Dried alginate-entrapped enzymes (DALGEEs) and their application to the production of fructooligosaccharides

A modification of the classical calcium alginate enzyme entrapment technique is described aiming to overcome some of the limitations of the former gel-based biocatalysts. Dried alginate entrapped enzymes (DALGEEs) were obtained dehydrating calcium alginate gel beads containing entrapped enzymes. A fructosyltransferase from Aspergillus aculeatus, present in Pectinex Ultra SP-L, was entrapped using this technique. The resulting DALGEEs were successfully tested both operating batchwise and in a continuous fixed-bed reactor for fructooligosaccharides (FOS) synthesis from sucrose. Interestingly, DALGEEs did not re-swell upon incubation in concentrated (600 g/L) sucrose solutions, probably due to the lowered water activity (a_w) of such media. Confocal laser scanning microscopy of DALGEEs revealed that the enzyme molecules accumulated preferably in the shell of the particles. DALGEEs showed an approximately 30-fold higher volumetric activity (300 U/mL) compared with the calcium alginate gel beads. Moreover, a significant enhancement (40-fold) of the space-time-yield of fixed-bed bioreactors was observed when using DALGEEs as biocatalyst compared with gel beads (4030 g/day L of FOS vs. 103 g/day L). The operational stability of fixed-bed reactors packed with DALGEEs was extraordinary, providing a nearly constant FOS composition of the outlet during at least 700 h. It was also noticeable their resistance against microbial attack, even after long periods of storage at room temperature. The DALGEEs immobilisation strategy may also be useful for other biotransformations, in particular when they take place in low a_w media.     

Granulovirus formulations efficiently protect stored and field potatoes from Phthorimaea operculella and Tecia solanivora in Costa Rica

The control efficiency of a Phthorimaea operculella granulovirus isolate from Costa Rica (PhopGV-CR1) against the concurrent insect pests P. operculella (Zeller) and Tecia solanivora (Povolny) (Lepidoptera: Gelechiidae) was evaluated. In warehouses, the best control efficiency was achieved with a powder formulate applied inside bags, which reduced injury over 70 % compared with the untreated controls. In the field, liquid and powder virus formulations significantly reduced injury between 56.2 and 81.7 % compared with the untreated controls, and were as efficient as chemical treatments. The efficiency of formulations stored at ?20 °C for six months (liquid) or at ambient temperature for three months (powder) was maintained, but higher temperatures and/or longer exposure times resulted in loss of pathogenicity. The data presented here favor the inclusion of granulovirus formulations of PhopGV-CR1 in Integrated Pest Management programs against tuberworms in Costa Rica and give clues on storage conditions for the growers in this country.     

Paired inspiratory-expiratory chest CT scans to assess for small airways disease in COPD

Background

Gas trapping quantified on chest CT scans has been proposed as a surrogate for small airway disease in COPD. We sought to determine if measurements using paired inspiratory and expiratory CT scans may be better able to separate gas trapping due to emphysema from gas trapping due to small airway disease.

Methods

Smokers with and without COPD from the COPDGene Study underwent inspiratory and expiratory chest CT scans. Emphysema was quantified by the percent of lung with attenuation < −950HU on inspiratory CT. Four gas trapping measures were defined: (1) Exp₋₈₅₆, the percent of lung < −856HU on expiratory imaging; (2) E/I MLA, the ratio of expiratory to inspiratory mean lung attenuation; (3) RVC_856-950, the difference between expiratory and inspiratory lung volumes with attenuation between −856 and −950 HU; and (4) Residuals from the regression of Exp₋₈₅₆ on percent emphysema.

Results

In 8517 subjects with complete data, Exp₋₈₅₆ was highly correlated with emphysema. The measures based on paired inspiratory and expiratory CT scans were less strongly correlated with emphysema. Exp₋₈₅₆, E/I MLA and RVC_856-950 were predictive of spirometry, exercise capacity and quality of life in all subjects and in subjects without emphysema. In subjects with severe emphysema, E/I MLA and RVC_856-950 showed the highest correlations with clinical variables.

Conclusions

Quantitative measures based on paired inspiratory and expiratory chest CT scans can be used as markers of small airway disease in smokers with and without COPD, but this will require that future studies acquire both inspiratory and expiratory CT scans.     

Residential Selection across the Life Course: Adolescent Contextual and Individual Determinants of Neighborhood Disadvantage in Mid-Adulthood

Background

Numerous cross-sectional studies have examined neighborhood effects on health. Residential selection in adulthood has been stressed as an important cause of selection bias but has received little empirical attention, particularly its determinants from the earlier life course. The present study aims to examine whether neighborhood, family, school, health behaviors and health in adolescence are related to socioeconomic disadvantage of one''s neighborhood of residence in adulthood.

Methods

Based on the prospective Northern Swedish Cohort (analytical N = 971, 90.6% retention rate), information was collected at age 16 years concerning family circumstances, school adjustment, health behaviors and mental and physical health. Neighborhood register data was linked to the cohort and used to operationalize aggregated measures of neighborhood disadvantage (ND) at age 16 and 42. Data was analyzed with linear mixed models, with ND in adulthood regressed on adolescent predictors and neighborhood of residence in adolescence as the level-2 unit.

Results

Neighborhood disadvantage in adulthood was clustered by neighborhood of residence in adolescence (ICC = 8.6%). The clustering was completely explained by ND in adolescence. Of the adolescent predictors, ND (b = .14 (95% credible interval = .07–.22)), final school marks (b = −.18 (−.26–−.10)), socioeconomic disadvantage (b = .07 (.01–.14)), and, with borderline significance, school peer problems (b = .07 (−.00–.13)), were independently related to adulthood ND in the final adjusted model. In sex-stratified analyses, the most important predictors were school marks (b = −.21 (−.32–−.09)) in women, and neighborhood of residence (ICC = 15.5%) and ND (b = .20 (.09–.31)) in men.

