Mesocestoides corti (syn. vogae, cestoda): characterization of genes encoding cysteine-rich secreted proteins (CRISP)

With the aim of identifying genes involved in development and parasite adaptation in cestodes, four coding sequences were isolated from the cyclophyllidean Mesocestoides corti larval stage (tetrathyridium). Genes showed significant similarity to the cysteine-rich secreted protein (CRISP) encoding genes, a large family that includes stage and tissue-specific genes from diverse organisms, many associated with crucial biological processes. The full-length McCrisp2 cDNA encodes a predicted protein of 202 residues in length, containing 10 cysteines and a putative signal peptide. The expression level of McCrisp2 was estimated by Real-time PCR, relative to GAPDH, showing an increase of 75% in segmented worms compared to tetrathyridia. By in situ hybridization, McCrisp2 expression was localized mainly at the larvae apical region of tetrathyridia and in the proglottids of segmented worms. Taken together our results suggest a possible role for M. corti CRISP proteins as ES products, potentially involved in differentiation processes as proposed for homologs in other organisms.     

Membrane cholesterol content modulates ClC-2 gating and sensitivity to oxidative stress

ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient. ClC-2 was found concentrated in detergent-insoluble membranes in basal conditions and relocalized to DSM upon cholesterol depletion by methyl-beta-cyclodextrin. As assessed by patch clamp recordings, relocalization was accompanied by acceleration of the activation kinetics of the channel. A similar distribution and activation pattern were obtained when cells were treated with the oxidant tert-butyl hydroperoxide and after ATP depletion. In both cases activation was prevented by cholesterol enrichment of cells. We conclude that the cholesterol environment regulates ClC-2 activity, and we provide evidence that the increase in ClC-2 activity in response to acute oxidative or metabolic stress involves relocalization of this channel to DSM.     

Mechanical response and conformational changes of alpha-actinin domains during unfolding: a molecular dynamics study

Alpha-actinin is a cytoskeleton-binding protein involved in the assembly and regulation of the actin filaments. In this work molecular dynamics method was applied to investigate the mechanical behaviour of the human skeletal muscle α-actinin. Five configurations were unfolded at an elongation speed of 0.1 nm/ps in order to investigate the conformational changes occurring during the extension process. Moreover, a sensitivity analysis at different velocities was performed for one of the R2–R3 spectrin-like repeat configuration extracted in order to evaluate the effect of the pulling speed on the mechanical behaviour of the molecule. Two different behaviours were recognized with respect to the pulling speed. In particular, at speed higher than 0.025 nm/ps a continuous rearrangement without evident force peaks was obtained, on the contrary at lower speed evident peaks in the range 500–750 pN were detected. R3 repeat resulted more stable than R2 during mechanical unfolding, due to the lower hydrophobic surface available to the solvent. The characterization of the R2–R3 units can be useful for the development of cytoskeleton network models based on stiffness values obtained by analyses performed at the molecular level.     

