Investigating luxS gene expression in lactobacilli along lab-scale cocoa fermentations.

Previous metagenomic analyses have suggested that lactobacilli present potential for Quorum Sensing (QS) in cocoa fermentation, and in the present research, laboratory scale fermentations were carried out to monitor the expression of luxS, a universal marker of QS. For that, 96 h-fermentations were studied, as follows: F0 (non inoculated control), F1 (inoculated with yeasts, lactic acid bacteria, and acetic acid bacteria), F2 (inoculated with yeasts and acetic acid bacteria), F3 (inoculated with yeasts only). The parameters evaluated were: plate counting, quantification of key enzymes and analysis of volatile organic compounds associated with key sensory descriptors, using headspace gas chromatography-mass spectrometry (GC-MS). Furthermore, QS was estimated by the quantification of the expression of luxS genes by Reverse Transcriptase Real-Time PCR. The results demonstrated that microbial succession occurred in pilot scale fermentations, but no statistical differences for microbial enumeration and α-diversity index were observed among experiments and control. Moreover, it was not possible to make conclusive correlations of enzymatic profile and fermenting microbiota, likely due to the intrinsic activity of plant hydrolases. Regarding to the expression of luxS genes, in Lactiplantibacillus plantarum they were active along the fermentation, but for Limosilactobacillus fermentum, luxS was expressed only at early and middle phases. Correlation analysis of luxS expression and production of volatile metabolites evidenced a possible negative association of Lp. Plantarum with fermentation quality. In conclusion, these data corroborate former shotgun metagenomic analysis by demonstrating the expression of luxS by lactobacilli in pilot scale cocoa fermentation and evidence Lp. Plantarum is the main lactic acid bacteria related to its expression.

Simplified magnetic anchor-guided endoscopic submucosal dissection: an ex vivo porcine model.

INTRODUCTION AND AIMS: Endoscopic submucosal dissection (ESD) is a well-established treatment for superficial gastrointestinal tumors and enables en bloc resection. Adequate tissue tension is important for safe and effective dissection. Simplified magnetic anchor-guided ESD (MAG-ESD) with a neodymium magnet has potential benefits, compared with other current traction methods. We evaluated the feasibility of simplified MAG-ESD in an ex vivo porcine model. MATERIALS AND METHODS: An experimental study was conducted, utilizing the standard ESD technique. An external magnet and an internal magnet, both neodymium magnets, were used for the magnetic anchoring. The internal magnet was attached to an arm of a hemoclip with a 2-0 silk suture. After the incision, the clip with the internal magnet was placed at the edge of the lesion. The external magnet was maneuvered around the surface to apply adequate tension. RESULTS: A total of 15 en bloc ESDs (5 with no magnetic anchoring and 10 with magnetic anchoring) were carried out. Traction and dissection were feasible in all cases and the procedures were completed in fewer than 90min. Lesion size ranged from 15 to 50mm (mean 30mm). Two cases in the group with magnetic anchoring presented with punctate perforation (13.3%). CONCLUSIONS: Our study demonstrated the feasibility of simplified MAG-ESD and en bloc resection in an ex vivo porcine model.

Simplified magnetic anchor-guided endoscopic submucosal dissection: an ex vivo porcine model. / Disección endoscópica de la submucosa asistida con anclaje magnético simplificado: modelo porcino ex vivo.

INTRODUCTION AND AIMS: Endoscopic submucosal dissection (ESD) is a well-established treatment for superficial gastrointestinal tumors and enables en bloc resection. Adequate tissue tension is important for safe and effective dissection. Simplified magnetic anchor-guided ESD (MAG-ESD) with a neodymium magnet has potential benefits, compared with other current traction methods. We evaluated the feasibility of simplified MAG-ESD in an ex vivo porcine model. MATERIALS AND METHODS: An experimental study was conducted, utilizing the standard ESD technique. An external magnet and an internal magnet, both neodymium magnets, were used for the magnetic anchoring. The internal magnet was attached to an arm of a hemoclip with a 2-0 silk suture. After the incision, the clip with the internal magnet was placed at the edge of the lesion. The external magnet was maneuvered around the surface to apply adequate tension. RESULTS: A total of 15 en bloc ESDs (five with no magnetic anchoring and 10 with magnetic anchoring) were carried out. Traction and dissection were feasible in all cases and the procedures were completed in fewer than 90 min. Lesion size ranged from 15 to 50 mm (mean 30 mm). Two cases in the group with magnetic anchoring presented with punctate perforation (13.3%). CONCLUSIONS: Our study demonstrated the feasibility of simplified MAG-ESD and en bloc resection in an ex vivo porcine model.