Conclusions

These findings show that factors from adolescence – which also may impact on adult health – could influence the neighborhood context in which one will live in adulthood. This indicates that residential selection bias in neighborhood effects on health research may have its sources in early life.     

New Metrics for Comparison of Taxonomies Reveal Striking Discrepancies among Species Delimitation Methods in Madascincus Lizards

Delimiting and describing species is fundamental to numerous biological disciplines such as evolution, macroecology, and conservation. Delimiting species as independent evolutionary lineages may and often does yield different outcomes depending on the species criteria applied, but methods should be chosen that minimize the inference of objectively erroneous species limits. Several protocols exploit single-gene or multi-gene coalescence statistics, assignment tests or other rationales related to nuclear DNA (nDNA) allele sharing to automatically delimit species. We apply seven different species delimitation protocols to a taxonomically confusing group of Malagasy lizards (Madascincus), and compare the resulting taxonomies with two newly developed metrics: the Taxonomic index of congruence C_tax which quantifies the congruence between two taxonomies, and the Relative taxonomic resolving power index R_tax which quantifies the potential of an approach to capture a high number of species boundaries. The protocols differed in the total number of species proposed, between 9 and 34, and were also highly incongruent in placing species boundaries. The Generalized Mixed Yule-Coalescent approach captured the highest number of potential species boundaries but many of these were clearly contradicted by extensive nDNA admixture between sympatric mitochondrial DNA (mtDNA) haplotype lineages. Delimiting species as phenotypically diagnosable mtDNA clades failed to detect two cryptic species that are unambiguous due to a lack of nDNA gene flow despite sympatry. We also consider the high number of species boundaries and their placement by multi-gene Bayesian species delimitation as poorly reliable whereas the Bayesian assignment test approach provided a species delimitation highly congruent with integrative taxonomic practice. The present study illustrates the trade-off in taxonomy between reliability (favored by conservative approaches) and resolving power (favored by inflationist approaches). Quantifying excessive splitting is more difficult than quantifying excessive lumping, suggesting a priority for conservative taxonomies in which errors are more liable to be detected and corrected by subsequent studies.     

Reproductive aspects of American minks (Neovison vison) in central Spain: Testing the effects of prey availability

Some reproductive components of fitness (breeding times, litter size and the proportion of breeding females per year) of two feral populations of American minks in central Spain were investigated by means of direct observation from 2006 to 2010. The effects of prey availability were explored. Mating season was from February to March. Parturition dates did not differ between the two sites and pooled births took place between April (33.3%) and July (2.8%), peaking in May (40.3%). Mean litter size (the number of small cubs following their mother) was 3.43 ± 1.01 cubs (±SD; n = 30) with statistical significant differences between the two study sites. The strong seasonality in births and differences in litter size were related to prey availability (i.e. these are food-limited life-history traits), but in a complex non-linear fashion. A minimum value of prey abundance is necessary to breed. While litter size rises with prey abundance, there is a point where increasing prey did not result in an increase in litter size. Up to 75% of the females in the breeding pool reared cubs each year in both areas, which represents a somewhat high pool of breeding females. Delayed implantation is shorter than a month or does not exist in these populations. Such a short time-span seems to emerge because longer delays would have an important fitness cost to females. The breeding calendar and the litter size were similar to that reported previously from other areas.     

MicroRNA signatures of iPSCs and endoderm-derived tissues

MicroRNAs (miRNAs), small non-coding RNAs that fine-tune gene expression, play multiple roles in the cell, including cell fate specification. We have analyzed the differential expression of miRNAs during fibroblast reprogramming into induced pluripotent stem cells (iPSCs) and endoderm induction from iPSCs upon treatment with high concentrations of Activin-A. The reprogrammed iPSCs assumed an embryonic stem cell (ESC)-like miRNA signature, marked by the induction of pluripotency clusters miR-290–295 and miR-302/367 and conversely the downregulation of the let-7 family. On the other hand, endoderm induction in iPSCs resulted in the upregulation of 13 miRNAs. Given that the liver and the pancreas are common derivatives of the endoderm, analysis of the expression of these 13 upregulated miRNAs in hepatocytes and pancreatic islets revealed a tendency for these miRNAs to be expressed more in pancreatic islets than in hepatocytes. These observations provide insights into how differentiation may be guided more efficiently towards the endoderm and further into the liver or pancreas. Moreover, we also report novel miRNAs enriched for each of the cell types analyzed.     

The Genetic Basis of Escherichia coli Pathoadaptation to Macrophages

Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity.     

Trichomonas vaginalis Exosomes Deliver Cargo to Host Cells and Mediate Host∶Parasite Interactions

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we use a combination of methodologies including cell fractionation, immunofluorescence and electron microscopy, RNA, proteomic and cytokine analyses and cell adherence assays to examine pathogenic properties of T. vaginalis. We have found that T.vaginalis produces and secretes microvesicles with physical and biochemical properties similar to mammalian exosomes. The parasite-derived exosomes are characterized by the presence of RNA and core, conserved exosomal proteins as well as parasite-specific proteins. We demonstrate that T. vaginalis exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. Moreover, exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells. In contrast, exosomes from poorly adherent strains had no measurable effect on parasite adherence. Exosomes from parasite strains that preferentially bind prostate cells increased binding of parasites to these cells relative to vaginal cells. In addition to establishing that parasite exosomes act to modulate host∶parasite interactions, these studies are the first to reveal a potential role for exosomes in promoting parasite∶parasite communication and host cell colonization.