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Find video protocols related to scientific articles indexed in Pubmed.
Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures.
Springerplus
PUBLISHED: 01-01-2013
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In spite of the large number of reports on fed-batch cultivation of E. coli, alternative cultivation/induction strategies remain to be more deeply exploited. Among these strategies, it could be mentioned the use of complex media with combination of different carbon sources, novel induction procedures and feed flow rate control matching the actual cell growth rate. Here, four different carbon source combinations (glucose, glycerol, glucose?+?glycerol and auto-induction) in batch media formulation were compared. A balanced combination of glucose and glycerol in a complex medium formulation led to: fast growth in the batch-phase; reduced plasmid instability by preventing early expression leakage; and protein volumetric productivity of 0.40 g.L(-1).h(-1). Alternative induction strategies were also investigated. A mixture of lactose and glycerol as supplementary medium fully induced a high biomass population, reaching a good balance between specific protein production (0.148 gprot.gDCW (-1)) and volumetric productivity (0.32 g.L(-1).h(-1)). The auto-induction protocol showed excellent results on specific protein production (0.158 gprot.gDCW (-1)) in simple batch cultivations. An automated feed control based on the on-line estimated growth rate was implemented, which allowed cells to grow at higher rates than those generally used to avoid metabolic overflow, without leading to acetate accumulation. Some of the protocols described here may provide a useful alternative to standard cultivation and recombinant protein production processes, depending on the performance index that is expected to be optimized. The protocols using glycerol as carbon source and induction by lactose feeding, or glycerol plus glucose in batch medium and induction by lactose pulse led to rSpaA production in the range of 6 g.L(-1), in short fed-batch processes (16 to 20 h) with low accumulation of undesired side metabolites.
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Anopheles atroparvus density modeling using MODIS NDVI in a former malarious area in Portugal.
J. Vector Ecol.
PUBLISHED: 12-02-2011
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Malaria is dependent on environmental factors and considered as potentially re-emerging in temperate regions. Remote sensing data have been used successfully for monitoring environmental conditions that influence the patterns of such arthropod vector-borne diseases. Anopheles atroparvus density data were collected from 2002 to 2005, on a bimonthly basis, at three sites in a former malarial area in Southern Portugal. The development of the Remote Vector Model (RVM) was based upon two main variables: temperature and the Normalized Differential Vegetation Index (NDVI) from the Moderate Resolution Imaging Spectroradiometer (MODIS) Terra satellite. Temperature influences the mosquito life cycle and affects its intra-annual prevalence, and MODIS NDVI was used as a proxy for suitable habitat conditions. Mosquito data were used for calibration and validation of the model. For areas with high mosquito density, the model validation demonstrated a Pearson correlation of 0.68 (p<0.05) and a modelling efficiency/Nash-Sutcliffe of 0.44 representing the models ability to predict intra- and inter-annual vector density trends. RVM estimates the density of the former malarial vector An. atroparvus as a function of temperature and of MODIS NDVI. RVM is a satellite data-based assimilation algorithm that uses temperature fields to predict the intra- and inter-annual densities of this mosquito species using MODIS NDVI. RVM is a relevant tool for vector density estimation, contributing to the risk assessment of transmission of mosquito-borne diseases and can be part of the early warning system and contingency plans providing support to the decision making process of relevant authorities.
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Species Composition and WNV Screening of Mosquitoes from Lagoons in a Wetland Area of the Algarve, Portugal.
Front Physiol
PUBLISHED: 10-17-2011
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The aim of this study was to evaluate mosquito abundance, species diversity, larval and adult population dynamics in seven lagoons integrated in the wetland coastal system of the Algarve, Portugal, in the summer of 2007, as well as the screening of these for West Nile virus (WNV). WNV has been isolated from mosquitoes in this region, in the summer of 2004, next to the putative area of infection of two linked human WN cases. Adult mosquitoes were collected with CDC traps baited with CO(2), and potential breeding sites were surveyed for immature stages. Morphological identification of 1,432 adult mosquitoes and 85 larvae revealed the presence of 10 species: Anopheles atroparvus, Anopheles algeriensis, Coquillettidia richiardii, Culex modestus, Culex pipiens, Culex theileri, Culex univittatus, Culiseta longiareolata, Aedes caspius, and Aedes detritus. Adult mosquito peak densities were recorded in July, contrasting with null larval breeding in the same month in the surveyed biotopes. Most abundant species were C. pipiens (52%), C. theileri (29%), and A. caspius (11%). Lagoon Salgados and Quinta das Salinas, exhibited the highest similarity of culicid fauna, despite being most distant from each other, Female mosquitoes (1,249 specimens) screened by RT-PCR, did not reveal WNV products. However, previous detection of WNV activity in this area, susceptible to re-introductions, demands for continued vigilance.
