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Producción Científica, Dunaliella salina has been recognized as an excellent biomass source of carotenoid, which can be used as a natural orange coloring agent for food products. The most eco-friendly approach for extracting carotenoid is through supercritical carbon dioxide extraction, as it yields highly concentrated extracts while preventing pigment thermal degradation. However, there are limitations when a lipophilic extract is considered a food ingredient, in particular very difficult handling and low solubility in water-based products. The aim of this study was to develop a hydrosoluble form of a natural carotenoid-rich extract recovered from algae biomass within a biorefinery concept to be incorporated in aqueous-based food products. A two-step process was developed, starting with the emulsification of the supercritical extract into a mixture of maltodextrin and gum arabic, using soy lecithin as an emulsifier. The emulsification was followed by a spray-drying step. The impact of process variables on the encapsulation yield, efficiency, emulsion properties, and particle characteristics was studied. The resulting particles exhibited an intense orange color and good water dispersibility, facilitating uniform yellow coloring when incorporated into an aqueous-based product. Overall, spray-drying emulsions containing carotenoids derived from Dunaliella salina prove to be a promising strategy for the global market demand for natural colorants., Fundación Portuguesa para la Ciencia y la Tecnología (FCT) - (grant SFRH/BPD/79533/2011), IF Starting Grant - GRAPHYT (IF/00723/2014), Laboratorio Asociado de Química Verde LAQV y Fondo Europeo de Desarrollo Regional (FEDER) - (UID/QUI/50006/2013, POCI-01-0145-FEDER–007265), European Knowledge-Based Bioeconom (KBBE) P7 - (project 613870), Fundación Portuguesa para la Ciencia y la Tecnología (FCT) - (PEst- OE/EQB/LA0004/2011), Fundación Portuguesa para la Ciencia y la Tecnología (FCT)/Ministerio de Ciencia y Enseñanza Superior de Portugal y Fondo Europeo de Desarrollo Regional (FEDER) - iNOVA4Health (LISBOA-01-0145-FEDER-007344)

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

The specific genes and molecules that drive physiological angiogenesis differ from those involved in pathological angiogenesis, suggesting distinct mechanisms for these seemingly related processes. Unveiling genes and pathways preferentially associated with pathologic angiogenesis is key to understanding its mechanisms, thereby facilitating development of novel approaches to managing angiogenesis-dependent diseases. To better understand these different processes, we elucidated the transcriptome of the mouse retina in the well-accepted oxygen-induced retinopathy (OIR) model of pathological angiogenesis. We identified 153 genes changed between normal and OIR retinas, which represent a molecular signature relevant to other angiogenesis-dependent processes such as cancer. These genes robustly predict the survival of breast cancer patients, which was validated in an independent 1,000-patient test cohort (40% difference in 15-year survival; p = 2.56 x 10-21). These results suggest that the OIR model reveals key genes involved in pathological angiogenesis, and these may find important applications in stratifying tumors for treatment intensification or for angiogenesis-targeted therapies.

The specific genes and molecules that drive physiological angiogenesis differ from those involved in pathological angiogenesis, suggesting distinct mechanisms for these seemingly related processes. Unveiling genes and pathways preferentially associated with pathologic angiogenesis is key to understanding its mechanisms, thereby facilitating development of novel approaches to managing angiogenesis-dependent diseases. To better understand these different processes, we elucidated the transcriptome of the mouse retina in the well-accepted oxygen-induced retinopathy (OIR) model of pathological angiogenesis. We identified 153 genes changed between normal and OIR retinas, which represent a molecular signature relevant to other angiogenesis-dependent processes such as cancer. These genes robustly predict the survival of breast cancer patients, which was validated in an independent 1,000-patient test cohort (40% difference in 15-year survival; p = 2.56 x 10-21). These results suggest that the OIR model reveals key genes involved in pathological angiogenesis, and these may find important applications in stratifying tumors for treatment intensification or for angiogenesis-targeted therapies.

