Radioiodination and characterization of the plasma membrane of sea urchin sperm

Two methods were used to radioiodinate sea urchin sperm: lactoperoxidase-glucose oxidase and Iodo-Gen. Following iodination the sperm are viable, they undergo the acrosome reaction, and they fertilize eggs. Of the radioactivity associated with the labeled sperm, 28–50% is presumed to be free ¹²⁵I^?, 37–47% is incorporated in lipid, and 8–15% is in trypsin-digestible material believed to be protein. Digestion of the labeled, living sperm with trypsin removes 95.6–99.5% of the macromolecular label (the cells are alive after digestion) suggesting that almost all the protein label is on the external surface of the cell. Thin-layer chromatography of the lipid fraction shows that the major membrane phospholipids and cholesterol are labeled. SDS-PAGE analysis shows the protein-incorporated ¹²⁵I is distributed among four glycoproteins of >250K, 84K, 64K, and 52K dalton apparent molecular weight. Twenty-eight percent of the total protein (trypsin-digestible) label is in the 84K component and 46% in the 64K band. Although both molecules contain much of the label, they are relatively minor components of the TX-100 extract of sperm. The methods outlined will be useful in determining the role of sperm surface components in fertilization.     

Non-zero mean alpha oscillations revealed with computational model and empirical data

Ongoing oscillations and evoked responses are two main types of neuronal activity obtained with diverse electrophysiological recordings (EEG/MEG/iEEG/LFP). Although typically studied separately, they might in fact be closely related. One possibility to unite them is to demonstrate that neuronal oscillations have non-zero mean which predicts that stimulus- or task-triggered amplitude modulation of oscillations can contribute to the generation of evoked responses. We validated this mechanism using computational modelling and analysis of a large EEG data set. With a biophysical model, we indeed demonstrated that intracellular currents in the neuron are asymmetric and, consequently, the mean of alpha oscillations is non-zero. To understand the effect that neuronal currents exert on oscillatory mean, we varied several biophysical and morphological properties of neurons in the network, such as voltage-gated channel densities, length of dendrites, and intensity of incoming stimuli. For a very large range of model parameters, we observed evidence for non-zero mean of oscillations. Complimentary, we analysed empirical rest EEG recordings of 90 participants (50 young, 40 elderly) and, with spatio-spectral decomposition, detected at least one spatially-filtred oscillatory component of non-zero mean alpha oscillations in 93% of participants. In order to explain a complex relationship between the dynamics of amplitude-envelope and corresponding baseline shifts, we performed additional simulations with simple oscillators coupled with different time delays. We demonstrated that the extent of spatial synchronisation may obscure macroscopic estimation of alpha rhythm modulation while leaving baseline shifts unchanged. Overall, our results predict that amplitude modulation of neural oscillations should at least partially explain the generation of evoked responses. Therefore, inference about changes in evoked responses with respect to cognitive conditions, age or neuropathologies should be constructed while taking into account oscillatory neuronal dynamics.     

Epicardial adipose tissue extent: relationship with age, body fat distribution, and coronaropathy

Epicardial fat is a relatively neglected component of the heart and could be an important risk factor of cardiac disease. The objective of our study was to assess the relationship between epicardial adipose tissue (EAT) extent, fat distribution, and coronaropathy in a group of adult victims of accidental or suspicious sudden death. In 56 cadavers, we performed 34 measurements of EAT from five computerized photographs of the heart (anterior and posterior faces, and three ventricle transversal slices) and analyzed their relationship with anthropometric markers of adiposity (BMI, waist and leg circumference, thickness of abdominal and thigh subcutaneous adipose tissue (SAT)), with the presence and staging of coronary artery disease (CAD), and with markers of myocardial hypertrophy. Simple linear regressions showed that EAT measurements are highly intercorrelated (r from 0.4 to 0.6, P < 0.001), and correlate with age, waist circumference, and heart weight, and to a lesser extent, with BMI, abdominal SAT thickness, and leg SAT thickness. Multiple regression showed that age, waist circumference, and heart weight significantly and independently correlate with EAT (P < 0.0001). No other anthropometric measurement was found independently correlated with EAT. The EAT/myocardium ratios correlated positively with age and waist circumference. Anterior and posterior areas of EAT were found significantly increased in patients with CAD and correlated positively with CAD staging (P = 0.0034, r = 0.38). Anterior EAT surface was found positively associated with CAD (P = 0.01), independently of age and other adiposity measurements. Prospective studies are needed to assess the risk of occurrence/progression of CAD that relate to EAT excess.     

