Citric acid production from sucrose by recombinant Yarrowia lipolytica using semicontinuous fermentation

The genetically modified yeast strain Yarrowia lipolytica H222‐S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild‐type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose‐peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose‐peptone. Furthermore, two process strategies (fed‐batch and repeated fed‐batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed‐batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.     

Electronic circular dichroism for the detection of microalbuminuria

Over the past decades, chiroptical spectroscopy has proved its incomparable ability to elucidate the structure and spatial arrangement of chiral molecules. Systematic analysis of biomolecules in the natural environment of biofluids, however, remains challenging. In this study, we used chiroptical spectroscopy to monitor urinary levels of human serum albumin. Not only severe proteinuria but even just a slightly increased urinary excretion of albumin (microalbuminuria) may indicate serious health complications, especially for diabetic individuals. Given the chiral nature of albumin and its typical spectral pattern, it may be easily observable by chiroptical spectroscopy, particularly electronic circular dichroism. The performed chiroptical analysis of urine not only allowed the detection of clinically confirmed microalbuminuria but was also able to reveal this pathological condition in cases beyond the diagnostic capability of common clinical procedures. Thus, our approach suggests that electronic circular dichroism is a useful tool for the fast and reliable qualitative monitoring of microalbuminuria with the potential for a quantitative analysis in the future.     

Parasite-host fitness trade-offs change with parasite identity: genotype-specific interactions in a plant-pathogen system

Simultaneous effects of host and parasite in determining quantitative traits of infection have long been neglected in theoretical and experimental investigations of host-parasite coevolution with the notable exception of gene-for-gene resistance studies. A cross-infection experiment, using five lines of the plant Arabidopsis thaliana and two strains of its oomycete pathogen Hyaloperonospora parasitica, revealed that three traits traditionally considered those of the parasite (number of infected leaves, transmission success, and time until 50% transmission), differed among specific combinations of host and parasite lines, being determined by the two protagonists of the infection. However, the two parasite strains did not differ significantly for most measured phenotypic traits of the infection. Globally, transmission increased with increasing virulence among the different host-parasite combinations, as assumed by most models of evolution of virulence. Surprisingly, however, there was no general relationship between parasite and host fitness, estimated respectively as transmission and seed production. Only one of the two strains showed the expected significant negative genetic correlation between these two variables. Our results thus highlight the importance of taking into account both host and parasite genetic variation because their interaction can lead to unexpected evolutionary outcomes.     

Stage-Specific Adhesion of Leishmania Promastigotes to Sand Fly Midguts Assessed Using an Improved Comparative Binding Assay

Background

The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s) that mediate binding is not fully understood.

Methodology/Principal Findings

To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica) to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti). The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed.

Conclusions/Significance

The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms), but is absent in the early blood meal and final stages (procyclic and metacyclic forms). Further they show that although gut binding may be necessary for parasite establishment, in several vector-parasite pairs the specificity of such in vitro binding alone is insufficient to explain overall vector specificity. Other significant barriers to development must exist in certain refractory Leishmania parasite-sand fly vector combinations. A re-appraisal of the specificity of the Leishmania-sand fly relationship is required.     

Individual flowering phenology shapes plant–pollinator interactions across ecological scales affecting plant reproduction

The balance of pollination competition and facilitation among co-flowering plants and abiotic resource availability can modify plant species and individual reproduction. Floral resource succession and spatial heterogeneity modulate plant–pollinator interactions across ecological scales (individual plant, local assemblage, and interaction network of agroecological infrastructure across the farm). Intraspecific variation in flowering phenology can modulate the precise level of spatio-temporal heterogeneity in floral resources, pollen donor density, and pollinator interactions that a plant individual is exposed to, thereby affecting reproduction. We tested how abiotic resources and multi-scale plant–pollinator interactions affected individual plant seed set modulated by intraspecific variation in flowering phenology and spatio-temporal floral heterogeneity arising from agroecological infrastructure. We transplanted two focal insect-pollinated plant species (Cyanus segetum and Centaurea jacea, n = 288) into agroecological infrastructure (10 sown wildflower and six legume–grass strips) across a farm-scale experiment (125 ha). We applied an individual-based phenologically explicit approach to match precisely the flowering period of plant individuals to the concomitant level of spatio-temporal heterogeneity in plant–pollinator interactions, potential pollen donors, floral resources, and abiotic conditions (temperature, water, and nitrogen). Individual plant attractiveness, assemblage floral density, and conspecific pollen donor density (C. jacea) improved seed set. Network linkage density increased focal species seed set and modified the effect of local assemblage richness and abundance on C. segetum. Mutual dependence on pollinators in networks increased C. segetum seed set, while C. jacea seed set was greatest where both specialization on pollinators and mutual dependence was high. Abiotic conditions were of little or no importance to seed set. Intra- and interspecific plant–pollinator interactions respond to spatio-temporal heterogeneity arising from agroecological management affecting wild plant species reproduction. The interplay of pollinator interactions within and between ecological scales affecting seed set implies a co-occurrence of pollinator-mediated facilitative and competitive interactions among plant species and individuals.     