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

The little-known Eurasian Sparrowhawk of Macaronesia (Accipiter nisus granti), also named as the Macaronesian sparrowhawk, is endemic to Madeira Island and the Canary Islands (North Atlantic Ocean) and has the smallest area of distribution of the sparrowhawk subspecies. We studied the breeding biology of the Macaronesian sparrowhawk for the first time on Madeira Island, Portugal. Specifically, we described nests, tree nests, nest sites, and nesting territories, and we estimated incubation, hatching and fledging dates. Moreover, we evaluated the influence of altitude on the date of the initiation of breeding and measured the number of fledglings and the factors influencing this parameter. Most nesting territories (88.6%) were located in forest patches where valleys with watercourses were present. Breeding success (73.2% ± 0.1 SE, n = 18) and the mean number of young fledged per nest with eggs (2.27 ± 0.04) are lower than the values for the Canary Islands. Altitude influenced the date of the initiation of breeding, with pairs in lowlands (<700 masl) initiating breeding earlier. However, pairs breeding earlier did not have higher reproductive rate than those breeding later. The number of fledglings per nest with eggs in mixed habitats was higher than in exotic and Laurel forests. The main cause of breeding failure was forest cutting. We believe that if the forestry industry does not consider the nesting areas, as well as the breeding phenology of this subspecies, and forest fires are not prevented, then its population in Madeira may be reduced in the near future., Nest

Sporadic and inflammatory forms of colorectal cancer (CRC) account for more than 80% of cases. Recent publications have shown mechanistic evidence for the involvement of gut bacteria in the development of both CRC-forms. Whereas, colon and rectal cancer have been routinely studied together as CRC, increasing evidence show these to be distinct diseases. Also, the common use of fecal samples to study microbial communities may reflect disease state but possibly not the tumor microenvironment. We performed this study to evaluate differences in bacterial communities found in tissue samples of 18 rectal-cancer subjects when compared to 18 non-cancer controls. Samples were collected during exploratory colonoscopy (non-cancer group) or during surgery for tumor excision (rectal-cancer group). High throughput 16S rRNA amplicon sequencing of the V4V5 region was conducted on the Ion PGM platform, reads were filtered using Qiime and clustered using UPARSE. We observed significant increases in species richness and diversity in rectal cancer samples, evidenced by the total number of OTUs and the Shannon and Simpson indexes. Enterotyping analysis divided our cohort into two groups, with the majority of rectal cancer samples clustering into one enterotype, characterized by a greater abundance of Bacteroides and Dorea. At the phylum level, rectal-cancer samples had increased abundance of candidate phylum OD1 (also known as Parcubacteria) whilst non-cancer samples had increased abundance of Planctomycetes. At the genera level, rectal-cancer samples had higher abundances of Bacteroides, Phascolarctobacterium, Parabacteroides, Desulfovibrio, and Odoribacter whereas non-cancer samples had higher abundances of Pseudomonas, Escherichia, Acinetobacter, Lactobacillus, and Bacillus. Two Bacteroides fragilis OTUs were more abundant among rectal-cancer patients seen through 16S rRNA amplicon sequencing, whose presence was confirmed by immunohistochemistry and enrichment verified by digital drop

Sporadic and inflammatory forms of colorectal cancer (CRC) account for more than 80% of cases. Recent publications have shown mechanistic evidence for the involvement of gut bacteria in the development of both CRC-forms. Whereas, colon and rectal cancer have been routinely studied together as CRC, increasing evidence show these to be distinct diseases. Also, the common use of fecal samples to study microbial communities may reflect disease state but possibly not the tumor microenvironment. We performed this study to evaluate differences in bacterial communities found in tissue samples of 18 rectal-cancer subjects when compared to 18 non-cancer controls. Samples were collected during exploratory colonoscopy (non-cancer group) or during surgery for tumor excision (rectal-cancer group). High throughput 16S rRNA amplicon sequencing of the V4V5 region was conducted on the Ion PGM platform, reads were filtered using Qiime and clustered using UPARSE. We observed significant increases in species richness and diversity in rectal cancer samples, evidenced by the total number of OTUs and the Shannon and Simpson indexes. Enterotyping analysis divided our cohort into two groups, with the majority of rectal cancer samples clustering into one enterotype, characterized by a greater abundance of Bacteroides and Dorea. At the phylum level, rectal-cancer samples had increased abundance of candidate phylum OD1 (also known as Parcubacteria) whilst non-cancer samples had increased abundance of Planctomycetes. At the genera level, rectal-cancer samples had higher abundances of Bacteroides, Phascolarctobacterium, Parabacteroides, Desulfovibrio, and Odoribacter whereas non-cancer samples had higher abundances of Pseudomonas, Escherichia, Acinetobacter, Lactobacillus, and Bacillus. Two Bacteroides fragilis OTUs were more abundant among rectal-cancer patients seen through 16S rRNA amplicon sequencing, whose presence was confirmed by immunohistochemistry and enrichment verified by digital drop

