Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.     

Notes on the distribution of phytoplankton in some Kenya waters

The distribution of the major plankton algae in a number of Kenya waters is described. The lakes and dams are shown to fall into ecological groups related to figures for the pH, conductivity and alkalinity of their waters and characterised by the dominance of certain types of alga. The composition of the phytoplankton collected at approximately monthly intervals from Sasumua and Ruiru reservoirs is described. Plankton periodicity is demonstrated for both waters and is shown to be related to rainfall. The periodicity of plankton in Lake Naivasha is noted.     

Removal of Prymnesium parvum (Haptophyceae) and its toxins using clay minerals

Laboratory experiments were conducted to examine the ability of several clay minerals from Sweden to remove the fish-killing microalga, Prymnesium parvum Carter, from suspension. In their commercial form (i.e. after incineration at 400 °C), seawater slurries (salinity = 26) of the three minerals tested were generally ineffective at removing P. parvum from culture within a range of 0.01 to 0.50 g/L, and after 2.5 h of flocculation and settling. Dry bentonite (SWE1) displayed the highest removal efficiency (RE) at 17.5%, with 0.50 g/L. Illite (SWE3) averaged only 7.5% RE between 0.10 to 0.50 g/L, while kaolinite (SWE2) kept the cells suspended instead of removing them. Brief mixing of the clay-cell suspension after SWE1 addition improved RE by a factor of 2.5 (i.e. 49% at 0.50 g/L), relative to no mixing. The addition of polyaluminum chloride (PAC, at 5 ppm) to 0.50 g/L SWE1 also improved RE to 50% relative to SWE1 alone, but only minor improvements in RE were seen with SWE2 and SWE2 combined with PAC. In further experiments, P. parvum grown in NP-replete conditions were removed in greater numbers than cells in N- or P-limited cultures, at 0.10–0.25 g/L of SWE1 and 5 ppm PAC. With 0.50 g/L, RE converged at 40% for all three culture conditions. The toxin concentration of NP-replete cultures decreased from 24.2 to 9.2 μg/mL (60% toxin RE) with 0.10–0.50 g/L SWE1 treatment and 5 ppm PAC. A strong correlation was found between cell and toxin RE (r²=0.995). For N-limited cultures, toxin RE ranged between 21 and 87% with the same clay/PAC concentrations, although the correlation between cell and toxin removal was more moderate (r²=0.746) than for NP-replete conditions. Interestingly, the toxin concentration within the clay-cell pellet increased dramatically after treatment, suggesting that clay addition may stimulate toxin production in N-stressed cells. For P-limited cultures, toxin concentration also decreased following clay/PAC treatment (i.e. 36% toxin RE), but toxin removal was poorly correlated to cell removal (r²=0.462). To determine whether incineration affected SWE1’s removal ability, a sample of its wet, unprocessed form was tested. The RE of wet bentonite (SWE4) was slightly better than that of SWE1 (31% versus 17%, respectively, at 0.50 g/L), but when 5 ppm PAC was added, RE increased from 10 to 64% with 0.05 g/L of SWE4, and increased further to 77% with 0.50 g/L. There were no significant differences in RE among NP-replete, N-limited and P-limited cultures using PAC-treated SWE4. Finally, RE varied with P. parvum concentration, reaching a maximum level at the lowest cell concentration (1×10³ cells/mL): 100% RE with 0.10 and 0.50 g/L SWE4 + 5 ppm PAC. RE dropped as cell concentration increased to 1×10⁴ and 5×10⁴ cells/mL, but rose again when concentration increased to 1×10⁵ cells/mL, the concentration used routinely for the removal experiments above. Based on these results, SWE4 with PAC was the most effective mineral sample against P. parvum. Overall, these studies demonstrated that clay flocculation can be effective at removing P. parvum and its toxins only under certain treatment conditions with respect to cell concentration, clay type and concentration, and physiological status.     