Fluorescence imaging in vivo: recent advances

In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the near-infrared (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addition of fluorescent organic, inorganic and biological nanoparticles. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resolution, multimodality and lifetime-based in vivo fluorescence imaging.     

Molecular profile of the NF‐κB signalling pathway in human colorectal cancer

The development and progression of colorectal cancer (CRC) have been associated with inflammation processes that involve the overactivation of the NF‐κB signalling pathway. The characterization of the NF‐κB expression profile in CRC is an important topic since the suppression of NF‐κB represents a potential therapeutic approach. In this study, we assessed the expression levels of 84 NF‐κB‐related genes in paired tumoral (T) and peritumoral (PT) tissues from 18 CRC patients and 18 normal colonic mucosae, and the expression levels of three miRNAs targeting the most dysregulated genes revealed by the case–control analysis. Comparing the gene expression profile of T and controls, 60 genes were dysregulated. The comparison of T and PT revealed 17 dysregulated genes in the tumoral tissues, with IL1B, CXCL8, IL1A, and CSF2 being the most upregulated. Notably, through a bioinformatics analysis, the differential gene expression of 11 out of the 17 genes was validated on a larger cohort of 308 CRC patients compared with 41 controls. Moreover, a decrease in the levels of RELA, NOD1, CASP8, BCL2L1, ELK1, and IKBKB was identified in poorly differentiated tumours compared to moderately differentiated tumours. The analysis of the three miRNAs targeting IL1B, CXCL8, IL1A, and CSF2 showed that miR‐182‐5p was upregulated in T compared with PT, whereas miR‐10b‐5p was downregulated in T compared with PT and control tissues. Our results may contribute to the design of new experimental therapeutic strategies based on endogenous molecules, such as miRNAs, to target the genetic key players of the NF‐ κB pathway.     

Cellular recognition of trimyristoylated peptide or enterobacterial lipopolysaccharide via both TLR2 and TLR4

Evidence for specific and direct bacterial product recognition through toll-like receptors (TLRs) has been emphasized recently. We analyzed lipopeptide analogues and enterobacterial lipopolysaccharide (eLPS) for their potential to activate cells through TLR2 and TLR4. Whereas bacterial protein palmitoylated at its N-terminal cysteine and N-terminal peptides derived thereof are known to induce TLR2-mediated cell activation, a synthetic acylhexapeptide mimicking a bacterial lipoprotein subpopulation for which N-terminal trimyristoylation is characteristic (Myr(3)CSK(4)) activated cells not only through TLR2 but also through TLR4. Conversely, highly purified eLPS triggered cell activation through overexpressed TLR2 in the absence of TLR4 expression if CD14 was coexpressed. Accordingly, TLR2(-/-) macrophages prepared upon gene targeting responded to Myr(3)CSK(4) challenge, whereas TLR2(-/-)/TLR4(d/d) cells were unresponsive. Through interferon-gamma (IFNgamma) priming, macrophages lacking expression of functional TLR4 and/or MD-2 acquired sensitivity to eLPS, whereas TLR2/TLR4 double deficient cells did not. Not only TLR2(-/-) mice but also TLR4(-/-) mice were resistant to Myr(3)CSK(4) challenge-induced fatal shock. d-Galactosamine-sensitized mice expressing defective TLR4 or lacking TLR4 expression acquired susceptibility to eLPS-driven toxemia upon IFNgamma priming, whereas double deficient mice did not. Immunization toward ovalbumin using Myr(3)CSK(4) as adjuvant was ineffective in TLR2(-/-)/TLR4(-/-) mice yet effective in wild-type, TLR2(-/-), or TLR4(-/-) mice as shown by analysis of ovalbumin-specific serum Ig concentration. A compound such as Myr(3)CSK(4) whose stimulatory activity is mediated by both TLR2 and TLR4 might constitute a preferable adjuvant. On the other hand, simultaneous blockage of both of the two TLRs might effectively inhibit infection-induced pathology.     