Fluoranthene-induced production of ethylene and formation of lysigenous intercellular spaces in pea plants cultivated in vitro

The effect of increasing concentration of polycyclic aromatic hydrocarbon (PAH) fluoranthene (FLT; 0.1, 1 and 5 mg l⁻¹) on the growth, ethylene production and anatomy of stems of 21-day-old pea plants cultivated in vitro in MS medium, with or without FLT, enriched with 0.1 mg l⁻¹ indole-3-acetic acid (IAA) or with combination of 0.1 mg l⁻¹ IAA + 0.1 mg l⁻¹ N⁶-benzyladenine (BA) were investigated. The low concentration of 0.1 mg l⁻¹ FLT, in both IAA- and IAA + BA-treated plants, significantly stimulated the growth of pea callus, while higher concentrations 1 mg l⁻¹ and especially 5 mg l⁻¹ FLT significantly inhibited it. Pea shoots were significantly influenced only after application of 5 mg l⁻¹ FLT in IAA treatment. Significantly increased production of ethylene was found in IAA + BA treatments in all concentrations of FLT, whereas in IAA treatments in 1 and 5 mg l⁻¹ FLT. The lysigenous aerenchyma formation in the cortex of pea stems significantly increased in all FLT treatments and its highest proportion was found in plants exposed to 1 mg l⁻¹ FLT.     

Individual quality and age but not environmental or social conditions modulate costs of reproduction in a capital breeder

Costs associated with reproduction are widely known to play a role in the evolution of reproductive tactics with consequences to population and eco‐evolutionary dynamics. Evaluating these costs as they pertain to species in the wild remains an important goal of evolutionary ecology. Individual heterogeneity, including differences in individual quality (i.e., among‐individual differences in traits associated with survival and reproduction) or state, and variation in environmental and social conditions can modulate the costs of reproduction; however, few studies have considered effects of these factors simultaneously. Taking advantage of a detailed, long‐term dataset for a population of feral horses (Sable Island, Nova Scotia, Canada), we address the question of how intrinsic (quality, age), environmental (winter severity, location), and social conditions (group size, composition, sex ratio, density) influence the costs of reproduction on subsequent reproduction. Individual quality was measured using a multivariate analysis on a combination of four static and dynamic traits expected to depict heterogeneity in individual performance. Female quality and age interacted with reproductive status of the previous year to determine current reproductive effort, while no effect of social or environmental covariates was found. High‐quality females showed higher probabilities of giving birth and weaning their foal regardless of their reproductive status the previous year, while those of lower quality showed lower probabilities of producing foals in successive years. Middle‐aged (prime) females had the highest probability of giving birth when they had not reproduced the year before, but no such relationship with age was found among females that had reproduced the previous year, indicating that prime‐aged females bear higher costs of reproduction. We show that individual quality and age were key factors modulating the costs of reproduction in a capital breeder but that environmental or social conditions were not, highlighting the importance of considering multiple factors when studying costs of reproduction.     

The role of the GX9GX3G motif in the gating of high voltage-activated Ca2+ channels

The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) Ca(V) channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in Ca(V)1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly(432) and Gly(436) in Ca(V)1.2) to form a triglycine motif unique to HVA Ca(V) channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca(2+) channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In Ca(V)1.2, alpha-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with Ca(V)1.2 wild type > G770A > G422A congruent with G436A > G432A (from the fastest to the slowest). Mutations at position Gly(432) produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly(436) to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX(3)G residues blunted Ca(2+)-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In Ca(V)2.3, mutation of the distal glycine Gly(352) impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX(3)G motif in the voltage-dependent activation and inactivation gating of HVA Ca(V) channels with the distal glycine residue being mostly involved in the inactivation gating.     

Cancer stem cells modulate patterns and processes of evolution in cancers

The clonal evolution (CE) model and the cancer stem cell (CSC) model are two independent models of cancers, yet recent data shows intersections between the two models. This article explores the impacts of the CSC model on the CE model. I show that CSC restriction, which depends on CSC frequency in cancer cell populations and on the probability of dedifferentiation of cancer non-stem cells (non-CSCs) into CSCs, can favor or impede some patterns of evolution (linear or branched evolution) and some processes of evolution (drift, evolution by natural selection, complex adaptations). Taking CSC restriction into account for the CE model thus has implications for the way in which we understand the patterns and processes of evolution, and can also provide new leads for therapeutic interventions.