Contrasting patterns of genetic differentiation in Macaronesian lineages of Ilex (Aquifoliaceae)

Islands offer an interesting framework in which to study the effect of geographical isolation on population genetic differentiation. For plant species with high dispersal abilities, however, oceanic barriers may not represent a factor promoting strong population structure. In this work, we analysed seven nuclear microsatellite loci in Ilex (Aquifoliaceae), a bird‐dispersed plant group, to infer patterns of genetic differentiation among Macaronesian taxa: I. canariensis, I. perado ssp. lopezlilloi, I. perado ssp. platyphylla (Canary Islands) and I. perado ssp. azorica (Azores). In agreement with current taxonomic classification, our results revealed a high genetic differentiation between Ilex lineages (I. canariensis and the I. perado complex), and also supported previous hypotheses that these are the result of independent dispersal events to the islands. In contrast, genetic differentiation between I. perado ssp. azorica and the two subspecies from the Canaries was high, suggesting that taxonomic revision may be necessary. Levels of genetic variation at microsatellite loci in ssp. azorica were, in addition, the lowest reported among Macaronesian bird‐dispersed taxa. Lastly, low genetic differentiation was observed between subspecies occurring on the same island (sspp. platyphylla and lopezlilloi). In summary, our results revealed contrasting patterns between Macaronesian Ilex lineages: I. canariensis displayed moderate population structure across islands, whereas the I. perado complex showed strong differentiation among populations sampled on different islands. Thus, the Macaronesian Ilex taxa show that long‐distance dispersal syndromes (ornithochory) do not always ensure genetic connectivity across large areas in island systems. Plant groups that successfully colonized the islands on multiple occasions may have found barriers to gene flow within certain lineages. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 258–268.     

Efficacy of a Food Safety Comic Book on Knowledge and Self-Reported Behavior for Persons Living with AIDS

Introduction

Persons living with AIDS are highly vulnerable to foodborne enteric infections with the potential for substantial morbidity and mortality. Educational materials about foodborne enteric infections intended for this immunocompromised population have not been assessed for their efficacy in improving knowledge or encouraging behavior change.

Methods/Results

AIDS patients in four healthcare facilities in Chicago, New Orleans, and Puerto Rico were recruited using fliers and word of mouth to healthcare providers. Those who contacted research staff were interviewed to determine food safety knowledge gaps and risky behaviors. A food safety educational comic book that targeted knowledge gaps was created, piloted, and provided to these patients who were instructed to read it and return at least 2 weeks later for a follow-up interview. The overall food safety score was determined by the number of the 26 knowledge/belief/behavior questions from the survey answered correctly. Among 150 patients who participated in both the baseline and follow-up questionnaire, the intervention resulted in a substantial increase in the food safety score (baseline 59%, post-intervention 81%, p<0.001). The intervention produced a significant increase in all the food safety knowledge, belief, and behavior items that comprised the food safety score. Many of these increases were from baseline knowledge below 80 percent to well above 90%. Most (85%) of the patients stated they made a change to their behavior since receiving the educational booklet.

Conclusion

This comic book format intervention to educate persons living with AIDS was highly effective. Future studies should examine to what extent long-term behavioral changes result.     

The Interacting Effects of Ungulate Hoofprints and Predatory Native Ants on Metamorph Cane Toads in Tropical Australia

Many invasive species exploit the disturbed habitats created by human activities. Understanding the effects of habitat disturbance on invasion success, and how disturbance interacts with other factors (such as biotic resistance to the invaders from the native fauna) may suggest new ways to reduce invader viability. In tropical Australia, commercial livestock production can facilitate invasion by the cane toad (Rhinella marina), because hoofprints left by cattle and horses around waterbody margins provide distinctive (cool, moist) microhabitats; nevertheless the same microhabitat can inhibit the success of cane toads by increasing the risks of predation or drowning. Metamorph cane toads actively select hoofprints as retreat-sites to escape dangerous thermal and hydric conditions in the surrounding landscape. However, hoofprint geometry is important: in hoofprints with steep sides the young toads are more likely to be attacked by predatory ants (Iridomyrmex reburrus) and are more likely to drown following heavy rain. Thus, anthropogenic changes to the landscape interact with predation by native taxa to affect the ability of cane toads in this vulnerable life-history stage to thrive in the harsh abiotic conditions of tropical Australia.     