Sporadic and inflammatory forms of colorectal cancer (CRC) account for more than 80% of cases. Recent publications have shown mechanistic evidence for the involvement of gut bacteria in the development of both CRC-forms. Whereas, colon and rectal cancer have been routinely studied together as CRC, increasing evidence show these to be distinct diseases. Also, the common use of fecal samples to study microbial communities may reflect disease state but possibly not the tumor microenvironment. We performed this study to evaluate differences in bacterial communities found in tissue samples of 18 rectal-cancer subjects when compared to 18 non-cancer controls. Samples were collected during exploratory colonoscopy (non-cancer group) or during surgery for tumor excision (rectal-cancer group). High throughput 16S rRNA amplicon sequencing of the V4V5 region was conducted on the Ion PGM platform, reads were filtered using Qiime and clustered using UPARSE. We observed significant increases in species richness and diversity in rectal cancer samples, evidenced by the total number of OTUs and the Shannon and Simpson indexes. Enterotyping analysis divided our cohort into two groups, with the majority of rectal cancer samples clustering into one enterotype, characterized by a greater abundance of Bacteroides and Dorea. At the phylum level, rectal-cancer samples had increased abundance of candidate phylum OD1 (also known as Parcubacteria) whilst non-cancer samples had increased abundance of Planctomycetes. At the genera level, rectal-cancer samples had higher abundances of Bacteroides, Phascolarctobacterium, Parabacteroides, Desulfovibrio, and Odoribacter whereas non-cancer samples had higher abundances of Pseudomonas, Escherichia, Acinetobacter, Lactobacillus, and Bacillus. Two Bacteroides fragilis OTUs were more abundant among rectal-cancer patients seen through 16S rRNA amplicon sequencing, whose presence was confirmed by immunohistochemistry and enrichment verified by digital drop

Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. Results: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research.

MotivationExtracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging.ResultsWe present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research.Availability and implementationThe web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention.

Citrus groves receiving nutritional sprays were compared with groves in the same areas managed with conventional fertilization treatments.  Fruit were harvested from healthy and Huanglongbing (HLB)-affected trees. Within HLB-affected trees, fruit were sorted into asymptomatic (HLB-a) and symptomatic (HLB-s) fruit.  Sensory tests were performed using the difference-from-control (DFC) method, where juice from HLB-affected trees was compared with juice from healthy trees.  Results show that panelists could detect differences between juice from HLB-affected and healthy trees in the 2009-2010 and 2010-2011 seasons, regardless of nutritional treatments, for Hamlin and Valencia. Like in previous years, those differences were perceived as more bitter or metallic for early harvests of HLB-affected Hamlin, but those differences were less and inconsistently perceived as more bitter, sweeter or more sour for late harvests or Valencia HLB-affected fruit. In the 2011-2012 season, there were much less differences between juice from healthy and HLB trees, possibly due to a season with high Brix/TA ratio, or due to later harvests.  Results will be discussed in relation to chemical analysis of sugars, acids, and limonoids.  Nutritional treatments that mitigate HLB symptoms on trees did not have a consistent effect on the HLB induced off-flavor of the fruit and juice, necessitating more seasons of study.