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Influence of environmental and genetic factors linked to celiac disease risk on infant gut colonization by Bacteroides species.
Appl. Environ. Microbiol.
PUBLISHED: 06-03-2011
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Celiac disease (CD) is an immune-mediated enteropathy involving genetic and environmental factors whose interaction might influence disease risk. The aim of this study was to determine the effects of milk-feeding practices and the HLA-DQ genotype on intestinal colonization of Bacteroides species in infants at risk of CD development. This study included 75 full-term newborns with at least one first-degree relative suffering from CD. Infants were classified according to milk-feeding practice (breast-feeding or formula feeding) and HLA-DQ genotype (high or low genetic risk). Stools were analyzed at 7 days, 1 month, and 4 months by PCR and denaturing gradient gel electrophoresis (DGGE). The Bacteroides species diversity index was higher in formula-fed infants than in breast-fed infants. Breast-fed infants showed a higher prevalence of Bacteroides uniformis at 1 and 4 months of age, while formula-fed infants had a higher prevalence of B. intestinalis at all sampling times, of B. caccae at 7 days and 4 months, and of B. plebeius at 4 months. Infants with high genetic risk showed a higher prevalence of B. vulgatus, while those with low genetic risk showed a higher prevalence of B. ovatus, B. plebeius, and B. uniformis. Among breast-fed infants, the prevalence of B. uniformis was higher in those with low genetic risk than in those with high genetic risk. Among formula-fed infants, the prevalence of B. ovatus and B. plebeius was increased in those with low genetic risk, while the prevalence of B. vulgatus was higher in those with high genetic risk. The results indicate that both the type of milk feeding and the HLA-DQ genotype influence the colonization process of Bacteroides species, and possibly the disease risk.
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Mosquito surveys and West Nile virus screening in two different areas of southern Portugal, 2004-2007.
Vector Borne Zoonotic Dis.
PUBLISHED: 09-22-2010
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Longitudinal mosquito surveys were carried out in southern Portugal from 2004 to 2007, in a wetland area (Comporta, District of Setúbal) and around the perimeter of a dam irrigation plant that created the largest artificial lake in Europe, 250 km(2) (Alqueva, Districts of Evora and Beja). Our aim was to study the diversity, abundance, and seasonal dynamics of mosquitoes, comparing these two different areas, to screen mosquitoes for West Nile Virus (WNV), an arboviral agent already detected in Portugal, because these areas are populated with abundant avian fauna. Monthly collections of adult mosquitoes were carried out by Centers for Disease Control light-traps with CO(2) and by indoor resting collections. Mosquitoes were identified and screened for arboviruses by reverse transcriptase (RT)-polymerase chain reaction directed toward amplification of a 217-bp fragment of the NS5 gene. Mosquito peak densities were observed in July-August in Comporta and May-June, with a plateau in July-October, in Alqueva. However, densities were far higher in Comporta area (220,821 specimens) than in Alqueva area (9442 specimens), with a clear difference in species distribution, as in Comporta the predominant species was Culex theileri (85%), followed by Aedes caspius (6%), Anopheles atroparvus (4%), and Culex pipiens sensu latu (s.l.) (3%), whereas in Alqueva the predominant species was Cx. pipiens s.l. (56%), followed by An. atroparvus (18%), Cx. theileri (14%), and Culiseta longiareolata (9%). Female mosquitoes (8842 in 175 pools) of the species Ae. caspius, An. atroparvus, Culex mimeticus, Cx. pipiens Sensu latu (s.l.), Cx. theileri, and Culex univittatus were screened and found to be negative for WNV genomic RNA. Although there was no detection of WNV sequences in mosquitoes, vigilance should continue as the circulation of virus has been previously detected more than once in Portugal, in humans, animals, and mosquitoes, and in other surrounding Mediterranean countries.