The Paradox of Clonality and the Evolution of Self-Incompatibility

In the January issue of New Phytologist Vallejo-Marín and O''Brien¹ documented that in the genus Solanum (Solanaceae) clonality and self-incompatibility, a common genetic mechanism enforcing cross-fertilization, co-occur more often than expected by chance. Using a phylogenetic approach the authors showed that the statistical association between clonality and self-incompatibility persists even after taking into account phylogenetic relationships among species, uncertainty in the phylogenetic reconstruction, and associations between clonality and life history (annual/perennial). Vallejo-Marín and O''Brien¹ suggest that clonality and self-incompatibility tend to co-occur because clonality, by allowing the persistence and propagation of a genotype in environments with limited pollinator or mate availability, reduces the selective pressure favoring the breakdown of self-incompatibility. In addition to promoting the maintenance of self-incompatibility, when clonality results in the spatial aggregation of genetically identical individuals, clonality may promote its breakdown by restricting pollen transfer between different genotypes. Here I call attention to these contradictory predictions of the effects of clonality on the evolution of self-incompatibility, and suggest that the outcome of this paradox depend on both the extent to which clonal propagation compensates for limited seed production, and on the extent to which clonality reduces pollen transfer between genotypes.Key Words: asexual reproduction, clonality, mating system, pollen limitation, reproductive assurance, reproductive compensation, SolanumFlowering plants display a variety of mechanisms preventing self-fertilization, among which self-incompatibility is one of the best studied.^2,3 In the genus Solanum (Solanaceae) ancestrally present self-incompatibility has broken down multiple independent times through out the evolutionary history of this group to give rise to self-compatible lineages.^4,5 The breakdown of outcrossing mechanisms, including self-incompatibility, is one of the most common and best studied evolutionary transitions in flowering plants.⁶Self-incompatibility is a genetic mechanism by which the maternal plant can recognize and reject pollen grains expressing alleles in common with the maternal genotype.⁷ The reproductive advantages of self-incompatibility can be understood as a balance between two forces. On one hand the rejection of pollen grains is a mechanism for the preferential support of outbred progeny which, generally, is of higher genetic quality. On the other hand, if pollen or pollinator availability is low, pollen grains that are rejected by the self-incompatibility mechanism may not be substituted, and thus some ovules would go unfertilized. The evolutionary maintenance of self-incompatibility depends on the relative benefit of producing higher quality offspring and the relative costs incurred by potential reduction in offspring number⁸Plant clonality can affect the relative benefits and costs of self-incompatibility through its effects on the persistence and spatial distribution of genotypes. For instance, Vallejo-Marín and O''Brien¹ suggest that clonality provides reproductive assurance in colonizing taxa by allowing genotypes to persist and propagate even in the absence of conditions conducive to seed production. The reproductive assurance conferred by clonality would then relief the evolutionary costs of self-incompatibility incurred through reduced seed number, favoring the maintenance of self-incompatibility (Fig. 1).^1,9,10 In contrast, clonality may increase the evolutionary costs of self-incompatibility, by restricting pollen transfer between genotypes.^11,12 In some forms of clonal growth, dispersal of asexual propagules is very localized, resulting in an aggregation or clumping of genotypes. If spatial clumping restricts pollen flow between distinct genetic individuals, plants may not receive enough compatible pollen to fertilize all ovules (incomplete reproductive compensation) thus resulting in reduced seed set, which may favor the breakdown of self-incompatibility (Fig. 1).^8,12,13 It is important to note that both the maintenance and the breakdown effects mentioned above are expected to occur in the same ecological conditions, namely when the availability of pollen, pollinators, or compatible mates limit seed set (collectively known as pollen limitation).Open in a separate windowFigure 1Potential consequences of clonality for the evolutionary maintenance of self-incompatibility (SI). Full reproductive compensation occurs when pollen grains rejected by the SI system can be replaced with other compatible pollen grains. Under full reproductive compensation, seed set is independent of the level of SI expression.⁸ When pollen receipt is limited, rejected pollen grains cannot be always substituted (incomplete reproductive compensation) and consequently seed set is negatively related to the level of SI expression.⁸ Notice that under similar ecological conditions, e.g., when pollen receipt is limited (outside arrows), clonality may both favor the maintenance of SI through reproductive assurance, and facilitate its breakdown through increasing within-genotype pollen transfer and reducing seed set.A contrasting ecological scenario, may in turn favor the maintenance of self-incompatibility in clonal taxa. When pollen is abundant, and pollinators transport pollen through larger distances, enough pollen might still be received to fertilize all ovules even after accounting for pollen rejected by the self-incompatibility system (full reproductive compensation; Fig. 1). In this case, seed number will not be affected by the expression of self-incompatibility, but seed quality may be increased through the rejection of inbred pollen.¹⁴ In other words, when pollen receipt does not limit seed number, the presence of clonality is expected to favor self-incompatibility as a mechanism to successfully screen inbred pollen originated from different individuals of the same genotype or clone.To summarize, under the same ecological conditions (i.e., pollen limitation) the co-occurrence of clonality and self-incompatibility may have contrasting effects (Fig. 1). On one hand clonality favors the maintenance of self-incompatibility by providing reproductive assurance. On the other hand clonality may favor the breakdown of self-incompatibility, because rejected pollen cannot be compensated for, ensuing in a reduction in seed number. The resolution of the paradox of clonality for the evolution of self-incompatibility is likely to depend on the degree to which clonal propagation compensates for limited reproduction through seeds (e.g., during population establishment), as well as on the extent to which clonality reduces pollen flow between established genotypes, which in turn is affected by characteristics such as clonal architecture, plant density, and pollinator type and availability.^12,15,16Vallejo-Marín and O''Brien''s data¹ suggest that in the colonizing genus Solanum, which is expected to experience pollen limiting conditions, clonality reduces the costs of self-incompatibility through reproductive assurance (maintenance effect) more than it increases the costs of self-incompatibility due to pollen flow within spatially clumped genotypes (breakdown effect). Future comparative studies of the association between self-incompatibility and clonality in other groups could help us determine the general conditions in which the maintenance effect outweighs the breakdown effect.     

