwork of cellular processes underneath the lining cells) is contantly seen in the central zone. The intermediate zone is lined by elongated microvillous ependymal cells, and the peripheral zone by typical ependyma. The large liquor-contacting area in the pineal recess region, as well as the peculiar organization of its surface, suggest a complex interrelationship between the liquor and the pineal gland of the opossum

The pineal recess of the opossum: a scanning and transmission electron microcospe study

Scanning and transmission electron microscope studies were performed in the pineal recess region of eighteen adult Didelphis albiventris opossums. With the scanning electron microscope, three concentric regions could be observed in the pineal recess: a central zone lined by cells free of cilia or microvilli, or by a meshwork of interdigitating cell processes; an intermediate zone lined by microvillous cells; and a peripheral zone, lined by ciliated cells. The transmission electron microscope revealed that the central zone is lined mainly by pinealocytes. The lining cells are frequently overlaid by a meshwork of pinealocytic and gliak oricesses. A <> (a meshwork of cellular processes underneath the lining cells) is contantly seen in the central zone. The intermediate zone is lined by elongated microvillous ependymal cells, and the peripheral zone by typical ependyma. The large liquor-contacting area in the pineal recess region, as well as the peculiar organization of its surface, suggest a complex interrelationship between the liquor and the pineal gland of the opossum (AU)