Citrus groves receiving nutritional sprays were compared with groves in the same areas managed with conventional fertilization treatments.  Fruit were harvested from healthy and Huanglongbing (HLB)-affected trees. Within HLB-affected trees, fruit were sorted into asymptomatic (HLB-a) and symptomatic (HLB-s) fruit.  Sensory tests were performed using the difference-from-control (DFC) method, where juice from HLB-affected trees was compared with juice from healthy trees.  Results show that panelists could detect differences between juice from HLB-affected and healthy trees in the 2009-2010 and 2010-2011 seasons, regardless of nutritional treatments, for Hamlin and Valencia. Like in previous years, those differences were perceived as more bitter or metallic for early harvests of HLB-affected Hamlin, but those differences were less and inconsistently perceived as more bitter, sweeter or more sour for late harvests or Valencia HLB-affected fruit. In the 2011-2012 season, there were much less differences between juice from healthy and HLB trees, possibly due to a season with high Brix/TA ratio, or due to later harvests.  Results will be discussed in relation to chemical analysis of sugars, acids, and limonoids.  Nutritional treatments that mitigate HLB symptoms on trees did not have a consistent effect on the HLB induced off-flavor of the fruit and juice, necessitating more seasons of study.

Background: Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges. Methodology/Principal Findings: We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at .1,000-fold faster rate, and reduce costs ,250-fold for generating 106 ligand sequences. Conclusions/Significance: Our analyses demonstrates that whereas this approach correlates with the traditional colonycounting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is sup

Background: Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges. Methodology/Principal Findings: We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at .1,000-fold faster rate, and reduce costs ,250-fold for generating 106 ligand sequences. Conclusions/Significance: Our analyses demonstrates that whereas this approach correlates with the traditional colonycounting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is sup

Background: Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges. Methodology/Principal Findings: We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at .1,000-fold faster rate, and reduce costs ,250-fold for generating 106 ligand sequences. Conclusions/Significance: Our analyses demonstrates that whereas this approach correlates with the traditional colonycounting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is sup

The objective of this study was to determine the impact of the smelter waste on plant species diversity and structure in the Tsumeb area. The most important sources of contamination are solid and gaseous emissions from the Tsumeb Smelter, airborne particles from the Tsumeb tailings pond and airborne particles from slag mill waste. Six sites: Inside Smelter Site, Outside Smelter Site, Abattoir Hill Site, Transnamib Site, Nomtsoub Site and Airport Site were selected and ten 10 m x 10 m plots were randomly demarcated at each site for soil and plant sampling. All 10 m x 10 m plots were used for sampling trees, nested within them were 5 m x 5 m and 2 m x 2 m plots which were used for sampling shrubs and grasses respectively. Top soils were sampled and analysed for concentration of arsenic, cadmium and lead. All trees (basal circumference 15 cm) as well as selected trees species, Combretum apiculatum and Terminalia prunioides were identified, counted and their heights as well as basal circumferences were measured. All shrubs, saplings and seedlings as well as a selected shrub, Dichrostachys cinerea, were identified, counted and their heights were measured. Grasses were identified and total grass cover was visually determined. Plant species diversity, richness, composition, woody cover and plant densities were determined. The results indicated high concentrations of arsenic, cadmium and lead at sites closer to the smelter. There were significant differences in plant species diversity (F=8.227, df=59, p0.001), richness (F, The hierarchical cluster analysis on species presence/absence data separated the vegetation into five types. Indirect gradient analysis indicated a complex interaction of gradients which have influence the pattern in species composition; however, the direct gradient analysis indicated that heavy metal pollution, disturbance and geology accounted for a significant variation in species composition. High heavy metal concentrations at the Inside Smelter Site and Outside Smelter Site were due to proximity of these sites to the smelter while high concentrations at Abattoir Hill Site were because this site was in the direction of the prevailing wind directions. Differences in species diversity, richness and composition were due to wood cutting and pollution. Differences in vegetation structure can be attributed to a complexity of factors including pollution and wood clearing. Heavy metal toxicity contributed to poor plant development, poor recruitment and as a result there were very few small trees at the polluted sites. Geology also contributed to difference in species composition. It was concluded that heavy metal pollution influenced species composition and vegetation structure.