Mutations at residues 282, 286, and 293 of phage lambda integrase exert pathway-specific effects on synapsis and catalysis in recombination

Bacteriophage lambda integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. The att sites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: the attL site probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA with attR.     

ATPase‐driven oligomerization of RIG‐I on RNA allows optimal activation of type‐I interferon

The cytosolic pathogen sensor RIG‐I is activated by RNAs with exposed 5′‐triphosphate (5′‐ppp) and terminal double‐stranded structures, such as those that are generated during viral infection. RIG‐I has been shown to translocate on dsRNA in an ATP‐dependent manner. However, the precise role of the ATPase activity in RIG‐I activation remains unclear. Using in vitro‐transcribed Sendai virus defective interfering RNA as a model ligand, we show that RIG‐I oligomerizes on 5′‐ppp dsRNA in an ATP hydrolysis‐dependent and dsRNA length‐dependent manner, which correlates with the strength of type‐I interferon (IFN‐I) activation. These results establish a clear role for the ligand‐induced ATPase activity of RIG‐I in the stimulation of the IFN response.     

Raloxifene attenuates Gas6 and apoptosis in experimental aortic valve disease in renal failure

Renal failure is associated with aortic valve calcification. Using our rat model of uremia-induced reversible aortic valve calcification, we assessed the role of apoptosis and survival pathways in that disease. We also explored the effects of raloxifene, an estrogen receptor modulator, on valvular calcification. Gene array analysis was performed in aortic valves obtained from three groups of rats (n = 7 rats/group): calcified valves obtained from rats fed with uremic diet, valves after calcification resolution following diet cessation, and control. In addition, four groups of rats (n = 10 rats/group) were used to evaluate the effect of raloxifene in aortic valve calcification: three groups as mentioned above and a fourth group fed with the uremic diet that also received daily raloxifene. Evaluation included imaging, histology, and antigen expression analysis. Gene array results showed that the majority of the altered expressed genes were in diet group valves. Most apoptosis-related genes were changed in a proapoptotic direction in calcified valves. Apoptosis and decreases in several survival pathways were confirmed in calcified valves. Resolution of aortic valve calcification was accompanied by decreased apoptosis and upregulation of survival pathways. Imaging and histology demonstrated that raloxifene significantly decreased aortic valve calcification. In conclusion, downregulation of several survival pathways and apoptosis are involved in the pathogenesis of aortic valve calcification. The beneficial effect of raloxifene in valve calcification is related to apoptosis modulation. This novel observation is important for developing remedies for aortic valve calcification in patients with renal failure.     

Symmetry of septin hourglass and ring structures

Septin filaments form ordered hourglass and ring-shaped structures in close apposition to the yeast bud-neck membrane. The septin hourglass scaffolds the asymmetric localization of many essential cell division proteins. However, it is unknown whether the septin structures have an overall polarity along the mother-daughter axis that determines the asymmetric protein localization. Here we engineered rigid septin- green fluorescent protein (GFP) fusions with various fluorescence dipole directions by changing the position of the GFP beta-barrel relative to the septin filament axis. We then used polarized fluorescence microscopy to detect potential asymmetries in the filament organization. We found that both the hourglass and ring filament assemblies have sub-resolution C(2) symmetry and lack net polarity along the mother-daughter axis. The hourglass filaments have an additional degree of symmetry relative to the ring filaments, most likely due to a twist in their higher-order structure. We previously reported that during the hourglass to rings transition septin filaments change their direction. Here we show that the filaments also undergo a change in their lateral organization, consistent with filament untwisting. The lack of net septin polarity along the mother-daughter axis suggests that there are no septin-based structural reasons for the observed asymmetry of other proteins. We discuss possible anisotropic processes that could break the septin symmetry and establish the essential bud-neck asymmetry.