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Ferric iron uptake genes are differentially expressed in the presence of copper sulfides in Acidithiobacillus ferrooxidans strain LR.
Antonie Van Leeuwenhoek
PUBLISHED: 08-20-2010
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Acidithiobacillus ferrooxidans is one of the most widely used microorganisms in bioleaching operations to recover copper from low-grade copper sulfide ores. This work aimed to investigate the relative expression of genes related to the iron uptake system when A. ferrooxidans LR was maintained in contact with chalcopyrite or bornite as the sole energy source. Real-time quantitative PCR analysis revealed that the presence of bornite had no effect on the expression of seven genes related to the siderophore-mediated Fe(III) uptake system, while in the presence of chalcopyrite the expression of the genes was up-regulated. Bioinformatic analysis of the genomic region where these genes were found revealed the existence of three new putative DNA-binding sequences for the ferric iron uptake transcriptional regulator (Fur). Electrophoretic mobility shift assays demonstrated that a purified A. ferrooxidans His-tagged Fur protein was able to bind in vitro to each of these putative Fur boxes, suggesting that Fur regulated the expression of these genes. The expression of fur and two known Fur-regulated genes, mntH and dsrK, was also investigated in the presence of chalcopyrite. While the expression of fur and mntH was up-regulated, the expression of dsrK was down-regulated. The low amount of ferrous iron in the medium was probably responsible for the up-regulation of fur and the genes related to the siderophore-mediated Fe(III) uptake system when A. ferrooxidans LR was kept in the presence of chalcopyrite. A homology model of the A. ferrooxidans Fur was constructed and revealed that the putative DNA-binding surface presents conserved positively charged residues, supporting a previously suggested mode of interaction with DNA. The up-regulation of fur and the siderophore-mediated Fe(III) uptake genes, and the down-regulation of dsrK suggest that in the presence of chalcopyrite Fur acts as a transcription inducer and repressor.
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Gene expression modulation by chalcopyrite and bornite in Acidithiobacillus ferrooxidans.
Arch. Microbiol.
PUBLISHED: 04-30-2010
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Acidithiobacillus ferrooxidans is a mesophilic, acidophilic, chemolithoautotrophic bacterium that obtains energy from the oxidation of ferrous iron (Fe2+), elemental sulfur and reduced sulfur compounds. The industrial interest in A. ferrooxidans resides in its capacity to oxidize insoluble metal sulfides into soluble metal sulfates, thus allowing the recovery of the desired metals from low-grade sulfide ores. In the present work, RNA arbitrarily primed PCR (RAP-PCR) was performed to identify cDNAs differentially expressed in A. ferrooxidans cells grown in the presence of Fe2+ and cells maintained for 24 h in the presence of the copper sulfides bornite and chalcopyrite. Eighteen cDNAs corresponding to genes with known function were identified, and their relative expression was further characterized by real-time quantitative PCR. Bornite had a mild effect on the expression of the 18 genes analyzed. None of these genes was down-regulated and among the few genes up-regulated, it is worth mentioning lepA and def-2 that are involved in protein synthesis. Chalcopyrite presented the most significant changes. Five genes related to protein processing were down-regulated, and another 5 genes related to the transport system were up-regulated. The up- and down-regulation of these genes in the presence of bornite and chalcopyrite could be due to alterations in the ideal pH, presence of copper ions in solution and nutrient limitation. The results suggest that gene expression modulation might be important for the A. ferrooxidans early response to copper sulfides.
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Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal.
BMC Evol. Biol.
PUBLISHED: 07-09-2009
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Culex pipiens L. is the most widespread mosquito vector in temperate regions. This species consists of two forms, denoted molestus and pipiens, that exhibit important behavioural and physiological differences. The evolutionary relationships and taxonomic status of these forms remain unclear. In northern European latitudes molestus and pipiens populations occupy different habitats (underground vs. aboveground), a separation that most likely promotes genetic isolation between forms. However, the same does not hold in southern Europe where both forms occur aboveground in sympatry. In these southern habitats, the extent of hybridisation and its impact on the extent of genetic divergence between forms under sympatric conditions has not been clarified. For this purpose, we have used phenotypic and genetic data to characterise Cx. pipiens collected aboveground in Portugal. Our aims were to determine levels of genetic differentiation and the degree of hybridisation between forms occurring in sympatry, and to relate these with both evolutionary and epidemiological tenets of this biological group.