Oxidative stress response in Paracoccidioides brasiliensis

Survival of pathogenic fungi inside human hosts depends on evasion from the host immune system and adaptation to the host environment. Among different insults that Paracoccidioides brasiliensis has to handle are reactive oxygen and nitrogen species produced by the human host cells, and by its own metabolism. Knowing how the parasite deals with reactive species is important to understand how it establishes infection and survives within humans. The initiative to describe the P. brasiliensis transcriptome fostered new approaches to study oxidative stress response in this organism. By examining genes related to oxidative stress response, one can evaluate the parasite's ability to face this condition and infer about possible ways to overcome this ability. We report the results of a search of the P. brasiliensis assembled expressed sequence tag database for homologous sequences involved in oxidative stress response. We described several genes coding proteins involved in antioxidant defense, for example, catalase and superoxide dismutase isoenzymes, peroxiredoxin, cytochrome c peroxidase, glutathione synthesis enzymes, thioredoxin, and the transcription factors Yap1 and Skn7. The transcriptome analysis of P. brasiliensis reveals a pathogen that has many resources to combat reactive species. Besides characterizing the antioxidant defense system in P. brasiliensis, we also compared the ways in which different fungi respond to oxidative damage, and we identified the basic features of this response.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis.

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.

Cloning of the chaperonin t-complex polypeptide 1 gene from Schistosoma mansoni and studies of its expression levels under heat shock and oxidative stress.

The protein TCP-1 (t-complex polypeptide 1) is a subunit of the hetero-oligomeric complex CCT (chaperonin containing TCP- 1) present in the eukaryotic cytosol. Chaperone function may be critical for the development and survival of the different life stages of Schistosoma mansoni, a parasite that is exposed to drastic environmental changes during its development. We isolated a full-length S. mansoni TCP-1 cDNA (SmTCP-1A) encoding a protein highly homologous with TCP-1. The deduced SmTCP-1A amino-acid sequence shows up to 65% identity with other eukaryotic CCT family members. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the mRNA expression levels of SmTCP-1A in adult S. mansoni were down-regulated in worms subjected to heat shock and oxidative stress conditions. This down-regulation of SmTCP-1A mRNA may reflect a switch in CCT subunits as an adaptive response to heat shock and oxidative stress conditions.

Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro.

Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH). We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.

The role of deer as a possible reservoir host of potosi virus, a newly recognized arbovirus in the United States.

Potosi (POT) virus (Bunyaviridae) was isolated from Aedes albopictus, an introduced Asian mosquito species, collected at a used tire yard in Potosi, Missouri (USA), in August and September, 1989. In September, 1990, small animals were trapped at the tire yard and six cattle were sampled at an adjacent farm; in November 1990 and 1991, blood samples were collected with filter paper strips from 364 hunter-killed, white-tailed deer (Odocoileus virginianus) in the region to determine the possible reservoir hosts of the virus. Deer specimens from Arkansas (n = 70), Colorado (n = 29), and Iowa (n = 763) (USA) were also analyzed. Specimens from 33 small vertebrates captured at the tire yard were negative for viruses. Only one eastern chipmunk (Tamias striatus) and none of six cattle had neutralizing (N) antibody against POT virus by the plaque-reduction serum neutralization test in Vero cell culture but 45 (25%) of 178 deer specimens in 1990 and 55 (30%) of 186 in 1991 were antibody positive. The 186 deer sera from 1991 were tested further and 29 (16%) were also N antibody positive to Cache Valley (CV) virus. From the 763 deer specimens tested from Iowa in 1993, 114 (15%) had N antibody to POT virus. Of 70 serum specimens from Arkansas deer in 1990, 33 (47%) had N antibody to POT and 15 (21%) to CV viruses; two (7%) of 29 CV negative serum specimens from Colorado deer in 1981 were serologically positive to POT virus. Three eastern chipmunks were experimentally inoculated with POT virus to determine their reservoir potential; none became viremic but all developed N antibody. Thus we propose that POT virus may be another virus regularly infecting wild deer populations but its impact on the health of these animals is unknown.

