TNFα increases resting potential in isolated fibres from rat peroneus longus by a PKC mediated mechanism: involvement in ICU acquired polyneuromyopathy

Background and aims

Our aim was to investigate the effect of TNFα on muscle resting potential (RP) and then in muscle excitability and to demonstrate another mechanism implicated in intensive care units (ICU) acquired polyneuromyopathy.

Methods

Experiments were carried out on adult female Wistar rats. After isolation of muscle fibres from peroneus longus, influence of TNFα was tested on RP by using intracellular microelectrodes. Digoxin and chelerythrin were used to determine the mechanism of TNFα action.

Results

First, we found that TNFα induced a concentration dependent increase of muscle RP and that this mechanism, which was blocked by digoxin, was due to an effect on the Na/K ATPase. As it was also blocked by chelerythrin it was concluded that this effect was mediated by PKC activation of the Na/K ATPase.

Conclusions

We demonstrated that TNFα leads to a PKC mediated increase in muscle RP. Depolarization needed to reach the threshold voltage for muscle action potential should then be higher and this could be involved in the decrease in muscle excitability observed in acquired polyneuromyopathy.     

KCNQ1 channels voltage dependence through a voltage-dependent binding of the S4-S5 linker to the pore domain

Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1–S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5–S6) is surrounded by four voltage sensor domains (S1–S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5_L) and S6 C terminus (S6_T). From these data, we hypothesized that S4S5_L behaves like a ligand specifically interacting with S6_T and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5_L and S6_T of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5_L peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5_L peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6_T, consistent with S4S5_L binding to S6_T. Val²⁵⁴ in S4S5_L is known to contact Leu³⁵³ in S6_T when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5_L binding to S6_T. Our results suggest a mechanistic model in which S4S5_L acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5_L away from S6_T, allowing channel opening.     

Biases and Power for Groups Comparison on Subjective Health Measurements

Subjective health measurements are increasingly used in clinical research, particularly for patient groups comparisons. Two main types of analytical strategies can be used for such data: so-called classical test theory (CTT), relying on observed scores and models coming from Item Response Theory (IRT) relying on a response model relating the items responses to a latent parameter, often called latent trait. Whether IRT or CTT would be the most appropriate method to compare two independent groups of patients on a patient reported outcomes measurement remains unknown and was investigated using simulations. For CTT-based analyses, groups comparison was performed using t-test on the scores. For IRT-based analyses, several methods were compared, according to whether the Rasch model was considered with random effects or with fixed effects, and the group effect was included as a covariate or not. Individual latent traits values were estimated using either a deterministic method or by stochastic approaches. Latent traits were then compared with a t-test. Finally, a two-steps method was performed to compare the latent trait distributions, and a Wald test was performed to test the group effect in the Rasch model including group covariates. The only unbiased IRT-based method was the group covariate Wald’s test, performed on the random effects Rasch model. This model displayed the highest observed power, which was similar to the power using the score t-test. These results need to be extended to the case frequently encountered in practice where data are missing and possibly informative.     

The human DNA ends proteome uncovers an unexpected entanglement of functional pathways

DNA ends get exposed in cells upon either normal or dysfunctional cellular processes or molecular events. Telomeres need to be protected by the shelterin complex to avoid junctions occurring between chromosomes while failing topoisomerases or clustered DNA damage processing may produce double-strand breaks, thus requiring swift repair to avoid cell death. The rigorous study of the great many proteins involved in the maintenance of DNA integrity is a challenging task because of the innumerous unspecific electrostatic and/or hydrophobic DNA—protein interactions that arise due to the chemical nature of DNA. We devised a technique that discriminates the proteins recruited specifically at DNA ends from those that bind to DNA because of a generic affinity for the double helix. Our study shows that the DNA ends proteome comprises proteins of an unexpectedly wide functional spectrum, ranging from DNA repair to ribosome biogenesis and cytoskeleton, including novel proteins of undocumented function. A global mapping of the identified proteome on published DNA repair protein networks demonstrated the excellent specificity and functional coverage of our purification technique. Finally, the native nucleoproteic complexes that assembled specifically onto DNA ends were shown to be endowed with a highly efficient DNA repair activity.     