The objective of this study was to determine the impact of the smelter waste on plant species diversity and structure in the Tsumeb area. The most important sources of contamination are solid and gaseous emissions from the Tsumeb Smelter, airborne particles from the Tsumeb tailings pond and airborne particles from slag mill waste. Six sites: Inside Smelter Site, Outside Smelter Site, Abattoir Hill Site, Transnamib Site, Nomtsoub Site and Airport Site were selected and ten 10 m x 10 m plots were randomly demarcated at each site for soil and plant sampling. All 10 m x 10 m plots were used for sampling trees, nested within them were 5 m x 5 m and 2 m x 2 m plots which were used for sampling shrubs and grasses respectively. Top soils were sampled and analysed for concentration of arsenic, cadmium and lead. All trees (basal circumference 15 cm) as well as selected trees species, Combretum apiculatum and Terminalia prunioides were identified, counted and their heights as well as basal circumferences were measured. All shrubs, saplings and seedlings as well as a selected shrub, Dichrostachys cinerea, were identified, counted and their heights were measured. Grasses were identified and total grass cover was visually determined. Plant species diversity, richness, composition, woody cover and plant densities were determined. The results indicated high concentrations of arsenic, cadmium and lead at sites closer to the smelter. There were significant differences in plant species diversity (F=8.227, df=59, p0.001), richness (F, The hierarchical cluster analysis on species presence/absence data separated the vegetation into five types. Indirect gradient analysis indicated a complex interaction of gradients which have influence the pattern in species composition; however, the direct gradient analysis indicated that heavy metal pollution, disturbance and geology accounted for a significant variation in species composition. High heavy metal concentrations at the Inside Smelter Site and Outside Smelter Site were due to proximity of these sites to the smelter while high concentrations at Abattoir Hill Site were because this site was in the direction of the prevailing wind directions. Differences in species diversity, richness and composition were due to wood cutting and pollution. Differences in vegetation structure can be attributed to a complexity of factors including pollution and wood clearing. Heavy metal toxicity contributed to poor plant development, poor recruitment and as a result there were very few small trees at the polluted sites. Geology also contributed to difference in species composition. It was concluded that heavy metal pollution influenced species composition and vegetation structure.

The objective of this study was to determine the impact of the smelter waste on plant species diversity and structure in the Tsumeb area. The most important sources of contamination are solid and gaseous emissions from the Tsumeb Smelter, airborne particles from the Tsumeb tailings pond and airborne particles from slag mill waste. Six sites: Inside Smelter Site, Outside Smelter Site, Abattoir Hill Site, Transnamib Site, Nomtsoub Site and Airport Site were selected and ten 10 m x 10 m plots were randomly demarcated at each site for soil and plant sampling. All 10 m x 10 m plots were used for sampling trees, nested within them were 5 m x 5 m and 2 m x 2 m plots which were used for sampling shrubs and grasses respectively. Top soils were sampled and analysed for concentration of arsenic, cadmium and lead. All trees (basal circumference 15 cm) as well as selected trees species, Combretum apiculatum and Terminalia prunioides were identified, counted and their heights as well as basal circumferences were measured. All shrubs, saplings and seedlings as well as a selected shrub, Dichrostachys cinerea, were identified, counted and their heights were measured. Grasses were identified and total grass cover was visually determined. Plant species diversity, richness, composition, woody cover and plant densities were determined. The results indicated high concentrations of arsenic, cadmium and lead at sites closer to the smelter. There were significant differences in plant species diversity (F=8.227, df=59, p0.001), richness (F, The hierarchical cluster analysis on species presence/absence data separated the vegetation into five types. Indirect gradient analysis indicated a complex interaction of gradients which have influence the pattern in species composition; however, the direct gradient analysis indicated that heavy metal pollution, disturbance and geology accounted for a significant variation in species composition. High heavy metal concentrations at the Inside Smelter Site and Outside Smelter Site were due to proximity of these sites to the smelter while high concentrations at Abattoir Hill Site were because this site was in the direction of the prevailing wind directions. Differences in species diversity, richness and composition were due to wood cutting and pollution. Differences in vegetation structure can be attributed to a complexity of factors including pollution and wood clearing. Heavy metal toxicity contributed to poor plant development, poor recruitment and as a result there were very few small trees at the polluted sites. Geology also contributed to difference in species composition. It was concluded that heavy metal pollution influenced species composition and vegetation structure.