Schistosoma mansoni: determination of the sex of cercariae using a nonradioactively labelled DNA probe.

Cercarial sex determination protocol which utilizes nonradioactive nucleic acid labelling with digoxigenin (DIG)-11-dUTR, offers a rapid "clean" identification of the sex of Schistosoma mansoni cercaria. DIG labelled pW1-DNA (plasmid female-specific genomic DNA clone--W1) hybridized with the native cercarial DNA, and hybridization signals are visualized with Lumigen PPD. Results obtained are comparable with those obtained with the 32P labelled pW1.

Extraction and partial characterization of non-histone nuclear proteins of Schistosoma mansoni.

A pool of nuclear proteins from adult worms of Schistosoma mansoni was analyzed for amino acid composition and found to be compatible with high mobility group (HMG) proteins. One of the schistosome HMG proteins was identified as HMG 2 by one-dimensional and two-dimensional PAGE. Stage-specific differences in the HMG-like protein composition were encountered when adult worms were compared to schistosomula, the larval form. Immobilization of the adult male and female nuclear proteins onto nitrocellulose, followed by hybridization against 32P-F-10, a schistosome sex specific gene encoding a major egg shell protein, revealed distinct banding patterns. On the other hand, a synthetic oligonucleotide, derived from the 3' untranslated end of the F-10 gene and possibly containing one regulatory element of the gene, bound mainly to male low MW proteins.

Landscape ecology of Lyme disease in a residential area of Westchester County, New York.

The landscape ecology of Lyme disease was studied in 1989 on 67 residences in an endemic area of Armonk, Westchester County, a northern suburb of New York City. Four main habitat types were defined, and each property was surveyed for immature and adult lxodes dammini ticks; 98.6% of 1,790 ticks collected were I. dammini. Overall, 67.3% were collected from woods, 21.6% from ecotone (unmaintained edge), 9.1% from ornamental vegetation, and 2% from lawns. Larval ticks were concentrated in woods, but nymphs and adults were widely dispersed in all habitats. Tick abundance was positively correlated with property size. Larger properties (greater than or equal to 0.5 acre) were more likely to have woodlots and, hence, more ticks. Dark-field and direct fluorescent microscopic examination of tick midgut tissues revealed that 29.6% of nymphs and 49.7% of adults were infected with Borrelia burgdorferi. Infected nymphs and adults were found on 36% and 60% of properties, respectively. These data indicate that the abundance of ticks capable of transmitting Lyme disease spirochetes is related to landscape features of the suburban residential environment.

Duration of adult female Ixodes dammini attachment and transmission of Borrelia burgdorferi, with description of a needle aspiration isolation method.

The relationship between the attachment duration of adult female Ixodes dammini and the transmission of Borrelia burgdorferi was studied. Sixteen rabbits were exposed to spirochete-infected female ticks for specified intervals. All five rabbits exposed to ticks that fed to repletion (greater than 120 h) became infected, as did two of three exposed for 48 h. In contrast, five rabbits exposed to a cumulative total of 53 infected female I. dammini for 36 h failed to become infected, as did three rabbits exposed for 24 h. A needle aspirate method facilitated the isolation of spirochetes from host skin.

Apparent influence of the stage of blood meal digestion on the efficacy of ground applied ULV aerosols for the control of urban Culex mosquitoes. I. Field evidence.

The impact of ULV resmethrin on urban Culex mosquitoes was evaluated in 4 field trials by monitoring daily oviposition rate. A well-defined oscillation of effect, with a period corresponding to the duration of the gonotrophic cycle, was observed. We postulate that this oscillation arises from changes in susceptibility following blood feeding and/or behavioral factors. The data indicate that a single treatment with ULV may be inadequate for the effective control of vector mosquitoes.

Apparent influence of the stage of blood meal digestion on the efficacy of ground applied ULV aerosols for the control of urban Culex mosquitoes. II. Laboratory evidence.