Analysis of Catharanthus roseus alkaloids by HPLC

Catharanthus roseus is a medicinal plant from which secondary metabolites used in chemotherapy to treat diverse cancers are extracted. The well known high value metabolites vincristine and vinblastine are just 2 of 130 alkaloids that can be found in C. roseus. However, only few (∼11) of this high number of chemical entities are frequently analyzed and even fewer (∼8) are available commercially. For more than 30 years, different analytical techniques have been developed to isolate and identify C. roseus metabolites, and then allowing revealing the therapeutic potential of C. roseus metabolites. Among few approaches, high performance liquid chromatography (HPLC) technique is still widely used for the separation and analysis of secondary metabolites such as those from C. roseus. This article thus reviews the most recent developments in HPLC analysis of alkaloids from C. roseus. Diverse considerations that are crucial to the efficiency of secondary metabolites separation and identification steps, such as biomass manipulation, extraction phase and protocols, HPLC separation and analysis protocols are reviewed in details. Examples of spectra obtained using the most common detectors are also shown and suggestions are made on how to proceed in developing efficient separation and identification methods at the analytical and semi-preparative scales.     

Congenital defects among liveborn infants with Down syndrome

BACKGROUND: Many infants with Down syndrome (DS) have co-occurring congenital malformations requiring intensive surgical and medical management. To anticipate the care needed by these infants, providers and parents require accurate information about birth defects that may be present. This article uses a unique national hospital discharge dataset to identify the rate at which structural birth defects are identified among liveborn infants with DS. METHODS: ICD-9-CM diagnosis codes for data from the Healthcare Cost and Utilization Project were used to identify infants with and without DS, and to classify birth defects. The study population consisted of liveborn infants discharged from the hospital from 1993 through 2002. ORs for the association between the occurrence of congenital malformations and the presence of DS were computed using logistic regression models for survey data. RESULTS: Discharge data included 11,372 DS and 7,884,209 non-DS births, representing national estimates of 43,463 DS and 39,716,469 non-DS births respectively. In addition to congenital heart defects that co-occurred most often in DS infants compared to infants without DS, the risks for gastrointestinal malformations (OR 67.07), genitourinary malformations (OR 3.62), orofacial malformations (OR 5.63), and abdominal wall malformations (OR 3.25) were also elevated in infants with DS. There was no difference in the risk of spina bifida between infants with and without DS. CONCLUSIONS: This is the first nationally representative compilation of the co-occurrence of congenital malformations associated with DS. This information may assist providers and parents in their attempts to understand and prepare for the true burden of this condition.     

Mixed-dimensional multi-scale poroelastic modeling of adipose tissue for subcutaneous injection

Biomechanics and Modeling in Mechanobiology - Subcutaneous injection of therapeutic monoclonal antibodies (mAbs) has gained increasing interest in the pharmaceutical industry. The transport,...