The objective of this study was to determine the impact of the smelter waste on plant species diversity and structure in the Tsumeb area. The most important sources of contamination are solid and gaseous emissions from the Tsumeb Smelter, airborne particles from the Tsumeb tailings pond and airborne particles from slag mill waste. Six sites: Inside Smelter Site, Outside Smelter Site, Abattoir Hill Site, Transnamib Site, Nomtsoub Site and Airport Site were selected and ten 10 m x 10 m plots were randomly demarcated at each site for soil and plant sampling. All 10 m x 10 m plots were used for sampling trees, nested within them were 5 m x 5 m and 2 m x 2 m plots which were used for sampling shrubs and grasses respectively. Top soils were sampled and analysed for concentration of arsenic, cadmium and lead. All trees (basal circumference 15 cm) as well as selected trees species, Combretum apiculatum and Terminalia prunioides were identified, counted and their heights as well as basal circumferences were measured. All shrubs, saplings and seedlings as well as a selected shrub, Dichrostachys cinerea, were identified, counted and their heights were measured. Grasses were identified and total grass cover was visually determined. Plant species diversity, richness, composition, woody cover and plant densities were determined. The results indicated high concentrations of arsenic, cadmium and lead at sites closer to the smelter. There were significant differences in plant species diversity (F=8.227, df=59, p0.001), richness (F, The hierarchical cluster analysis on species presence/absence data separated the vegetation into five types. Indirect gradient analysis indicated a complex interaction of gradients which have influence the pattern in species composition; however, the direct gradient analysis indicated that heavy metal pollution, disturbance and geology accounted for a significant variation in species composition. High heavy metal concentrations at the Inside Smelter Site and Outside Smelter Site were due to proximity of these sites to the smelter while high concentrations at Abattoir Hill Site were because this site was in the direction of the prevailing wind directions. Differences in species diversity, richness and composition were due to wood cutting and pollution. Differences in vegetation structure can be attributed to a complexity of factors including pollution and wood clearing. Heavy metal toxicity contributed to poor plant development, poor recruitment and as a result there were very few small trees at the polluted sites. Geology also contributed to difference in species composition. It was concluded that heavy metal pollution influenced species composition and vegetation structure.

The objective of this study was to determine the impact of the smelter waste on plant species diversity and structure in the Tsumeb area. The most important sources of contamination are solid and gaseous emissions from the Tsumeb Smelter, airborne particles from the Tsumeb tailings pond and airborne particles from slag mill waste. Six sites: Inside Smelter Site, Outside Smelter Site, Abattoir Hill Site, Transnamib Site, Nomtsoub Site and Airport Site were selected and ten 10 m x 10 m plots were randomly demarcated at each site for soil and plant sampling. All 10 m x 10 m plots were used for sampling trees, nested within them were 5 m x 5 m and 2 m x 2 m plots which were used for sampling shrubs and grasses respectively. Top soils were sampled and analysed for concentration of arsenic, cadmium and lead. All trees (basal circumference 15 cm) as well as selected trees species, Combretum apiculatum and Terminalia prunioides were identified, counted and their heights as well as basal circumferences were measured. All shrubs, saplings and seedlings as well as a selected shrub, Dichrostachys cinerea, were identified, counted and their heights were measured. Grasses were identified and total grass cover was visually determined. Plant species diversity, richness, composition, woody cover and plant densities were determined. The results indicated high concentrations of arsenic, cadmium and lead at sites closer to the smelter. There were significant differences in plant species diversity (F=8.227, df=59, p0.001), richness (F, The hierarchical cluster analysis on species presence/absence data separated the vegetation into five types. Indirect gradient analysis indicated a complex interaction of gradients which have influence the pattern in species composition; however, the direct gradient analysis indicated that heavy metal pollution, disturbance and geology accounted for a significant variation in species composition. High heavy metal concentrations at the Inside Smelter Site and Outside Smelter Site were due to proximity of these sites to the smelter while high concentrations at Abattoir Hill Site were because this site was in the direction of the prevailing wind directions. Differences in species diversity, richness and composition were due to wood cutting and pollution. Differences in vegetation structure can be attributed to a complexity of factors including pollution and wood clearing. Heavy metal toxicity contributed to poor plant development, poor recruitment and as a result there were very few small trees at the polluted sites. Geology also contributed to difference in species composition. It was concluded that heavy metal pollution influenced species composition and vegetation structure.