The susceptibility of adult Culex pipiens s.l., Culiseta melanura and Aedes aegypti to insecticide aerosols in wind tunnel exposures varied with time, depending on the stage of blood meal digestion. Greater than 2-fold differences were observed in the concentrations of malathion and synergized resmethrin required to kill test mosquitoes, depending on whether they had been given a blood meal and, if they had, the length of time following the blood meal. The period of lowest susceptibility varied from 24 h after feeding in Ae. aegypti to 72 h in Cs. melanura. The greatest variability occurred during the period when undigested blood was present. Data from tests with a malathion-tolerant strain of Cx. pipiens s.l. suggested little change in susceptibility regardless of blood feeding and the associated weight changes that occur from ingestion of blood.

Apparent influence of the stage of blood meal digestion on the efficacy of ground applied ULV aerosols for the control of urban Culex mosquitoes. III. Results of a computer simulation.

When posttreatment response to ultra low volume (ULV) application of insecticide has been followed for periods approaching a week, a damped oscillation in oviposition rates is observed, probably because females who have been recently blood-fed are more resistant to insecticides than their unfed siblings. We describe a simple model (ULVSIM) that incorporates physiologic changes in insecticide susceptibility and accounts for much of our field data. The model follows 30 cohorts over 30 days following insecticidal treatment. Multiple treatments or short-term residual activity can be evaluated. The model predicts that oviposition will follow a pattern of damped oscillations after an adulticidal treatment. The model gave a good fit to oviposition data obtained in 2 field trials of resmethrin for 7 to 9 days after treatment. It can be used to evaluate the effect of single and multiple treatments on the total female Culex population and on numbers of infected females surviving for different periods following an infective blood meal.

Aedes albopictus and other container-inhabiting mosquitoes in the United States: results of an eight-city survey.

Extensive surveys were conducted in 1987 in Baytown, TX; Lafayette, Shreveport and Baton Rouge, LA; Memphis, TN; Kansas City, MO; Evansville, IN; and Jacksonville, FL. The program objective was to determine the intensity of Aedes albopictus infestations, to evaluate the degree to which Ae. albopictus had spread into residential areas, to document habitat selection and to obtain background information for possible suppression or eradication projects. This report describes the survey methods and presents a preliminary analysis of the data. Larvae, pupae and adult mosquitoes were collected from container habitats in a randomized selection of urban premises as well as at and around sites known to be at high risk for introduction of Ae. albopictus. Adult or larval mosquitoes were collected from 24.4% of 5,728 premises inspected, and there were an average of 3.27 positive containers per positive premise. Several known disease vectors, especially Culex pipiens (s.l.), were frequently found in urban container habitats. The large numbers of specimens collected during the surveys and the detailed information available for each collection make this a useful database for comparison in future studies.

Antibody prevalence of St. Louis encephalitis virus in avian hosts in Los Angeles, California, 1986.

A study was conducted to determine the pattern of St. Louis encephalitis (SLE) virus activity in the avian populations of the Los Angeles metropolitan area in 1986. In total, 679 birds of 42 species were captured at 7 study sites. The overall prevalence of SLE neutralizing (N) antibody of 3% indicated enzootic transmission. Antibody prevalences were higher in birds sampled in the central part of the metropolitan area, which was consistent with other epidemiologic data. The use of specific avian species as sentinels for future surveillance of SLE virus activity was suggested.

Importation of Aedes albopictus and other exotic mosquito species into the United States in used tires from Asia.

Between May 18 and December 4, 1986, 79 seagoing containers and their contents of 22,051 used tires were inspected for adult mosquitoes as well as eggs and larvae. Of the total inspected, 5,507 tires (25%) contained significant amounts of water. No adults or eggs were found. Fifteen tires contained mosquito larvae that were identified as Ae. albopictus, Ae. togoi, Culex pipiens complex, Tripteroides bambusa and Uranotaenia bimaculata. The infestation rate for all species was 6.8 infested tires per 10,000 tires (wet and dry) inspected. Aedes albopictus larvae were most frequently collected, occurring at a rate of 20 infested wet tires per 10,000 inspected.