Non-linear dynamics in muscle fatigue and strength model during maximal self-perceived elbow extensors training

Our aim was to determine the dynamics in muscle strength increase and fatigue development during repetitive maximal contraction in specific maximal self-perceived elbow extensors training program. We will derive our functional model for m. triceps brachii in spirit of traditional Hill’s two-component muscular model and after fitting our data, develop a prediction tool for this specific training system. Thirty-six healthy young men (21±1.0 y, BMI 25.4±7.2 kg/m²), who did not take part in any formal resistance exercise regime, volunteered for this study. The training protocol was performed on the isoacceleration dynamometer, lasted for 12 weeks, with a frequency of five sessions per week. Each training session included five sets of 10 maximal contractions (elbow extensions) with a 1 min resting period between each set. The non-linear dynamic system model was used for fitting our data in conjunction with the Levenberg–Marquardt regression algorithm. As a proper dynamical system, our functional model of m. triceps brachii can be used for prediction and control. The model can be used for the predictions of muscular fatigue in a single series, the cumulative daily muscular fatigue and the muscular growth throughout the training process. In conclusion, the application of non-linear dynamics in this particular training model allows us to mathematically explain some functional changes in the skeletal muscle as a result of its adaptation to programmed physical activity—training.     

Kinetics of proton transport into influenza virions by the viral M2 channel

M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pK(m) of ～4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.     

Up-regulation of the SOX3 gene expression by retinoic acid: characterization of the novel promoter-response element and the retinoid receptors involved

Sox3/SOX3 gene is considered to be one of the earliest neural markers in vertebrates and it is implicated in the genetic cascades that direct brain formation. We have previously shown that early phases of differentiation and neural induction of NT2/D1 embryonal carcinoma cells by retinoic acid (RA) involve up-regulation of the SOX3 gene expression. Here, we present identification of a novel positive regulatory promoter element involved in RA-dependent activation of the SOX3 gene expression in NT2/D1 cells. This element represents a direct repeat 3-like motif that directly interacts with retinoid X receptor (RXR) alpha in a sequence-specific manner. It is capable of independently mediating the RA effect in a heterologous promoter context and its disruption caused significant reduction of RA/RXR transactivation of the SOX3 promoter. Furthermore, by using synthetic antagonists of retinoid receptors, we have shown for the first time, that RA-induced SOX3 gene expression could be significantly down-regulated by the synthetic antagonist of RXR. Also, this data showed that RXRs, but not RA receptors, are mediators of RA effect on the SOX3 gene up-regulation in NT2/D1 cells. Presented data will be valuable for future investigation of SOX3 gene expression, not only in NT2/D1 model system, but also in diverse developmental, physiological and pathological settings.     

Identification of Novel Genetic Alterations in Samples of Malignant Glioma Patients

Glioblastoma is the most frequent and malignant human brain tumor. High level of genomic instability detected in glioma cells implies that numerous genetic alterations accumulate during glioma pathogenesis. We investigated alterations in AP-PCR DNA profiles of 30 glioma patients, and detected specific changes in 11 genes not previously associated with this disease: LHFPL3, SGCG, HTR4, ITGB1, CPS1, PROS1, GP2, KCNG2, PDE4D, KIR3DL3, and INPP5A. Further correlations revealed that 8 genes might play important role in pathogenesis of glial tumors, while changes in GP2, KCNG2 and KIR3DL3 should be considered as passenger mutations, consequence of high level of genomic instability. Identified genes have a significant role in signal transduction or cell adhesion, which are important processes for cancer development and progression. According to our results, LHFPL3 might be characteristic of primary glioblastoma, SGCG, HTR4, ITGB1, CPS1, PROS1 and INPP5A were detected predominantly in anaplastic astrocytoma, suggesting their role in progression of secondary glioblastoma, while alterations of PDE4D seem to have important role in development of both glioblastoma subtypes. Some of the identified genes showed significant association with p53, p16, and EGFR, but there was no significant correlation between loss of PTEN and any of identified genes. In conclusion our study revealed genetic alterations that were not previously associated with glioma pathogenesis and could be potentially used as molecular markers of different glioblastoma subtypes.     

Non-contiguous finished genome sequence and contextual data of the filamentous soil bacterium Ktedonobacter racemifer type strain (SOSP1-21)

Ktedonobacter racemifer corrig. Cavaletti et al. 2007 is the type species of the genus Ktedonobacter, which in turn is the type genus of the family Ktedonobacteraceae, the type family of the order Ktedonobacterales within the class Ktedonobacteria in the phylum 'Chloroflexi'. Although K. racemifer shares some morphological features with the actinobacteria, it is of special interest because it was the first cultivated representative of a deep branching unclassified lineage of otherwise uncultivated environmental phylotypes tentatively located within the phylum 'Chloroflexi'. The aerobic, filamentous, non-motile, spore-forming Gram-positive heterotroph was isolated from soil in Italy. The 13,661,586 bp long non-contiguous finished genome consists of ten contigs and is the first reported genome sequence from a member of the class Ktedonobacteria. With its 11,453 protein-coding and 87 RNA genes, it is the largest prokaryotic genome reported so far. It comprises a large number of over-represented COGs, particularly genes associated with transposons, causing the genetic redundancy within the genome being considerably larger than expected by chance. This work is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining

In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can potentially be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies suggest that these two mechanistically distinct repair pathways can compete for DNA ends. Because HR and NHEJ differ with respect to error susceptibility, generation of chromosome rearrangements, which are potentially carcinogenic products of DSB repair, may depend on the pathway choice. To investigate this hypothesis, the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations in G2 phase cells was investigated. SW-1573 and RKO cells were treated with mild (41 °C) hyperthermia in order to disable HR and/or NU7441/cisplatin to inactivate NHEJ and frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) were studied using premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH).It is shown here that temporary inhibition of HR by hyperthermia results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and that this effect is abrogated by NU7441- or cisplatin-mediated inhibition of NHEJ. The results suggest that in the absence of HR, DSB repair is shifted to the error-prone NHEJ pathway resulting in increased frequencies of chromosomal rearrangements. These results might be of consequence for clinical cancer treatment approaches that aim at inhibition of one or more DSB repair pathways.     

Hollow fiber bioreactor: new development for the study of contrast agent transport into hepatocytes by magnetic resonance imaging

The aim of our study was to develop a magnetic resonance (MR)-compatible in vitro model containing freshly isolated rat hepatocytes to study the transport of hepatobiliary contrast agents (CA) by MR imaging (MRI). We set up a perfusion system including a perfusion circuit, a heating device, an oxygenator, and a hollow fiber bioreactor (HFB). The role of the porosity and surface of the hollow fiber (HF) as well as the perfusate flow rate applied on the diffusion of CAs and O2 was determined. Hepatocytes were isolated and injected in the extracapillary space of the HFB (4 x 10(7) cells/mL). The hepatocyte HFB was perfused with an extracellular CA, gadopentetate dimeglumine (Gd-DTPA), and gadobenate dimeglumine (Gd-BOPTA), which also enters into hepatocytes. The HFB was imaged in the MR room using a dynamic T1-weighed sequence. No adsorption of CAs was detected in the perfusion system without hepatocytes. The use of a membrane with a high porosity (0.5 microm) and surface (420 cm2), and a high flow rate perfusion (100 mL/min) resulted in a rapid filling of the HFB with CAs. The cellular viability of hepatocytes in the HFB was greater than 85% and the O2 consumption was maintained over the experimental period. The kinetics of MR signal intensity (SI) clearly showed the different behavior of Gd-BOPTA that enters into hepatocytes and Gd-DTPA that remains extracellular. Thus, these results show that our newly developed in vitro model is an interesting tool to investigate the transport kinetics of hepatobiliary CAs by measuring the MR SI over time.     

Dynamic lateral organization of opioid receptors (kappa,muwt and muN40D) in the plasma membrane at the nanoscale level

Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited. Since these dynamic properties are important determinants of receptor function, it is crucial to define them. Herein, the nanoscale lateral dynamics and spatial organization of kappa opioid receptor (KOP), wild type mu opioid receptor (MOP_wt), and its naturally occurring isoform (MOP_N40D) were quantitatively characterized using fluorescence correlation spectroscopy and photoactivated localization microscopy. Obtained results, supported by ensemble‐averaged Monte Carlo simulations, indicate that these opioid receptors dynamically partition into different domains. In particular, significant exclusion from GM1 ganglioside‐enriched domains and partial association with cholesterol‐enriched domains was observed. Nanodomain size, receptor population density and the fraction of receptors residing outside of nanodomains were receptor‐specific. KOP‐containing domains were the largest and most densely populated, with the smallest fraction of molecules residing outside of nanodomains. The opposite was true for MOP_N40D. Moreover, cholesterol depletion dynamically regulated the partitioning of KOP and MOP_wt, whereas this effect was not observed for MOP_N40D.      

Recruitment of cellular clathrin to viral factories and disruption of clathrin-dependent trafficking

The viral factories of mammalian reovirus (MRV) are cytoplasmic structures that serve as sites of viral genome replication and particle assembly. A 721-aa MRV non-structural protein, μNS, forms the factory matrix and recruits other viral proteins to these structures. In this report, we show that μNS contains a conserved C-proximal sequence (711-LIDFS-715) that is similar to known clathrin-box motifs and is required for recruitment of clathrin to viral factories. Clathrin recruitment by μNS occurs independently of infecting MRV particles or other MRV proteins. Ala substitution for a single Leu residue (mutation L711A) within the putative clathrin-binding motif of μNS inhibits clathrin recruitment, but does not prevent formation or expansion of viral factories. Notably, clathrin-dependent cellular functions, including both endocytosis and secretion, are disrupted in cells infected with MRV expressing wild-type, but not L711A, μNS. These results identify μNS as a novel adaptor-like protein that recruits cellular clathrin to viral factories, disrupting normal functions of clathrin in cellular membrane trafficking. To our knowledge, this is the only viral or bacterial protein yet shown to interfere with clathrin functions in this manner. The results additionally establish a new approach for studies of clathrin functions, based on μNS-mediated sequestration.