Computational biomechanics of a lumbar motion segment in pure and combined shear loads

Anterior shear has been implicated as a risk factor in spinal injuries. A 3D nonlinear poroelastic finite element model study of a lumbar motion segment L4-L5 was performed to predict the temporal shear response under various single and combined shear loads. Effects of nucleotomy and facetectomy as well as changes in the posture and facet gap distance were analyzed as well.     

Body size as an important factor determining trophic niche partitioning in three syntopic rhinolophid bat species

We investigated a community of syntopically occurring horseshoe bats (Rhinolophus hipposideros, R. euryale, R. ferrumequinum) in southern Slovakia. The faecal pellets of these bat species were collected in the field and later analysed under a dissecting microscope. The three species studied are known to be very similar as far as their ecology, echolocation and preferred habitats are concerned, but they diverge significantly in their body sizes. In this study, all three species fed predominantly on moths [59–80 percentage frequency (%f); 87–95 percentage volume (%vol)], but their diet compositions differed in the size of individuals consumed. The smallest bat species (R. hipposideros) fed only on the smallest moths (%f = 59; %vol = 87), the medium-sized species (R. euryale) mainly on medium-sized moths (%f = 60; %vol = 74) and the largest one (R. ferrumequinum) especially on the largest moths (%f = 54; %vol = 89). Despite similar preferred habitat and the main prey category, the rates of trophic niche overlap were surprisingly low. The trophic niche percentage overlap was 7–31% (computed from %f data) and 1–20% (computed from %vol data), respectively and suggests an extraordinary importance of mere divergences of bats in their body sizes for trophic niche partitioning and stable species coexistence.     

Cynaropicrin targets the trypanothione redox system in Trypanosoma brucei

In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei—the causative agent of Human African Trypanosomiasis—by a so far unknown mechanism. We hypothesized that CYNs α,β-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)₂), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)₂ redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC–MS/MS analysis was established to quantify intra-cellular CYN, T(SH)₂, GSH, as well as GS-CYN and T(S-CYN)₂ adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)₂ pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)₂ redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)₂ redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism.     

Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line’s sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.     

Spontaneous Atopic Dermatitis-Like Symptoms in a/a ma ft/ma ft/J Flaky Tail Mice Appear Early after Birth

Loss-of-function mutations in human profilaggrin gene have been identified as the cause of ichthyosis vulgaris (IV), and as a major predisposition factor for atopic dermatitis (AD). Similarly, flaky tail (a/a ma ft/ma ft/J) mice were described as a model for IV, and shown to be predisposed to eczema. The aim of this study was to correlate the flaky tail mouse phenotype with human IV and AD, in order to dissect early molecular events leading to atopic dermatitis in mice and men, suffering from filaggrin deficiency. Thus, 5-days old flaky tail pups were analyzed histologically, expression of cytokines was measured in skin and signaling pathways were investigated by protein analysis. Human biopsies of IV and AD patients were analyzed histologically and by real time PCR assays. Our data show acanthosis and hyperproliferation in flaky tail epidermis, associated with increased IL1β and thymic stromal lymphopoietin (TSLP) expression, and Th2-polarization. Consequently, NFκB and Stat pathways were activated, and IL6 mRNA levels were increased. Further, quantitative analysis of late epidermal differentiation markers revealed increased Small proline-rich protein 2A (Sprr2a) synthesis. Th2-polarization and Sprr2a increase may result from high TSLP expression, as shown after analysis of 5-days old K14-TSLP tg mouse skin biopsies. Our findings in the flaky tail mouse correlate with data obtained from patient biopsies of AD, but not IV. We propose that proinflammatory cytokines are responsible for acanthosis in flaky tail epidermis, and together with the Th2-derived cytokines lead to morphological changes. Accordingly, the a/a ma ft/ma ft/J mouse model can be used as an appropriate model to study early AD onset associated with profilaggrin deficiency.     

Agrobacterium-mediated transformation of Guignardia citricarpa: An efficient tool to gene transfer and random mutagenesis

Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone – AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant–pathogen interaction.     