Two Homologous EF-G Proteins from Pseudomonas aeruginosa Exhibit Distinct Functions

Genes encoding two proteins corresponding to elongation factor G (EF-G) were cloned from Pseudomonas aeruginosa. The proteins encoded by these genes are both members of the EFG I subfamily. The gene encoding one of the forms of EF-G is located in the str operon and the resulting protein is referred to as EF-G1A while the gene encoding the other form of EF-G is located in another part of the genome and the resulting protein is referred to as EF-G1B. These proteins were expressed and purified to 98% homogeneity. Sequence analysis indicated the two proteins are 90/84% similar/identical. In other organisms containing multiple forms of EF-G a lower degree of similarity is seen. When assayed in a poly(U)-directed poly-phenylalanine translation system, EF-G1B was 75-fold more active than EF-G1A. EF-G1A pre-incubate with ribosomes in the presence of the ribosome recycling factor (RRF) decreased polymerization of poly-phenylalanine upon addition of EF-G1B in poly(U)-directed translation suggesting a role for EF-G1A in uncoupling of the ribosome into its constituent subunits. Both forms of P. aeruginosa EF-G were active in ribosome dependent GTPase activity. The kinetic parameters (K _M) for the interaction of EF-G1A and EF-G1B with GTP were 85 and 70 μM, respectively. However, EF-G1B exhibited a 5-fold greater turnover number (observed k _cat) for the hydrolysis of GTP than EF-G1A; 0.2 s^-1 vs. 0.04 s^-1. These values resulted in specificity constants (k _cat ^obs/K _M) for EF-G1A and EF-G1B of 0.5 x 10³ s^-1 M^-1 and 3.0 x 10³ s^-1 M^-1, respectively. The antibiotic fusidic acid (FA) completely inhibited poly(U)-dependent protein synthesis containing P. aeruginosa EF-G1B, but the same protein synthesis system containing EF-G1A was not affected. Likewise, the activity of EF-G1B in ribosome dependent GTPase assays was completely inhibited by FA, while the activity of EF-G1A was not affected.     

Combined MRI and 31P-MRS Investigations of the ACTA1(H40Y) Mouse Model of Nemaline Myopathy Show Impaired Muscle Function and Altered Energy Metabolism

Nemaline myopathy (NM) is the most common disease entity among non-dystrophic skeletal muscle congenital diseases. Mutations in the skeletal muscle α-actin gene (ACTA1) account for ∼25% of all NM cases and are the most frequent cause of severe forms of NM. So far, the mechanisms underlying muscle weakness in NM patients remain unclear. Additionally, recent Magnetic Resonance Imaging (MRI) studies reported a progressive fatty infiltration of skeletal muscle with a specific muscle involvement in patients with ACTA1 mutations. We investigated strictly noninvasively the gastrocnemius muscle function of a mouse model carrying a mutation in the ACTA1 gene (H40Y). Skeletal muscle anatomy (hindlimb muscles and fat volumes) and energy metabolism were studied using MRI and ³¹Phosphorus magnetic resonance spectroscopy. Skeletal muscle contractile performance was investigated while applying a force-frequency protocol (from 1–150 Hz) and a fatigue protocol (80 stimuli at 40 Hz). H40Y mice showed a reduction of both absolute (−40%) and specific (−25%) maximal force production as compared to controls. Interestingly, muscle weakness was associated with an improved resistance to fatigue (+40%) and an increased energy cost. On the contrary, the force frequency relationship was not modified in H40Y mice and the extent of fatty infiltration was minor and not different from the WT group. We concluded that the H40Y mouse model does not reproduce human MRI findings but shows a severe muscle weakness which might be related to an alteration of intrinsic muscular properties. The increased energy cost in H40Y mice might be related to either an impaired mitochondrial function or an alteration at the cross-bridges level. Overall, we provided a unique set of anatomic, metabolic and functional biomarkers that might be relevant for monitoring the progression of NM disease but also for assessing the efficacy of potential therapeutic interventions at a preclinical level.