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The Culex pipiens complex in continental Portugal: distribution and genetic structure.
J. Am. Mosq. Control Assoc.
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Portugal is a southern European country that displays favorable ecological conditions for the establishment of West Nile virus (WNV) transmission cycles. Competent mosquito vector species are present throughout the country. Among the species with reported cases of WNV isolation in Portugal, Culex pipiens is the most ubiquitous and abundant mosquito. This species exhibits two biological forms with differences in host preferences. The molestus form has a greater tendency to feed upon humans and other mammals whereas the pipiens form prefers avian hosts. In northern latitudes, both forms are physically separated, with molestus occupying underground habitats and pipiens being found aboveground. However, the warmer climatic conditions of southern regions such as Portugal may favor the sympatric occurrence of both forms hence promoting interform hybridization. Genetic introgression between molestus and pipiens forms may result in a higher propensity for admixed populations to serve as bridge-vectors of WNV between humans and birds. Here we revise our present knowledge on the distribution, role in WNV transmission and genetic structure of the Cx. pipiens complex in continental Portugal. We focus on recent findings of sympatric molestus and pipiens populations that display considerable levels of hybridization and discuss the epidemiological repercussions of this occurrence.
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Cloning, auto-induction expression, and purification of rSpaA swine erysipelas antigen.
Curr. Microbiol.
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This work reports the cloning, expression, and purification of a 42-kDa fragment of the SpaA protein from Erysipelothrix rhusiopathiae, the main antigenic candidate for a subunit vaccine against swine erysipelas. The use of an auto-induction protocol to improve heterologous protein expression in recombinant Escherichia coli cultures was also investigated. The cellular growth pattern and metabolite formation were evaluated under different induction conditions. The His-tagged protein was over-expressed as inclusion bodies, and was purified by a single chromatography step under denaturing conditions. Auto-induction conditions were shown to be an excellent process strategy, leading to a high level of rSpaA expression (about 25 % of total cellular protein content) in a short period of time.
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Influence of breastfeeding versus formula feeding on lymphocyte subsets in infants at risk of coeliac disease: the PROFICEL study.
Eur J Nutr
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In addition to genetic risk, environmental factors might influence coeliac disease (CD) development. We sought to assess the effect of the interaction between milk-feeding practices and the HLA-DQ genotype on peripheral lymphocyte subsets and their activation markers in infants at familial risk for CD.
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Influence of milk-feeding type and genetic risk of developing coeliac disease on intestinal microbiota of infants: the PROFICEL study.
PLoS ONE
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Interactions between environmental factors and predisposing genes could be involved in the development of coeliac disease (CD). This study has assessed whether milk-feeding type and HLA-genotype influence the intestinal microbiota composition of infants with a family history of CD. The study included 164 healthy newborns, with at least one first-degree relative with CD, classified according to their HLA-DQ genotype by PCR-SSP DQB1 and DQA1 typing. Faecal microbiota was analysed by quantitative PCR at 7 days, and at 1 and 4 months of age. Significant interactions between milk-feeding type and HLA-DQ genotype on bacterial numbers were not detected by applying a linear mixed-model analysis for repeated measures. In the whole population, breast-feeding promoted colonization of C. leptum group, B. longum and B. breve, while formula-feeding promoted that of Bacteroides fragilis group, C. coccoides-E. rectale group, E. coli and B. lactis. Moreover, increased numbers of B. fragilis group and Staphylococcus spp., and reduced numbers of Bifidobacterium spp. and B. longum were detected in infants with increased genetic risk of developing CD. Analyses within subgroups of either breast-fed or formula-fed infants indicated that in both cases increased risk of CD was associated with lower numbers of B. longum and/or Bifidobacterium spp. In addition, in breast-fed infants the increased genetic risk of developing CD was associated with increased C. leptum group numbers, while in formula-fed infants it was associated with increased Staphylococcus and B. fragilis group numbers. Overall, milk-feeding type in conjunction with HLA-DQ genotype play a role in establishing infants gut microbiota; moreover, breast-feeding reduced the genotype-related differences in microbiota composition, which could partly explain the protective role attributed to breast milk in this disorder.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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