The objective of this study was to determine the impact of the smelter waste on plant species diversity and structure in the Tsumeb area. The most important sources of contamination are solid and gaseous emissions from the Tsumeb Smelter, airborne particles from the Tsumeb tailings pond and airborne particles from slag mill waste. Six sites: Inside Smelter Site, Outside Smelter Site, Abattoir Hill Site, Transnamib Site, Nomtsoub Site and Airport Site were selected and ten 10 m x 10 m plots were randomly demarcated at each site for soil and plant sampling. All 10 m x 10 m plots were used for sampling trees, nested within them were 5 m x 5 m and 2 m x 2 m plots which were used for sampling shrubs and grasses respectively. Top soils were sampled and analysed for concentration of arsenic, cadmium and lead. All trees (basal circumference 15 cm) as well as selected trees species, Combretum apiculatum and Terminalia prunioides were identified, counted and their heights as well as basal circumferences were measured. All shrubs, saplings and seedlings as well as a selected shrub, Dichrostachys cinerea, were identified, counted and their heights were measured. Grasses were identified and total grass cover was visually determined. Plant species diversity, richness, composition, woody cover and plant densities were determined. The results indicated high concentrations of arsenic, cadmium and lead at sites closer to the smelter. There were significant differences in plant species diversity (F=8.227, df=59, p0.001), richness (F, The hierarchical cluster analysis on species presence/absence data separated the vegetation into five types. Indirect gradient analysis indicated a complex interaction of gradients which have influence the pattern in species composition; however, the direct gradient analysis indicated that heavy metal pollution, disturbance and geology accounted for a significant variation in species composition. High heavy metal concentrations at the Inside Smelter Site and Outside Smelter Site were due to proximity of these sites to the smelter while high concentrations at Abattoir Hill Site were because this site was in the direction of the prevailing wind directions. Differences in species diversity, richness and composition were due to wood cutting and pollution. Differences in vegetation structure can be attributed to a complexity of factors including pollution and wood clearing. Heavy metal toxicity contributed to poor plant development, poor recruitment and as a result there were very few small trees at the polluted sites. Geology also contributed to difference in species composition. It was concluded that heavy metal pollution influenced species composition and vegetation structure.

The IVTS project demonstrates the feasibility of applying to military training applications the video game technology developed for and marketed in consumer video games. The IVTS/CEV is a conceptual/breadboard-level classroom interactive training system designed to train Combat Engineer Vehicle (CEV) gunners in target acquisition and engagement with the main gun. The concept demonstration consists of two units: a gunner station and a display module. The gunner station has optics and gun controls replicating those of the CEV gunner station. The display module contains a standard large-screen color video monitor and a video tape player. The gunner's sight picture/position, vis a vis the video frame, is controlled by gun control handle movements; sight picture movement rate vs handle movement is modeled on actual equipment characteristics. A series of video-recorded targets are presented on the monitor and viewed through the sight. A Digital Data Module compares sight aiming point (derived from electrical sight position sensors) and known-location of target on the monitor; target location data is coded on a video tape sound track. The Digital Data Module scores trainee performance and provides the instructor with a measure of trainee proficiency. Both fixed and moving targets were used to demonstrate the effectiveness of this approach. A concept evaluation conducted by the Armor-Engineer Test Board indicates that the system fully demonstrated its ability to yield a high degree of knowledge transfer.