A Clinical Evaluation of Statin Pleiotropy: Statins Selectively and Dose-Dependently Reduce Vascular Inflammation

Statins are thought to reduce vascular inflammation through lipid independent mechanisms. Evaluation of such an effect in atherosclerotic disease is complicated by simultaneous effects on lipid metabolism. Abdominal aortic aneurysms (AAA) are part of the atherosclerotic spectrum of diseases. Unlike atherosclerotic occlusive disease, AAA is not lipid driven, thus allowing direct evaluation of putative anti-inflammatory effects. The anti-inflammatory potency of increasing doses (0, 20 or 40 mg/day) simvastatin or atorvastatin was evaluated in 63 patients that were at least 6 weeks on statin therapy and who underwent open AAA repair. A comprehensive analysis using immunohistochemistry, mRNA and protein analyses was applied on aortic wall samples collected during surgery. The effect of statins on AAA growth was analyzed in a separate prospective study in incorporating 142 patients. Both statins equally effectively and dose-dependently reduced aortic wall expression of NFκB regulated mediators (i.e. IL-6 (P<0.001) and MCP-1 (P<0.001)); shifted macrophage polarization towards a M2 phenotype (P<0.0003); selectively reduced macrophage-related markers such as cathepsin K and S (P<0.009 and 0.0027 respectively), and ALOX5 (P<0.0009), and reduced vascular wall NFκB activity (40 mg/day group, P<0.016). No effect was found on other cell types. Evaluation of the clinical efficacy of statins to reduce AAA progression did not indicate an effect of statins on aneurysm growth (P<0.337). Hence, in the context of AAA the clinical relevance of statins pleiotropy appears minimal.     

Tracking Fungal Community Responses to Maize Plants by DNA- and RNA-Based Pyrosequencing

We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age) in pots associated with four maize cultivars, including two genetically modified (GM) cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA). The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most “active” fungi (as recovered via RNA). Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production). Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.     

Glucose Augments Killing Efficiency of Daptomycin Challenged Staphylococcus aureus Persisters

Treatment of Staphylococcus aureus in stationary growth phase with high doses of the antibiotic daptomycin (DAP) eradicates the vast majority of the culture and leaves persister cells behind. Despite resting in a drug-tolerant and dormant state, persister cells exhibit metabolic activity which might be exploited for their elimination. We here report that the addition of glucose to S. aureus persisters treated with DAP increased killing by up to five-fold within one hour. This glucose-DAP effect also occurred with strains less sensitive to the drug. The underlying mechanism is independent of the proton motive force and was not observed with non-metabolizable 2-deoxy-glucose. Our results are consistent with two hypotheses on the glucose-DAP interplay. The first is based upon glucose-induced carbohydrate transport proteins that may influence DAP and the second suggests that glucose may trigger the release or activity of cell-lytic proteins to augment DAP’s mode of action.     

Is individual consistency in body mass and reproductive decisions linked to individual specialization in foraging behavior in a long‐lived seabird?

Individual specialization in diet or foraging behavior within apparently generalist populations has been described for many species, especially in polar and temperate marine environments, where resource distribution is relatively predictable. It is unclear, however, whether and how increased environmental variability – and thus reduced predictability of resources – due to global climate change will affect individual specialization. We determined the within‐ and among‐individual components of the trophic niche and the within‐individual repeatability of δ¹³C and δ¹⁵N in feathers and red blood cells of individual female southern rockhopper penguins (Eudyptes chrysocome) across 7 years. We also investigated the effect of environmental variables (Southern Annular Mode, Southern Oscillation Index, and local sea surface temperature anomaly) on the isotopic values, as well as the link between stable isotopes and female body mass, clutch initiation dates, and total clutch mass. We observed consistent red blood cell δ¹³C and δ¹⁵N values within individuals among years, suggesting a moderate degree of within‐individual specialization in C and N during the prebreeding period. However, the total niche width was reduced and individual specialization not present during the premolt period. Despite significant interannual differences in isotope values of C and N and environmental conditions, none of the environmental variables were linked to stable isotope values and thus able to explain phenotypic plasticity. Furthermore, neither the within‐individual nor among‐individual effects of stable isotopes were found to be related to female body mass, clutch initiation date, or total clutch mass. In conclusion, our results emphasize that the degree of specialization within generalist populations can vary over the course of 1 year, even when being consistent within the same season across years. We were unable to confirm that environmental variability counteracts individual specialization in foraging behavior, as phenotypic plasticity in δ¹³C and δ¹⁵N was not linked to any of the environmental variables studied.