Seeds,browse, and tooth wear: a sheep perspective

While grazing as a selective factor towards hypsodont dentition on mammals has gained a lot of attention, the importance of fruits and seeds as fallback resources for many browsing ungulates has caught much less attention. Controlled‐food experiments, by reducing the dietary range, allow for a direct quantification of the effect of each type of items separately on enamel abrasion. We present the results of a dental microwear texture analysis on 40 ewes clustered into four different controlled diets: clover alone, and then three diets composed of clover together with either barley, corn, or chestnuts. Among the seed‐eating groups, only the barley one shows higher complexity than the seed‐free group. Canonical discriminant analysis is successful at correctly classifying the majority of clover‐ and seed‐fed ewes. Although this study focuses on diets which all fall within a single dietary category (browse), the groups show variations in dental microwear textures in relation with the presence and the type of seeds. More than a matter of seed size and hardness, a high amount of kernels ingested per day is found to be correlated with high complexity on enamel molar facets. This highlights the high variability of the physical properties of the foods falling under the browsing umbrella.     

Ecdysteroids from the medicinal fern Microsorum scolopendria (Burm. f.)

Fronds of the fern Microsorum scolopendria are widely used in traditional medicine in the Society Islands. They were investigated for the presence of ecdysteroids, which might be responsible for at least some of their medicinal properties. M. scolopendria represents an excellent source of ecdysone (0.16% of dry weight) and 20‐hydroxyecdysone (0.20%), and also contains significant amounts (0.01–0.02%) of makisterones A and C, inokosterone and amarasterone A, together with lower amounts of poststerone and of a compound tentatively identified as 24,28‐diepi‐cyasterone. During this study, three new minor phytoecdysteroids, namely 20‐deoxymakisterone A, a 25(?)‐epimer of amarasterone A and 25‐deoxyecdysone 22‐glucoside were also isolated by a combination of normal‐ and reversed‐phase HPLC and subsequently identified by NMR. Copyright © 2007 John Wiley & Sons, Ltd.     

Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation

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PAMP (Pathogen-associated Molecular Pattern)-induced Changes in Plasma Membrane Compartmentalization Reveal Novel Components of Plant Immunity

Plasma membrane compartmentalization spatiotemporally regulates cell-autonomous immune signaling in animal cells. To elucidate immediate early protein dynamics at the plant plasma membrane in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin (flg22) we employed quantitative mass spectrometric analysis on detergent-resistant membranes (DRMs) of Arabidopsis thaliana suspension cells. This approach revealed rapid and profound changes in DRM protein composition following PAMP treatment, prominently affecting proton ATPases and receptor-like kinases, including the flagellin receptor FLS2. We employed reverse genetics to address a potential contribution of a subset of these proteins in flg22-triggered cellular responses. Mutants of three candidates (DET3, AHA1, FER) exhibited a conspicuous defect in the PAMP-triggered accumulation of reactive oxygen species. In addition, these mutants showed altered mitogen-activated protein kinase (MAPK) activation, a defect in PAMP-triggered stomatal closure as well as altered bacterial infection phenotypes, which revealed three novel players in elicitor-dependent oxidative burst control and innate immunity. Our data provide evidence for dynamic elicitor-induced changes in the membrane compartmentalization of PAMP signaling components.     

Can Dental Microwear Textures Record Inter-Individual Dietary Variations?

Background

Dental microwear analyses are commonly used to deduce the diet of extinct mammals. Conventional methods rely on the user identifying features within a 2D image. However, recent interdisciplinary research has lead to the development of an advanced methodology that is free of observer error, based on the automated quantification of 3D surfaces by combining confocal microscopy with scale-sensitive fractal analysis. This method has already proved to be very efficient in detecting dietary differences between species. Focusing on a finer, intra-specific scale of analysis, the aim of this study is to test this method''s ability to track such differences between individuals from a single population.

Methodology/Principal Findings

For the purposes of this study, the 3D molar microwear of 78 individuals from a well-known population of extant roe deer (Capreolus caprelous) is quantified. Multivariate statistical analyses indicate significant seasonal and sexual differences in individual dental microwear design. These are probably the consequence of seasonal variations in fruit, seed and leaf availability, as well as differences in feeding preference between males and females due to distinct energy requirements during periods of rutting, gestation or giving birth. Nevertheless, further investigations using two-block Partial Least-Squares analysis show no strong relationship between individual stomach contents and microwear texture. This is an expected result, assuming that stomach contents are composed of food items ingested during the last few hours whereas dental microwear texture records the physical properties of items eaten over periods of days or weeks.

Conclusions/Significance

Microwear 3D scale-sensitive fractal analysis does detect differences in diet ranging from the inter-feeding styles scale to the intra-population between-season and between-sex scales. It is therefore a possible tool, to be used with caution, in the further exploration of the feeding biology and ecology of extinct mammals.